Virus-producing cells determine the host protein profiles of HIV-1 virion cores

  • Steven Santos1,

    Affiliated with

    • Yuri Obukhov2, 3,

      Affiliated with

      • Sergei Nekhai2, 3,

        Affiliated with

        • Michael Bukrinsky1 and

          Affiliated with

          • Sergey Iordanskiy1Email author

            Affiliated with

            Retrovirology20129:65

            DOI: 10.1186/1742-4690-9-65

            Received: 20 April 2012

            Accepted: 15 July 2012

            Published: 13 August 2012

            Abstract

            Background

            Upon HIV entry into target cells, viral cores are released and rearranged into reverse transcription complexes (RTCs), which support reverse transcription and also protect and transport viral cDNA to the site of integration. RTCs are composed of viral and cellular proteins that originate from both target and producer cells, the latter entering the target cell within the viral core. However, the proteome of HIV-1 viral cores in the context of the type of producer cells has not yet been characterized.

            Results

            We examined the proteomic profiles of the cores purified from HIV-1 NL4-3 virions assembled in Sup-T1 cells (T lymphocytes), PMA and vitamin D3 activated THP1 (model of macrophages, mMΦ), and non-activated THP1 cells (model of monocytes, mMN) and assessed potential involvement of identified proteins in the early stages of infection using gene ontology information and data from genome-wide screens on proteins important for HIV-1 replication. We identified 202 cellular proteins incorporated in the viral cores (T cells: 125, mMΦ: 110, mMN: 90) with the overlap between these sets limited to 42 proteins. The groups of RNA binding (29), DNA binding (17), cytoskeleton (15), cytoskeleton regulation (21), chaperone (18), vesicular trafficking-associated (12) and ubiquitin-proteasome pathway-associated proteins (9) were most numerous. Cores of the virions from SupT1 cells contained twice as many RNA binding proteins as cores of THP1-derived virus, whereas cores of virions from mMΦ and mMN were enriched in components of cytoskeleton and vesicular transport machinery, most probably due to differences in virion assembly pathways between these cells. Spectra of chaperones, cytoskeletal proteins and ubiquitin-proteasome pathway components were similar between viral cores from different cell types, whereas DNA-binding and especially RNA-binding proteins were highly diverse. Western blot analysis showed that within the group of overlapping proteins, the level of incorporation of some RNA binding (RHA and HELIC2) and DNA binding proteins (MCM5 and Ku80) in the viral cores from T cells was higher than in the cores from both mMΦ and mMN and did not correlate with the abundance of these proteins in virus producing cells.

            Conclusions

            Profiles of host proteins packaged in the cores of HIV-1 virions depend on the type of virus producing cell. The pool of proteins present in the cores of all virions is likely to contain factors important for viral functions. Incorporation ratio of certain RNA- and DNA-binding proteins suggests their more efficient, non-random packaging into virions in T cells than in mMΦ and mMN.

            Background

            HIV-1 viral particles released from infected cells have been shown to incorporate many cellular proteins during the assembly and budding steps of morphogenesis. Findings from earlier studies, summarized in a web-based database (http://​web.​ncifcrf.​gov/​research/​avp/​protein_​db.​asp), identified more than three hundred cellular proteins in HIV-1 particles. HIV-1, as well as other lentiviruses, incorporates components of the cellular endosomal sorting machinery and cytoskeleton proteins involved in the process of particle assembly [15], surface proteins captured with the plasma membrane during budding [6, 7], RNA-binding proteins associated with incorporated viral RNA and RNA-Gag complexes [4, 810], chaperones [11], and multiple concomitant proteins (reviewed in [12]) whose functions in viral morphogenesis and infectivity are still unknown.

            Upon fusion of an HIV-1 particle with a target cell, viral cores are released into the cytoplasm and rearranged into sub-viral particles called reverse transcription complexes (RTCs), which subsequently mature into pre-integration complexes (PICs). These nucleoprotein structures support reverse transcription and also protect and transport viral cDNA to the site of integration. RTCs are composed of both viral and cellular proteins. Since RTCs are formed from the viral cores, their initial composition is identical to that of viral cores. Other than the key enzymatic components, reverse transcriptase (RT) and integrase (IN), at least five other viral proteins involved in structural organization, cytoplasmic trafficking and nuclear import (matrix [MA], nucleocapsid [NC], capsid [CA], Nef and viral protein R [Vpr]), have been identified as components of HIV-1 RTCs [1319], reviewed in [2022].

            Although the key early steps of HIV replication, reverse transcription and integration, are relatively autonomic, the participation of cellular proteins in early infection events has been demonstrated in previous studies [2325]. After release from the viral particles, RTCs are still encapsulated in the shells formed by p24CA molecules which are stable in the cytoplasm for at least several hours [26, 27]. The shell is believed to protect the reverse transcription machinery and all encapsulated proteins from the cytoplasmic environment to provide optimal conditions for their functional activity [28] and may contribute to the nuclear import of PICs [19, 2931]. The shell likely limits the access of host cell proteins to the RTC interior. Thus, most cellular factors which may contribute to the functional competence of early RTCs should be expected to get into the complexes from the cores of infecting virions.

            The cellular proteins, which are known to be hijacked by assembling virus particles from virus-producing cells and are involved in the early post-entry stages of HIV-1 infection, can be grouped into the following categories. (1) Factors involved in the spatial organization and correct folding of viral proteins in the virion and probably RTC: clathrin [5, 32] and heat shock proteins (Hsp70, Hsc70, Hsp60) [11, 33] are probably critical for spatial organization of Gag and Pol proteins and regulation of proteolytic processing and folding of the Pol products – RT and integrase; thioltransferase is found in HIV-1 virions and may be important for dimerization and activation of the viral protease [34], (reviewed in [12]); staufen1, an RNA-binding protein is packaged into virions and is involved in incorporation of HIV-1 RNA [8]. Interaction of staufen1 with Pr55Gag zing finger motifs may also be important for Gag multimerization and formation of the viral capsids [35]. (2) Proteins which have an effect on cDNA synthesis/accumulation: lysyl-tRNA synthetase is incorporated through the interaction with Gag and is critical for the priming of reverse transcription [36]; uracil DNA glycosylase 2 (UNG2), a cellular DNA repair enzyme that binds HIV-1 integrase and Vpr [37, 38] . The role of this enzyme in early post-entry steps of infection remains controversial. The hypothesis that the catalytic activity of Vpr-associated UNG could modulate virus mutation rate and APOBEC3G-mediated G-to-A hypermutations [3941] was not supported by subsequent studies [42]. However, recently published work of Guenzel and co-authors showed that the virion-incorporated nuclear form of UNG2 facilitated reverse transcription through a non-enzymatic mechanism involving direct interaction with the p32 subunit of the replication protein A (RPA) complex [43]. RNA helicase A (RHA or DHX9) is packaged into HIV-1 virions probably through the interaction with an RNA or Gag polyprotein and facilitates reverse transcription [10]. The protein INI1/hSNF5, a member of SWI/SNF chromatin remodeling complex, has been shown to be packaged into virions through the direct binding to integrase [44], and is involved in the synthesis of reverse transcription products [45]. Later studies demonstrated that INI1/hSNF5 selectively recruits into HIV-1 virions the components of Sin3a-HDAC1 cellular complex, whose presence is critical for the early reverse transcription stage [46]. Furthermore, interaction of HIV-1 integrase with INI1 has been shown to be essential for the nucleosome remodeling of host chromatin and hence overcoming the structural nucleosome barrier for viral integration [47]. (3) Proteins involved in RTC formation, protection and transport: cyclophilin A, which is incorporated into virions via binding to the CA domain of Pr55Gag [23]. The role of CA-bound cyclophilin A in the viral life cycle is still unclear [48] Recently published data showed this protein to be critical for protection and stabilization of HIV-1 cores [49]. It may also be involved in PIC nuclear transport [31]. (4) Restriction factors of the early stages of HIV-1 infection: members of the APOBEC3 family of DNA/RNA editing cytidine deaminases, APOBEC3G (A3G) and APOBEC3F (A3F), are incorporated in Vif-negative virus particles (a small amount of these factors may be present also in Vif-positive virions) and then restrict reverse transcription by carrying out hypermutation of newly synthesized HIV-1 DNA [41, 5052]. Numerous studies have shown that A3G molecules from the target cell have no effect on cDNA deamination, and only virion-incorporated A3G affects viral DNA (reviewed in [53]), suggesting that the cDNA synthesis and accumulation machineries are effectively isolated from the environment of the target cell cytoplasm, but can be affected by factors which are encapsulated in cores and found within RTCs. Initiation of uncoating or disintegration of the capsid shell is believed to be dependent on the completion of reverse transcription [27, 28]. Uncoated complexes containing viral cDNA are capable of interacting with numerous factors of the target cell, which are necessary for nuclear import and probably facilitation of viral genome integration (reviewed [21, 54]).

            Although numerous studies, including recently published proteomic analyses of whole highly-purified retroviral virions or virus-like particles, contain broad information about profiles of cellular proteins in viral particles [4, 10, 55, 56], the host proteins associated specifically with the cores of mature HIV-1 virions have not been characterized. To identify the proteomic profiles of HIV-1 cores , we performed LC-MS/MS analysis of the cores isolated from virus particles of HIV-1 NL4-3 produced by Sup-T1 cells (T lymphocytes), PMA and vitamin D3 activated THP1 (model of monocyte-derived macrophages), and non-activated THP1 cells (model of monocytes), and Western blot analysis of selected proteins in the cores and producer cells. Potential involvement of identified proteins in the early stages of HIV-1 infection was assessed using gene ontology information and data from published genome-wide screens on proteins important for HIV-1 replication [5759]. Our study revealed 202 proteins associated with HIV-1 cores. More than 20% of these proteins were detected in the cores of virions from all cell types, suggesting that this group contains cellular proteins potentially involved in viral replication. We found that some members of this group, which belong to subfamilies of RNA and DNA helicases, are packaged into the virions from producing T cells more efficiently than into virions from monocyte or MDM model cells, indicating that the mechanism of their incorporation is nonrandom.

            Results

            Preparation of HIV-1 core structures for proteomic analysis

            Since HIV-1 is a highly variable virus, its different subtypes and individual variations may interact differently with cellular proteins [60]. Therefore, we selected the NL4-3 isolate of HIV-1 subtype B as a model virus for infection of different cell types: T lymphocytes and the model of monocytes and MDM. NL4-3 is a CXCR4-tropic isolate of HIV-1 that normally does not infect monocytes and macrophages, so to infect THP1 cells we used virus pseudotyped with amphotropic murine leukemia virus (MLV) Env glycoprotein. For isolation of viral cores and subsequent LC-MS/MS analysis, we used the virus particles produced by infected Sup-T1 cells (T lymphocyte cell line), THP1 (monocytic leukemia cells) and THP1 cells treated with phorbol 12-myristate 13-acetate (PMA) and vitamin D3. This treatment activates cell differentiation that results in acquisition of the biochemical and morphological characteristics of MDM. Thus, the activated THP1 cells may be considered as a model of macrophages [61, 62].

            To isolate core structures from HIV-1 virions produced by infected cells, we engaged a technique of “spin-thru” equilibrium density gradient sedimentation described earlier [6366]. This method of ultra-speed centrifugation of previously concentrated HIV-1 virions through a sucrose density gradient overlaid with a detergent layer (1% Triton X-100) allows for the purification of mature lentiviral cores whose density varies from 1.23 to 1.27 g/ml [67, 68] (Figure 1A, lower panel), whereas intact viral particles display buoyant density 1.18-1.20 mg/ml (Figure 1A, upper panel). To establish the purity of our viral core preparations from cellular vesicles, which have density similar to that of virions (1.14-1.20 g/ml) and may contaminate virus preparations [69], and to compare maturation of capsid cores in the viruses produced by T lymphocytes and MDM model cells, we engaged electron microscopy and Western blot analysis. Examination of the negatively stained concentrated virion samples in a transmission electron microscope revealed the presence of both extracellular vesicles and viral particles with diameter from 120 to 130 nm (Figure 1B1-B3). Analysis of ultrathin sections of the viral particles used for core isolation showed that the population represented a mix of immature (Figure 1B4, black double arrows) and mature virions (Figure 1B4, single arrow). The lipid membrane-covered structures were not found in purified viral core preparations. Only the conical-shape and oval structures with length about 80–110 nm were found in the samples of purified viral cores, indicating that the preparations after “spin-thru” purification contained only mature capsid cores (Figure 1B5-B8). Previous studies of cores from HIV-1 [64] and HIV-2 virions [63] isolated by “spin-thru” centrifugation method showed the presence of mature products of Gag and GagPol proteolytic processing (CA, MA, Vpx [for HIV-2] and RT with high enzymatic activity). Immunoblotting of our core preparations obtained after “spin-thru” purification of the same amounts of Sup-T1- and THP1-derived viral particles, carried out with human IgG prepared from pooled plasma of HIV antibody positive donors, revealed similar amounts of major products of Gag and GagPol processing, such as MA, CA, IN and RT proteins (Figure 1C). The Pr55Gag and Pr41Gag (MA + CA) have also been identified in the core samples from both viruses, suggesting that mature cores may contain some amounts of unprocessed Gag polyprotein. Taken together, our data indicate that the selected method of purification allows for the isolation of mostly mature cores from the pools of virions produced by both T lymphocytes and MDM model cells.
            http://static-content.springer.com/image/art%3A10.1186%2F1742-4690-9-65/MediaObjects/12977_2012_2899_Fig1_HTML.jpg
            Figure 1

            “Spin-thru” purification isolates mature cores from HIV-1 virions. The cores were isolated from the HIV-1 virions concentrated from culture media of infected Sup-T1, PMA-activated and non-activated THP1 cells by the “spin-thru” purification. A – CA p24Gag profiles of 30-70% sucrose gradients after centrifugation of concentrated HIV-1 virions (upper panel) and “spin-thru”purified viral cores (lower panel). The 0.4 ml sucrose gradient fractions were collected, dialyzed against PBS and subjected to p24 ELISA. B – Electron microscopy of uranyl acetate negatively stained HIV-1 virions concentrated through 30% sucrose cushion (B1-B3), ultrathin sections of virions harvested from infected Sup-T1 cells (B4) and negatively stained core preparations after “spin-thru” purification (B5-B8). The negatively stained viral particles are indicated by single white arrows; extracellular vesicle contaminants in the preparations of concentrated virions are indicated by double white arrows; mature virions in the sections of viral preparatins are indicated by single black arrows, immature particles – by double black arrows. C. The cores of viruses produced by Sup-T1 and activated THP1 cells do not have differences in the profile of the GagPol processing products. The “spin-thru” purified and p24Gag normalized cores were analyzed by Western blotting using the human HIV immunoglobulin (HIV-IG) from NIH AIDS Research & Reference Reagent Program. The bands of HIV-1 Gag and Pol processing products are indicated on the left.

            We also performed Western blotting of our samples using the anti-CD45 antibody (Figure 2A). CD45 is known to be abundant in microvesicles, but is apparently excluded from HIV-1 virions [70]; the lack of this protein in our core preparations would confirm their purity from the vesicular fraction. Indeed, we did not detect CD45 in the samples of cores from HIV-1 virions produced by both Sup-T1 and THP1 cells (Figure 2A, two right lanes), whereas the specimens of culture media from these cells concentrated only through a 30% sucrose cushion contained detectable amounts of CD45. Western blotting of the samples of culture media from untransfected and NL4-3 proviral clone-transfected 293 T/17 cells using anti-RNA helicase A (RHA or DHX9) antibody showed presence of this DEAD box RNA helicase in the preparations of media from both transfected and untransfected cells after purification through 30% sucrose. However, in the “spin-thru” purified samples, RHA was detected only in the preparations of media containing HIV-1 (Figure 2B). Since RHA is known to be present in both vesicles [71] and HIV-1 virions [10, 72, 73], our analysis confirmed that the method of “spin-thru” centrifugation removed extracellular membranous structures from the 30% sucrose-concentrated cell culture supernatants, but retained intravirion core structures.
            http://static-content.springer.com/image/art%3A10.1186%2F1742-4690-9-65/MediaObjects/12977_2012_2899_Fig2_HTML.jpg
            Figure 2

            “Spin-thru” purification allows isolation of viral cores for proteomic analysis. A, B – “Spin-thru” purification separates viral cores from extracellular vesicles. Western blot analysis with anti-CD45 mouse monoclonal antibody of the virus samples before (left two lanes) and after (right two lanes) “spin-thru” purification. CD45 was used as an extracellular vesicle marker, p24Gag as a reference viral protein (A). Western blot of culture media from uninfected 293 T/17 cells and suspension of virus harvested from pNL4-3 transfected 293 T/17 cells before and after “spin-thru” centrifugation with anti-RHA rabbit polyclonal antibody (B). C – “Spin-thru” purification separates viral cores from the envelope: Western blot analysis of VSV-G glycoprotein in the samples of VSV-G-pseudotyped HIV-1 before and after “spin-thru” centrifugation. D – Representative SDS-PAGE profiles of the preparations of purified virion cores. Coomassie blue-stained preparative SDS-PAGE 12.5% gels of HIV-1 viral cores and the cell culture supernatants of non-infected cells after “spin-thru” centrifugation were sectioned as shown by the dashed lines. Infected samples are marked as (+) and uninfected as (−). The molecular mass markers are indicated on the left and the gel fractions are specified on the right. Positions of HIV-1 core proteins p24CA and p31IN are shown on the left side.

            To further prove that “spin-thru” centrifugation purifies cores from intact virions, we tested the presence of VSV-G envelope protein in the samples of VSV-G-pseudotyped HIV-1 produced by co-transfected 293 T/17 cells after concentration through 30% sucrose cushion and “spin-thru” centrifugation (Figure 2C). The VSV-G was clearly detected in the samples of concentrated pseudotyped virus, but was not found in the core samples after “spin-thru” purification, confirming purity of the core preparations from the envelope glycoproteins.

            The SDS-PAGE separation of our core preparations (Figure 2D) revealed major bands corresponding to proteins with molecular weights of 24 and 31 kDa (corresponding to HIV-1 CA and IN, respectively), indicating the presence of mature viral Gag and GagPol products in the analyzed core structures. On the other hand, multiple bands corresponding to the polypeptides of different molecular weights, which do not represent known HIV-1 proteins, suggest incorporation of many cellular proteins in the core structures of the viral particles produced by different cell types. The data of proteomic analysis (shown below) confirmed this suggestion. The staining of SDS-PAGE with Coomassie also revealed multiple protein bands in the control preparations, suggesting that the culture media from uninfected THP1 and especially Sup-T1 cells contained protein-rich, non-viral, non-membranous particles with buoyant density ≥1.23 mg/ml, probably the products of disintegrated dead cells (Figure 2D). Thus, to obtain proteomic profiles of the host proteins associated with HIV-1 viral cores, both viral cores and uninfected control preparations from each cell type were subjected to SDS-PAGE protein separation, trypsin digestion and subsequent LC-MS/MS analysis. The protein profile of each viral core sample was then compared with the corresponding control sample. Overlapping proteins were eliminated from the protein spectra of the viral cores, except the proteins whose scores were >5-fold higher in the preparations of viral cores than in control samples (proteins such as chaperones Hsp70 and Hsp90, and cytoskeletal proteins β actin, α and β tubulin, whose presence in core samples was confirmed by Western blot [Figure 4A]). As a result, a total of 202 cellular proteins were found to be associated with the cores of HIV-1 virions.
            http://static-content.springer.com/image/art%3A10.1186%2F1742-4690-9-65/MediaObjects/12977_2012_2899_Fig3_HTML.jpg
            Figure 3

            Profiles of host proteins associated with the cores of HIV-1 virions from different producer cells – data of LC-MS/MS analysis. A, B – Venn Diagrams depicting the number of overlapping cellular proteins within viral cores among the three producer cell types (A) and within the cores of viruses produced by the same cell type (Sup-T1) infected with HIV-1 NL4-3 strain pseudotyped with MLV Env (yellow) or VSV-G (green) envelope glycoproteins (B). The number of non-overlapping proteins and percent of these proteins within all cellular proteins identified in the core are shown as a numerator and denominator respectively. C – Categories of cellular proteins (by function) depicted as a percentage of the total proteins that were identified within the cores of virions produced by Sup-T1 (C1, n = 125), THP1 cells activated with PMA and vitamin D3 (C2, n = 110), and non-activated THP1 cells (C3, n = 90). The diagram of overlapping cellular proteins detected in all core preparations is shown on C4 (n = 42).

            Proteomic profiling of HIV-1 viral cores

            The proteins obtained from LC-MS/MS analysis of peptide preparations and filtered as described above and in Materials and Methods were categorized according to their functions and subcellular localization using NCBI protein database (http://​www.​ncbi.​nlm.​nih.​gov/​sites/​entrez?​db=​Protein), NCBI RefSeq database (http://​www.​ncbi.​nlm.​nih.​gov/​RefSeq/​) and DAVID Bioinformatics Resources 6.7 (NIAID NIH) (http://​david.​abcc.​ncifcrf.​gov). We compared our data with published results of the genome-wide analyses of cellular proteins involved in HIV-1 infection [5759, 74] and with global network of HIV-human protein–protein interactions [75] to assess the potential role of identified proteins in HIV-1 infection and putative mechanisms of their incorporation into the virion. The HIV-1 proteins identified in the viral cores are summarized in Table 1, the cellular proteins – in Tables2 and S1 in Additional file 1.
            Table 1

            Viral proteins that were detected in viral cores of the virions produced by different types of infected cells a

            Protein

            Contained in samples from:

             

            Sup-T1

            Act. THP1

            Non-act. THP1

            Pr 160 GagPol polyprotein-precursor (MA, CA, NC, p6, PR, RT, IN domains)

            Pol TF (p6*, PR, RT, IN domains)

            Pol TF (PR, RT domains)

             

            RT p66 subunit

            Pr55 Gag polyprotein-precursor ( MA, CA, sp2, NC, sp1, p6 domains) and probably partially-processed Gag 48 kDa polyprotein (MA, CA, sp2, NC domains)

            RT p51 subunit

            gp41 TM Env glycoprotein

             

            Gag 41 kDa partially-processed polyprotein (MA, CA, sp2 domains)

              

            IN p31 Pol

            CA p24 Gag

            Nef

            Vif

              

            MA p17 Gag

            RNase H 15 kDa Pol polypeptide

            15 kDa partially-processed Gag precursor (NC, sp1, p6)

              

            Rev

              

            PR p10 Pol

            NC p7 Gag

            aProteins are indicated as being within the core from a specific producer cell type by a check symbol (✓).

            Pol TF, polymerase trans-frame.

            Table 2

            Cellular Proteins in HIV-1 cores a

            Locationb

            Gene Namec

            Accession Numberd

            Protein Namee

            Contained in cores produced by:

                

            SupT1

            Act. THP1

            N-Act. THP1

            DNA Binding: Chromatin Organization, Replication, Topoisomerases

            N,C

            RUVBL1

            4506753

            TATA binding protein interacting protein 49 kDa

            M,C,N

            MCM5

            1232079

            Minichromosome maintenance complex component 5

            N

            RCC2

            11360295

            Regulator of chromosome condensation protein 2

             

            N,C

            RUVBL2

            5730023

            TBP-interacting protein, 48-KD

             

            N

            TOP2B

            288565

            DNA topoisomerase II

              

            N,C

            SSRP1

            4507241

            Structure specific recognition protein 1

              

            N

            H2AFY

            32492946

            H2A histone family, member Y

              

            N,C

            ERVK-6

            3600071

            Reverse transcriptase encoded by human endogenous HERV-K retrovirus

              

            N,C

            MCM7

            2134885

            Replication licensing factor MCM7

              

            N,M

            TEP1

            1848277

            telomerase-associated protein 1; TP-1

              

            N

            HIST1H1B

            4885381

            H1 histone family, member 5; H1b

              

            N

            HIST1H1C

            4885375

            H1 histone family, member 2; H1d

              

            Number of Proteins per Cell Type:

            6

            3

            4

            DNA Damage Repair

            N

            XRCC5

            35038

            Nuclear factor IV - KU80, ATP-dependant DNA helicase II

            N

            PRKDC

            1362789

            DNA-activated protein kinase

             

            N

            DDB1

            12643730

            DNA damage binding protein 1

             

             

            Number of Proteins per Cell Type:

            1

            3

            2

            DNA Binding: Transcription Regulation

            N

            POLR2B

            23270691

            Polymerase (RNA) II (DNA directed) polypeptide B

              

            N

            RHOX11

            27715523

            Similar to Paired-like homeobox protein OTEX

              

            Number of Proteins per Cell Type:

            1

            0

            1

            RNA Binding: Structure Organization, Modification, Transport, Splicing

            N

            UPF1

            1575536

            Regulator of nonsense transcript stability

            N

            DHX9

            3915658

            ATP-dependent RNA helicase A - DEAD box protein 9

            N

            EFTUD2

            24474791

            Small nuclear ribonucleoprotein component

            N

            SNRNP200

            14043179

            Helicase hBrr2 200 kDa

            N

            HNRNPM

            14141154

            Heterogeneous nuclear ribonucleoprotein M isoform b

             

            N,C

            EIF3A

            32449796

            Eukaryotic translation initiation factor 3, subunit A

             

            N,C

            PABPC1

            29743688

            Poly(A) binding protein, cytoplasmic 1

             

            N

            PRPF8

            17999537

            U5 snRNP-specific protein

             

            N,C

            HNRNPH1

            5031753

            Heterogeneous nuclear ribonucleoprotein H1

             

            N

            PDCD7

            4416183

            ES18 - U12-type spliceosome component

             

            N,C

            FLII

            2135121

            Flightless-I homolog

             

            C

            DDX3X

            13514813

            Helicase like protein 2 - DEAD/H box polypeptide 3

              

            N,C,Mit

            DDX17

            5453840

            RNA-dependent helicase p72 - DEAD box polypeptide 17 isoform 1

              

            N

            RENT1

            1575536

            Rregulator of nonsense transcript stability

              

            N,C

            SYNCRIP

            26454828

            Synaptotagmin-binding, cytoplasmic RNA-interacting protein

              

            N,C

            PABP2

            12229876

            Polyadenylate-binding protein 2

              

            N,C

            HNRNPR

            13629286

            Heterogeneous nuclear ribonucleoprotein R

              

            N

            SNRNP200

            14043179

            Small nuclear ribonucleoprotein 200 kDa (U5)

              

            C

            ACO1

            9802308

            Iron regulatory protein 1

              

            C,M

            FHL1

            6942193

            Four and a half LIM domains 1 protein isoform C

              

            N

            PCBP1

            444021

            Poly(rC) binding protein 1

              

            N,C

            HNRNPF

            16876910

            Heterogeneous nuclear ribonucleoprotein F

              

            N,C

            SRSF3

            4506901

            Splicing factor, arginine/serine-rich 3

              

            N

            RALY

            27689091

            Autoantigenic RNA binding protein

             

             

            N

            DDX21

            11890755

            RNA helicase II/Gu protein - DEAD box polypeptide 21

             

             

            N,C,Mit

            RTCD1

            4506589

            RNA 3'-terminal phosphate cyclase

             

             

            N,C

            HNRNPA1

            30157273

            Heterogeneous nuclear ribonucleoprotein A1

             

             

            V,N,C

            EIF5A2

            9966867

            Eukaryotic translation initiation factor 5A2; eIF-5A2 protein

             

             

            N,C

            SNUPN

            6730226

            D3b subcomplex of human core snRNP domain

              

            Number of Proteins per Cell Type:

            24

            11

            9

            Cytoskeleton

            C

            MYH10

            1346640

            Cellular myosin type B, heavy chain

            C

            MYH9

            29436380

            Cellular myosin, heavy polypeptide 9

            C

            TUBA1A

            135395

            Tubulin α 1

            C

            TUBA1C

            1438930

            Tubulin α 6

            C

            TUBB

            1280489

            Tubulin β 5

            C

            TUBB3

            1204535

            Tubulin β 3

            C

            ACTBL2

            2973662

            β Actin

            C

            TUBB4

            2748197

            β Tubulin 4Q

             

            C

            TUBB1

            13562114

            Tubulin β 1

            C

            TEKT1

            16753231

            Tektin 1

              

            C

            DYNC1H1

            30581065

            Dynein Heavy Chain, cytosolic

             

            C

            MYO1F

            1924940

            Myosin-IF

             

            N,M

            MYO1G

            14269502

            Unconventional myosin IG valine form

             

             

            C,M

            TUBG1

            31543831

            Tubulin γ 1

              

            C

            TPM2

            6573280

            Tropomyosin 2 (β)

              

            Number of Proteins per Cell Type:

            10

            11

            12

            Cytoskeleton Regulation

            C

            HSPB1

            662841

            Heat shock protein 27

            M,N

            MSN

            14625824

            Moesin

            C

            CORO1A

            5902134

            Coronin, actin binding protein, 1A

            M,C,V

            CNP

            180687

            2',3'-cyclic-nucleotide 3'-phosphodiesterase; CNPase

             

            N,C

            FLII

            2135121

            Flightless-I homolog

             

            M,C

            TTLL10

            27663488

            Tubulin tyrosine ligase-like family, member 10

              

            C

            ACTR2

            27500905

            Human ARP2 actin-related protein 2 homolog (yeast)

              

            C,N

            8-SEP

            12654963

            SEPT11 - Septin filament-forming cytoskeletal GTPase family member 11

              

            N,C

            PFN1

            3891601

            Human platelet profilin chain A

              

            C,N

            ACTR3

            5031573

            Human ARP3 actin-related protein 3 homolog (yeast)

             

             

            C

            DNAJA1

            219588

            DnaJ (Hsp40) homolog, subfamily A, member 1

             

             

            C

            RHOA

            12654251

            RAS homolog gene family, member A

             

             
             

            ARPC4-TTLL3

            10436409

            Actin related protein 2/3 complex, subunit 4-tubulin tyrosine ligase-like family, member 3 read-through fusion protein

             

             

            C

            ARPC4

            15214920

            Actin related protein 2/3 complex, subunit 4

             

             

            C

            RAC2

            4506381

            RAS-related C3 botulinum toxin substrate 2

             

             

            M,V,C

            TLN1

            6739602

            Talin

              

            M,C

            FLNA

            1203969

            Filamin

              

            M

            SPG8

            20070788

            Strumpellin

              

            C

            NCKAP1L

            32425702

            HEM1 protein

              

            C

            TBCD

            13111855

            Tubulin folding cofactor D

              

            M,C

            CAP1

            29739285

            Human adenylyl cyclase-associated CAP protein homolog 1 (S. cerevisiae, S. pombe)

              

            Number of Proteins per Cell Type:

            9

            10

            10

            Cell Signaling

            N,C

            GNB2L1

            5174447

            Guanine nucleotide binding protein (G protein), beta polypeptide 2-like 1

             

            MYADM

            27730943

            Myeloid-associated differentiation marker

             

            C,V

            PTPRC

            10999057

            Protein tyrosine phosphatase

             

            M

            SEMA7A

            3551779

            Semaphorin L

              

            M,C

            PKN1

            1085381

            Serine/threonine protein kinase

              

            C,M

            MMP14

            1705985

            Matrix metalloproteinase 14

             

             

            C

            TIMP3

            1304484

            Tissue inhibitor of metalloproteinases-3

             

             

            Number of Proteins per Cell Type:

            3

            5

            3

            Nuclear Import

            N,C

            TNPO1

            27681051

            Karyopherin β2; importin β 2; transportin; transportin 1

             

            V,N

            TNPO3

            6912734

            Transportin-SR; importin 12; transportin-SR2

              

            N,C

            KPNB1

            19923142

            Karyopherin β1; importin 90; importin β-1

              

            N

            KPNA2

            1354365

            Karyopherin α2; RAG cohort 1, importin α1

              

            Number of Proteins per Cell Type:

            1

            1

            3

            Nuclear Export

            N

            XPO5

            12407633

            RANBP21/exportin 5

             

            N,C

            XPOT

            17367977

            Exportin T (tRNA exportin)

             

            Number of Proteins per Cell Type:

            2

            0

            2

            Apoptosis

            C

            HP95

            13375569

            Programmed cell death 6 interacting protein

            Number of Proteins per Cell Type:

            1

            1

            1

            Extracellular Signaling

            V,C,M

            PTPRC

            10999057

            Protein tyrosine phosphatase, receptor type, C

             

             

            C

            AIMP1

            27065983

            Aminoacyl tRNA synthetase complex-interacting multifunctional protein 1

              

            M

            CLEC1A

            30159086

            C-type lectin domain family 1, member A

              

            Number of Proteins per Cell Type:

            2

            1

            0

            Intracellular Transport

            C,M

            CLTC

            30353925

            Clathrin, heavy chain (Hc)

            M,C

            RAB8A

            234746

            RAB8A, member RAS oncogene family

            M

            CPNE1

            4503013

            Copine I

             

            C,N

            TNIP1

            1800305

            TNFAIP3 interacting protein 1; HIV-1 Nef interacting protein

              

            M,N,C

            GC

            18655422

            Vitamin D binding protein

             

             

            M,C

            AP2B1

            33504652

            Adaptor-related protein complex 2, β1 subunit

              

            Number of Proteins per Cell Type:

            3

            4

            4

            RNA Stability Regulation

            C,N

            RNH1

            15029922

            Ribonuclease/angiogenin inhibitor 1

              

            Number of Proteins per Cell Type:

            1

            0

            0

            Nuclear Lamina, intranuclear components

            N

            LMNB1

            15126742

            lamin B1

              

            N

            NCL

            21750187

            Nucleolin

              

            N

            DKC1

            14602859

            Dyskerin; dyskeratosis congenita 1

              

            N

            RPL3

            18606060

            Ribosomal protein L3

              

            Number of Proteins per Cell Type:

            4

            0

            0

            Cell adhesion

            M,C

            PCDHGA7

            14196477

            Protocadherin gamma subfamily A, 7

              

            M

            HABP2

            4758502

            Hyaluronan binding protein 2

              

            Number of Proteins per Cell Type:

            2

            0

            0

            Mitochondria

            Mit

            SLC25A6

            113463

            Solute carrier family 25 (mitochondrial carrier; adenine nucleotide translocator), member 6

             

             

            Number of Proteins per Cell Type:

            0

            1

            0

            Lipid Biosynthesis

            C,N

            FDFT1

            11514495

            Farnesyl-diphosphate farnesyltransferase 1

              

            M,C

            FASN

            15779138

            Fatty acid synthase

             

             

            Number of Proteins per Cell Type:

            1

            1

            0

            Transmembrane Ion Transport

            N,M

            SLC4A10

            7513341

            Sodium bicarbonate cotransport protein 2

              

            C,M

            ATP6V0E2

            542837

            ATPase, H + transporting V0 subunit e2

             

             

            Number of Proteins per Cell Type:

            1

            1

            0

            Cell-Cell Transport

            M,C

            ESYT1

            7512911

            Extended synaptotagmin-like protein 1

             

             

            Number of Proteins per Cell Type:

            0

            1

            0

            Ribosomal

            R

            RPL7A

            4506661

            60 S-L7A

            R

            RPL18A

            11415026

            60 S-L18A

            R

            RPL7A

            17456110

            60 S-L7

             

            R

             

            27483402

            40 S-S2

             

            R

            RPL7L1

            27498574

            60 S-L7Like1

             

            R

            RPL13

            15431295

            60 S-L13

             

            R

            RPL24

            4506619

            60 S-L24

             

            R

            RPS8

            4506743

            40 S-S8

              

            Number of Proteins per Cell Type:

            7

            5

            5

            Ubiquitin/Proteasome

            C,N

            UBA1

            24485

            Ubiquitin-like modifier activating enzyme 1

            C

            BFAR

            27675450

            Bifunctional apoptosis regulator

            N,C

            PSMC3

            107855

            Proteasome (prosome, macropain) 26 S subunit, ATPase, 3

            C

            PSMD7

            2134660

            Proteasome (prosome, macropain) 26 S subunit, non-ATPase, 7

              

            M,C

            PSMB1

            12653473

            Proteasome (prosome, macropain) subunit, β type, 1

              

            N,C

            PSMD11

            2150046

            Proteasome (prosome, macropain) 26 S subunit, non-ATPase, 11

              

            C,N

            PAAF1

            33150632

            Proteasomal ATPase-associated factor 1

             

             

            C,N

            RPN1

            14124942

            Ribophorin I

             

             

            N,C

            PSMD3

            16550621

            Proteasome (prosome, macropain) 26 S subunit, non-ATPase, 3

             

             

            Number of Proteins per Cell Type:

            6

            6

            3

            Metabolism, Metabolism Regulation

            M

            PFKP

            11321601

            Phosphofructokinase, platelet

            C,N,M

            ACLY

            13623199

            ATP citrate lyase

             

            M,C

            PYGL

            10120741

            Chain A, Human Liver Glycogen Phosphorylase A

             

            N,C

            GPT

            1507680

            Glutamic-pyruvate transaminase (alanine aminotransferase)

              

            C,M

            PKM2

            125604

            Pyruvate kinase, M2 isozyme

              

            C

            SLC2A1

            3387905

            Solute carrier family 2 (facilitated glucose transporter), member 1

              

            N,C

            TPI1

            16877874

            Triosephosphate isomerase 1

             

             

            Number of Proteins per Cell Type:

            4

            4

            3

            Cell Cycle Regulation/Cell Differentiation

            N

            CCNB3

            14719420

            Cyclin B3 isoform 3

            C

            PPP2R1A

            21749746

            Protein phosphatase 2A, regulatory subunit A, α

             

            C,M

            RAP1B

            12751117

            RAP1B, member of RAS oncogene family

             

            C,N

            PPP2R3B

            7019501

            Protein phosphatase 2A, regulatory subunit B'', β; PP2A B''

              

            N,C

            CDK11A

            16357490

            Cyclin-dependent kinase 11A

              

            C

            PRDX3

            32483377

            Peroxiredoxin 3 isoform b

             

             

            C

            TIMP3

            1304484

            TIMP metallopeptidase inhibitor 3

             

             

            Number of Proteins per Cell Type:

            4

            4

            3

            Nucleotide Biosynthesis

            C

            CAD

            18105007

            Carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase

              

            Number of Proteins per Cell Type:

            1

            0

            0

            Amino Acid Biosynthesis

            M,C,N

            PHGDH

            5771521

            3-phosphoglycerate dehydrogenase

              

            C

            ASS1

            16950633

            Argininosuccinate synthase 1

             

             

            C

            GCN1L1

            2282576

            HsGCN1; GCN1 general control of amino-acid synthesis 1-like 1 (human homolog of yeast)

              

            Number of Proteins per Cell Type:

            1

            1

            1

            Vesicular Transport

            C,M

            CLTC

            30353925

            Clathrin

            C,M,V

            COPA

            4758030

            Coatomer protein complex, subunit α; alpha coat protein; xenin

            V,C

            MVP

            19913410

            Major Vault Protein

            C,V

            TFRC

            4507457

            Transferrin receptor (p90, CD71)

            C,V

            RAB7A

            1709999

            RAB7A, member RAS oncogene family

            C

            PDCD6IP

            13375569

            Programmed cell death 6 interacting protein, HP95, AIP1/ALIX

            C

            RAB5C

            4759020

            RAB5C, member RAS oncogene family

             

            C,V

            EHD4

            7212811

            EH-domain containing 4

             

             

            C

            SAR1A

            21634445

            GTP-binding protein Sara; SAR1 homolog A (S. cerevisiae)

             

             

            M,V

            ARFRP1

            1065361

            Chain A, Human Adp-ribosylation factor related protein 1

             

             

            V,N

            ATP6V0A1

            1638835

            ATPase, H + transporting, lysosomal V0 subunit a1

             

             

            C

            RAB11A

            4758984

            RAB11A, member RAS oncogene family

              

            Number of Proteins per Cell Type:

            5

            10

            7

            Aminoacyl tRNA synthetases

            C,N

            IARS

            31873336

            Isoleucyl-tRNA synthetase

            C,N

            MARS

            15929104

            Methionyl-tRNA synthetase

             

            C,N

            RARS

            2118344

            Arginyl-tRNA synthetase

              

            C,N

            GARS

            3845409

            Glycyl-tRNA synthetase

              

            C,Mit

            WARS

            8439415

            Tryptophanyl-tRNA synthetase

              

            C,N

            DARS

            4557513

            Aspartyl-tRNA synthetase

             

             

            C,N

            QARS

            11493441

            Glutaminyl-tRNA synthetase

             

             

            Number of Proteins per Cell Type:

            5

            3

            2

            Translation and Translation Regulation

            N,C

            EIF3A

            32449796

            Eukaryotic translation initiation factor 3, subunit A

             

            C

            EEF2

            4503483

            Eukaryotic translation elongation factor 2; polypeptidyl-tRNA translocase

              

            C

            CC2D1B

            27715655

            Coiled-coil and C2 domain containing 1B

              

            C

            CYFIP1

            24307969

            Cytoplasmic FMR1 interacting protein 1 (Sra1)

             

             

            C

            AIMP2

            27662300

            Aminoacyl tRNA synthetase complex-interacting multifunctional protein 2

             

             

            V,N,C

            EIF5A2

            9966867

            Eukaryotic translation initiation factor 5A2; eIF-5A2 protein

             

             

            Number of Proteins per Cell Type:

            3

            4

            0

            Protein Post-Translation Modification

            C,M

            PAFAH1B

            4505587

            Platelet-activating factor acetylhydrolase 1b, catalytic subunit 3

              

            N,C

            PARP1

            130781

            Poly [ADP-ribose] polymerase-1

              

            C

            USP14

            4827050

            Ubiquitin specific protease 14

              

            C,N

            SERPINC1

            4502261

            Serpin peptidase inhibitor, clade C (antithrombin), member 1

              

            M,C

            LPL

            15030193

            Lipoprotein lipase

             

             

            M,C

            NMT1

            345862

            N-myristoyltransferase 1

             

             

            C,M

            CPD

            21903712

            Carboxypeptidase D

             

             

            Number of Proteins per Cell Type:

            4

            3

            0

            Protein Degradation (non-proteasomal)

            M

            ANPEP

            28678

            Alanyl (membrane) aminopeptidase

             

             

            Number of Proteins per Cell Type:

            0

            1

            1

            Chaperones/Molecular Folding

            C

            DNAJC13

            7513063

            DnaJ (Hsp40) homolog, subfamily C, member 13

            C

            HSP90AB1

            20149594

            Heat shock protein 90 kDa α (cytosolic), class B member 1

            C,N

            HSPA8

            24234686

            Heat shock 70 kDa protein 8

            C,M, N

            CCT3

            2136253

            Chaperonin containing t-complex polypeptide 1 (TCP1), subunit 3 (γ); TCP1 ring complex protein TRiC5

            C,M

            CCT6A

            4502643

            Chaperonin containing t-complex polypeptide 1 (TCP1), subunit 6A (ζ1)

            C,M

            CCT6B

            22654293

            Chaperonin containing t-complex polypeptide 1 (TCP1), subunit ζ2

            C

            HSPB1

            662841

            Heat shock 27 kDa protein 1

            C

            PPIA

            2624881

            Human Cyclophilin A

            N

            NAP1L4

            5174613

            Nucleosome assembly protein 1-like 4; nucleosome assembly protein 2

             

            N,M,C

            TCP1

            13540473

            t-complex polypeptide 1 (TCP1)

             

            N,C

            CCT5

            12804225

            Chaperonin containing t-complex polypeptide 1 (TCP1), subunit 5 (ϵ)

              

            C

            P4HB

            20070125

            Prolyl 4-hydroxylase, β polypeptide

             

             

            C

            CCT7

            5453607

            Chaperonin containing t-complex polypeptide 1 (TCP1), subunit η

             

             

            C

            SERPINH1

            123576

            Serpin peptidase inhibitor, clade H (heat shock protein 47), member 1

             

             

            C

            HYOU1

            5453832

            Oxygen regulated protein 1

             

             

            C

            CANX

            10716563

            Calnexin

             

             

            Mit

            HSPA9

            12653415

            Heat shock 70 kDa protein 9 (mortalin)

             

             

            C,N

            CCT4

            2559008

            Chaperonin containing t-complex polypeptide 1 (TCP1), subunit δ

              

            Number of Proteins per Cell Type:

            10

            13

            10

            a Cellular proteins were detected in the cores from the virions produced by different types of infected cells. Proteins are listed by functional category and indicated as being within the core from a specific producer type by a check symbol (✓). Bolded proteins were found in viral cores produced by all producer cells.

            b The locations of listed proteins are abbreviated as follows: C – Cytoplasmic, N – Nucleus, M – Plasma Membrane, Mit – Mitochondria, R – Endoplasmic Reticulum, V – Vesicles.

            c Official Gene Symbol as listed by HGNC.

            d Swiss-Prot Protein Accession number.

            e Full Protein name as listed in NCBI Protein Database (http://​www.​ncbi.​nlm.​nih.​gov/​protein).

            Viral proteins

            The major proteins constituting the HIV-1 nucleocapsid core, CA, NC, IN and RT (both p51 and p66 subunits) were identified in the preparations from virions generated by all cell types (Table 1). All core samples also contained MA protein, which is located in retroviral virions mostly outside the capsid and forms a matrix between the viral capsid and envelope [76, 77]. However, presence of MA in the RTCs and PICs [13, 14, 17, 78, 79] suggests that this protein is physically associated with the cores of HIV-1 virions. Comparison of our list of identified viral proteins with the MS/MS analysis data of whole MDM-produced HIV-1 particles [4] shows characteristic differences between the protein profiles of viral cores and whole virions. The gp120Env glycoprotein detected in whole virions is absent in our samples; however, the gp41 transmembrane (TM) Env product was identified in the cores from virions assembled in T lymphocytes and MDM-like activated THP1 cells. Since gp41 has been found to be associated with MA during virion assembly and probably in mature viral particles [80, 81] (reviewed in [82]), a low amount of this glycoprotein in detergent-purified core preparations was expected. The fact that we could not identify gp41 in the cores of virions from non-activated THP1 cells by LC-MS/MS confirms our suggestion that the concentration of this glycoprotein in our preparations is negligible. The absence of gp120Env in our samples confirms purity of isolated core structures from the viral envelope and intact virions, shown also in Figure 2C.

            The Tat, Nef and Vif proteins were identified earlier in the viral particles produced by infected MDM using proteomic methods [4]. We did not detect Tat in our core preparations, but MS/MS analysis revealed Nef in the viral cores from all analyzed cell types. Earlier studies indicated that Nef is incorporated into viral particles, stably associates with virion cores and facilitates reverse transcription and early steps of replication [16, 64, 83, 84]. The Vif and Rev proteins were identified with low scores only in the cores from T lymphocyte-derived virus, suggesting their very low concentrations in the cores. HIV-1 Vif has been shown to interact with Pr55Gag and viral protease during the assembly of virus particles [85]. Rev protein can be incorporated into cores in the complex with viral RNA. Surprisingly, we did not identify Vpr in the viral core samples. Numerous studies revealed Vpr in HIV-1 virions [8688] and RTCs [15, 8991]. However, while traditional methods of identification using specific antibodies recognize Vpr in HIV-1 particles, LC-MS/MS analysis performed by Chertova and co-authors [4] also did not reveal this protein in the preparations of whole highly-purified virions. The visibility of protein for LC-MS is defined by a few factors, including affinity to C18 column (hydrophobicity) and ionization efficiency of the peptides. Typically, about 10% of tryptic peptides of a long protein are visible. Thus, some proteins could be invisible for LC-MS/MS in spite of overall high sensitivity of a method. Moreover, although trypsin cleaves Vpr to 14 peptides, most of them are not charged or have a negative total charge in solution with neutral pH (analyzed using Innovagen Peptide property calculator tool http://​www.​innovagen.​se/​), which may cause additional problem with their detection by mass spectrometry.

            HIV-1 protease (PR) was observed in all our core preparations. At the same time, detection of Gag and GagPol polyprotein-precursors in respective gel fractions suggests that not all precursor molecules are subjected to the proteolytic cleavage during the virion assembly and a subset of unprocessed polyproteins is present in the mature viral cores.

            Cellular proteins

            Within 202 unique cellular proteins revealed in our preparations of purified cores from HIV-1 virions, the samples from Sup-T1-derived virus included 125 proteins, while the virion cores from activated and non-activated THP1 cells contained 110 and 90 proteins, respectively (Table 2). A similar number of common proteins was found between cores from viruses produced by activated and non-activated THP1 cells (51), non-activated THP1 and Sup-T1 cells (63), and activated THP1 and Sup-T1 (52) (Figure 3A). Forty two proteins were common to all viral cores, which equates to 34%, 38% and 47% of the proteins found in the cores of viruses derived from Sup-T1, activated and non-activated THP1 cells, respectively.

            We identified 125 host cell proteins in the cores of virions assembled in Sup-T1 cells; cores of the viruses from activated THP1 cells contained 12% less host proteins, whereas the number of host proteins in the cores of non-activated THP1-derived virus was 28% less than in the cores of virus from Sup-T1. Interestingly, only 20% of all cellular proteins identified in the cores of virus from non-activated monocytic cells were unique, other proteins were also found in the cores of the virions derived from Sup-T1 or activated THP1 cells. The cores of activated THP1 and Sup-T1-derived viruses, in contrast, contained 45% and 42% of unique host proteins, respectively (Figure 3A). Since the T lymphocytes and MDM (activated THP1 is a model of MDM) naturally support productive HIV-1 infection, whereas the non-differentiated monocytes may serve as HIV-1 reservoirs, but do not support active replication (reviewed in [92]), the incorporation of a smaller number of cellular proteins by the virions released from non-activated THP1 is probably related to the lower efficiency of virion assembly in this type of cells.

            To validate the credibility of differences in protein composition between viral cores from different cell lines, we performed proteomic analysis of two core preparations purified from the same cell line. Cores of virions harvested from Sup-T1 cells infected with MLV Env-pseudotyped and VSV-G-pseudotyped NL4-3 viruses were digested by trypsin without gel separation and subjected to LC-MS/MS. Per one hundred high scoring overlapping proteins we found only 18 unique cellular proteins incorporated in the viral cores from the MLV Env-pseudotyped virus-infected Sup-T1 (15% of all incorporated host proteins) and 10 proteins (9%) in cores of the virus from Sup-T1 cells infected with VSV-G-pseudotyped NL4-3 strain (Figure 3B; Additional file 1: Table S1). Since the minimal difference in the spectra of host proteins between cores of virions harvested from different cell lines was not less than 20%, and was equal to 42% and 45% for the viral cores from Sup-T1 and activated THP1 cells, respectively, relative to cores from non-activated THP1 cells, we believe that the detected differences between the profiles of cellular proteins in the viral cores from various cell lines are significant.

            All the cellular proteins in HIV-1 cores were classified into 31 functional categories (Table 2). The most numerous categories were as follows: RNA-binding proteins (29), components of the cytoskeleton (15) and cytoskeleton regulators (21), chaperones (18), DNA-binding proteins (17), proteins involved in vesicular transport (12) and components of the ubiquitin-proteasome system (9). Although these protein groups were numerous in all preparations, the viruses assembled in different types of producer cells demonstrated diversity in the protein spectra and number of proteins within each group (Figure 3C).

            Within the forty two cellular proteins which were present in all core preparations, the spectra of molecular chaperones (7), cytoskeleton components (7), and vesicular transport-associated proteins (5) were the most numerous (Figure 3C4). These functional groups of proteins have been shown to be involved in the folding of viral proteins and HIV-1 virion assembly (reviewed in [12, 93]). Some of these proteins (clathrin, transferrin receptor 1, RAB7, RAB5C, EHD4, Hsp70, Hsp90, cyclophilin A, β actin, tubulin α1) are very typical for HIV-1 virions and were registered earlier in the samples of purified viral particles (summarized in the database of Host Proteins in HIV-1 http://​web.​ncifcrf.​gov/​research/​avp/​protein_​db.​asp). Probably some of these proteins, such as β actin, α and β tubulin, moesin and major vault protein 1 are incorporated into HIV-1 virions non-specifically, due to their close proximity to a budding site, as suggested earlier [4, 94], whereas other cellular proteins may be incorporated due to specific interactions with viral proteins, such as Gag and GagPol [11, 23, 33], viral genomic RNA [8], or tRNALys3 primer [73]. These host proteins, called “Captives” in a recent review [12], may be involved in the virion assembly and budding process or be important for post-assembly steps of HIV-1 life cycle. Below we attempt to assess the packaging of selected cellular proteins in the cores of HIV-1 virions depending on the type of producer cells.

            Semi-quantitative analysis of selected cellular proteins packaging into the cores of HIV-1 virions assembled in different types of cells

            Although the spectra of the cellular proteins incorporated in the viral cores depended on the type of virus-producing cells, the quantitative differences within the group of overlapping proteins could also be observed. We compared the abundance of certain cellular proteins, selected from the group of 42 common proteins, in the viral cores and lysates of the producer cells. Since the group of 42 cellular proteins identified in all viral cores included the members which are likely involved in viral replication (Table 3), we selected 7 proteins from different functional categories for analysis by Western blot. Within the group of RNA-binding proteins, we analyzed incorporation of RNA helicase A, because the function of this protein in HIV-1 replication has been shown earlier [10, 95], and small nuclear ribonucleoprotein 200 kDa (U5) (SNRNP200 or HELIC2), which currently has no known role in HIV replication but has been detected with a high score in our MS/MS preparations. Within DNA-binding group of proteins, we analyzed the Minichromosome maintenance complex component 5 (MCM5) involved in initiation of DNA replication, 80-kilodalton subunit of the Ku heterodimer protein or ATP-dependant DNA helicase II (Ku80 or XRCC5), which is involved in the repair of DNA double-strand breaks and telomerase function [96, 97], Pontin52 (RUVBL1) and Reptin52 (RUVBL2) DNA helicases, both being the components of several high molecular weight protein complexes involved in chromatin remodeling, transcription regulation, DNA damage sensing and repair [98]. All these DNA-binding proteins were detected with high scores by LC-MS/MS in the cores of virions from both Sup-T1 and THP1 cells. Beta tubulin was selected as one of the major cytoskeletal proteins found earlier in HIV-1 particles (http://​web.​ncifcrf.​gov/​research/​avp/​protein_​db.​asp). All preparations were normalized to CA p24Gag; the cell lysates were additionally normalized by the cell count and β globin DNA count using quantitative real-time PCR. Cytoskeletal proteins, such as actin or β tubulin, could not be used for normalization of the cell lysates, because they are differently expressed in uninfected Sup-T1 and THP1 cells (Figure 4A).
            Table 3

            Previously discovered viral proteins with a known role in HIV-1 Replication a

             Proteinb

             Gene namec

            Score in virion cores fromd

            Known role in HIV-1 replicatione

              

            Sup-T1

            Act. THP1

            N-Act. THP1

             

            DNA Binding: Chromatin Organization, Replication, Topoisomerases

            Regulator of chromosome condensation 2

            RCC2

            3.32

            1.78

            -

            RCC proteins interact with Rac1 and Arf6 subnetworks and limit signaling required for membrane protrusion and delivery [99]; RCC2 acts as a Rac1 guanine nucleotide exchange factor (GEF); RCC1 is involved in HIV-1 RNA nuclear export through activation of RanGAP [100] and hence facilitates dissociation of RNA nuclear export complex [101].

            RNA Binding: Structure Organization, Modification, Splicing, Transport

            Helicase like protein 2 - DEAD/H box polypeptide 3

            DDX3X

            2.05

            -

            -

            Member of DEAD box RNA helicases that is implicated in alteration of RNA secondary structure such as translation initiation, nuclear and mitochondrial splicing, and ribosome and spliceosome assembly. RNAi knockdown of DDX3 suppresses HIV-1 viral replication [102]; interaction of DDX3 with Rev/CRM1 is important for nuclear export of non-spliced HIV-1 RNA [103].

            RNA-dependent helicase p72 - DEAD box polypeptide 17 isoform 1

            DDX17

            6.36

            -

            -

            Members of ATP-dependent DEAD box RNA helicases, potentially involved in interaction with HIV-1 RNA.

            RNA helicase II/Gu protein - DEAD box polypeptide 21

            DDX21

            -

            2.07

            -

            ATP-dependent RNA helicase A - DEAD box protein 9

            DHX9

            6.36

            6.09

            27.18

            RNA helicases that catalyze ATP-dependent unwinding of double-stranded RNA and DNA-RNA complexes; localize in both nucleus and cytoplasm and function as transcriptional regulators; may also be involved in expression and nuclear export of retroviral RNAs, particularly in post-transcriptional regulation of HIV-1 [104]. DHX9 is packaged in HIV-1 particles and contributes to particle assembly and reverse transcription [10]; it also facilitates tRNALys3 binding and initiation of reverse transcription [73]

            Heterogeneous nuclear ribonucleoprotein A1

            HNRNPA1

            -

            1.92

            -

            RNA binding proteins that complex with heterogeneous nuclear RNA (hnRNA) and are involved in pre-mRNA processing in the nucleus: alternative splicing regulation, polyadenylation, nucleo-cytoplasmic transport and other aspects of mRNA metabolism and transport; hnRNP-A1 is involved in HIV-1 mRNA splicing [105, 106]; hnRNP-A2 is found to be important for trafficking of HIV-1 mRNA out of the nucleus and through the cytoplasm [105]; hnRNP H and hnRNP K interact directly with HIV-1 RNA and are involved in alternative splicing [107].

            Heterogeneous nuclear ribonucleoprotein F

            HNRNPF

            3.47

            -

            -

            Heterogeneous nuclear ribonucleoprotein H1

            HNRNPH1

            3.04

            -

            8.26

            Heterogeneous nuclear ribonucleoprotein M isoform b

            HNRNPM

            1.9

            -

            --

            Heterogeneous nuclear ribonucleoprotein R

            HNRNPR

            6.31

            -

            --

            Nonsense-mediated decay (NMD) factor

            UPF1

            5.65

            6.48

            6.32

            ATP-dependent RNA helicase, the HIV-1 RNP component, positively influences HIV-1 RNA translatability. Important for stability of unspliced viral RNA and translation of Gag polypeptide in producer cells [108]

            Cytoskeleton

            β Actin

            ACTBL2

            60.12

            35.05

            99.59

            Actin microfilaments are important for RTC formation and RTC transport in cytoplasm [1]; interaction with NC domain of Gag is required for HIV-1 assembly [109].

            Tubulin α 1

            TUBA1A

            69.39

            58.56

            66.49

            Microtubules are shown to be important for RTC cytoplasmic trafficking [18, 110]assembly of Gag polyprotein molecules [111] and viral genomic RNA trafficking [112].

            Tubulin α 6

            TUBA1C

            57.11

            41.39

            61.72

            Tubulin β 5

            TUBB

            29.82

            27.85

            49.68

            Tubulin β 3

            TUBB3

            13.84

            9.07

            30.56

            Tubulin β 1

            TUBB1

            13.3

            -

            22.5

            Tubulin γ 1

            TUBG1

            -

            -

            4.96

            Dynein

            DYNC1H1

            -

            11.68

            10.11

            Dynein motor and late endosomes are involved in viral RNA trafficking [112] and transport of RTC toward the nucleus [18].

            Cytoskeleton Regulation

            ARP3 actin-related protein 3 homolog (yeast)

            ACTR3

            -

            9.52

            -

            Major constituent of the ARP2/3, a 7 subunit complex, responsible for actin polymerization [113]. The complex is required for early phase of HIV-1 replication [114].

            Nuclear Import

            Transportin 3; importin-SR; importin 12; transportin SR2

            TNPO3

            1.0

            -

            -

            Impotin-β family member, binds catalytic core domain close to the N terminus of IN and promotes nuclear entry of PICs [115, 116]; might serve as a chaperone that associates with PIC post-entry to guide it through nuclear pore [117].

            Karyopherin α2; importin α1)

            KPNA2

            -

            -

            7.91

            Directly interacts with central core domain of HIV-1 integrase, facilitates PIC nuclear import [118120].

            Karyopherin β2; importin β2

            TNPO1

            -

            3.63

            1.95

            Importin α/β heterodimer interacts with HIV-1 integrase and probably MA protein and Vpr to translocate PIC into the nucleus [121]

            Karyopherin β1; nuclear factor p97; importin 90

            KPNB1

            -

            -

            8.02

            Nuclear Export

            RANBP21/exportin 5 (Exp5)

            XPO5

            5.5

            -

            6.87

            Association of RanBP1 and 2 with Rev-CRM1-RanGTP complex has been shown [122], thus RanBP is required for dissociation of nuclear export complex during HIV-1 RNA nuclear export [101].

            Vesicular Transport

            CLTC protein - clathrin

            CLTC

            37.6

            7.7

            7

            Clathrin is incorporated in HIV-1 particles probably through interaction with Pol, especially IN domain [32]; it facilitates the accurate morphogenesis of infectious particles probably by contribution to spatial organization of Gag and Pol proteins and proteolytic processing of virion components during particle assembly [5].

            Rab5C GTP protease

            RAB5C

            -

            6.78

            6.58

            Rab GTP proteases are important for vesicular trafficking. They are activated by guanine nucleotide exchange factor (GEF), RCC2 protein revealed in HIV-1 cores can act as a GEF. Rab11 is important for HIV-1 production [123]; Rab1 potentially associates with HIV-1 Rev and is involved in nuclear export of viral RNA [124]; Rab9 is required for Gag trafficking to the site of assembly [123]; Rab7-interacting lysosomal protein promotes vRNA clustering at the MTOC [112]; Rab6 is probably involved in viral entry [58].

            Rab7A GTP protease

            RAB7A

            8.89

            9.08

            21.97

            Rab8A GTP protease

            RAB8A

            7.91

            6.78

            6.58

            Rab11A GTP protease

            RAB11A

            -

            -

            9.31

            Programmed cell death 6 interacting protein; HP95; AIP1/ALIX

            PDCD6IP

            6.43

            13.62

            29.94

            Alix/HP95 is a protein implicated in endosomal organization and virus budding; overexpression results in cytoplasmic vacuolization, which may be partially responsible for protection against cell death. AIP1/ALIX is a binding partner for HIV-1 Gag L-domain and other budding network proteins (Tsg101) functioning in virus budding [125].

            Intracellular Trafficking

            TNFAIP3 interacting protein 1; HIV-1 Nef interacting protein; NAF1

            TNIP1

            1.0

            -

            -

            An ERK2 binding protein, Naf1, attenuates EGF/ERK2 nuclear signaling, binds HIV-1 Nef and increases cell surface CD4 expression [126]. ERK2 interacts with HIV-1 matrix, packaged into virions and responsible for MA phosphorylation [24].

            Chaperones/Molecular Folding

            Hsp70 protein 8

            HSPA8

            7.85

            26.4

            31.35

            Heat shock protein 70 family members are shown to be incorporated in HIV-1 particles. This is important for subsequent viral cDNA synthesis [11, 127]; they can also interfere with Vpr in HIV-1 nuclear import in macrophages [128, 129].

            Hsp70 protein 9 (mortalin)

            HSPA9

            -

            5.75

            -

            Cyclophilin A, CyPA

            PPIA

            22.35

            20.00

            7.68

            incorporates into virions via binding to the CA domain of Pr55Gag [23]. The role of CA-bound CyPA is still unclear [48] It is critical for protection and stabilization of HIV-1 cores as a chaperone [49] and is probably involved in PIC nuclear transport [31]

            a Proteins are listed by function category.

            b Full Protein name as listed in NCBI Protein database (http://​www.​ncbi.​nlm.​nih.​gov/​protein).

            c Official Gene Symbol as listed by HGNC.

            d The Xcorr values of each protein in different viral core samples are shown; if protein was not present in the virion core from a specific producer cell type, the Xcorr is not shown.

            e Protein function within the cell is listed, along with known implicated function(s) in HIV-1 replication.

            http://static-content.springer.com/image/art%3A10.1186%2F1742-4690-9-65/MediaObjects/12977_2012_2899_Fig4_HTML.jpg
            Figure 4

            Incorporation of certain RNA- and DNA-binding cellular proteins into HIV-1 viral cores does not correlate with abundance of these proteins in infected cells. A – Western blot detection of the cytoskeleton proteins actin and β tubulin in uninfected Sup-T1, activated and non-activated THP1 cells. Lysates were normalized according to cell counts and then according to the count of β globin DNA using quantitative real-time PCR, and subjected to SDS-PAGE and Western blot analysis. B – Western blot detection of cellular RNA-binding proteins (DHX9, SNRNP200), DNA- binding proteins (MCM5, XRCC5, RUVBL1, RUVBL2), cytoskeleton protein β tubulin, and viral protein CA p24Gag in the lysates of virus-producing cells (left bands) and in “spin-thru” purified viral cores (right bands). Virus was harvested at 72 h p.i. from Sup-T1, activated and non-activated THP1 cells infected with MLV Env-pseudotyped HIV-1 NL4-3, normalized to CA p24Gag and subjected to the “spin-thru” core isolation. Lysates of infected cells were normalized according to total protein count and β globin DNA count as described in A. Cellular and viral core preparations were analyzed by Western blotting. C – Quantification of Western blotting results. Western blotting data were quantified using ImageJ software. Results are presented as percentage of the peak value for each protein in the cellular and viral core preparations. D – Quantification of viral genomic RNA in the cores of virions. Viral cores were prepared as described in Figure 1. RNA was isolated from CA p24Gag-normalized core samples, subjected to reverse transcription with oligo-dT primer and then to quantitative real-time PCR with the primer set specific for positive-strand HIV-1 DNA. The data represents analysis of three independent preparations. Each point shows mean RNA copy number ± SD per 1 ng of p24CA in the viral core sample.

            Analysis of selected proteins using Western blot with subsequent quantification of the band intensity using ImageJ software did not reveal differences in the abundance of β tubulin between the infected cells and the cores of virions produced by these cells (Figure 4B,C). The same ratio of protein concentrations between cells and viral cores was also detected for RUVBL1 and RUVBL2 DNA helicases. However, other analyzed DNA- and RNA-binding proteins demonstrated either moderate (XRCC5, SNRNP200) or high prevalence (MCM5, DHX9) in the cores of virions assembled in Sup-T1 cells, whereas in the cores of THP1-derived virions (both activated and non-activated) these proteins were less abundant. Incorporation of these proteins in the viral cores did not correlate with their abundance in the producer cells: XRCC5, MCM5 and SNRNP200 were presented in all types of cells at a similar level, whereas the RNA helicase A (DHX9) was abundant in dividing Sup-T1 and non-activated THP1 cells and decreased in non-dividing activated THP1 (Figure 4B,C). Interestingly, the incorporation of RNA helicases DHX9 and SNRNP200 into the cores did not correlate with the packaging of viral genomic RNA, as quantitative RT-PCR of the RNA isolated from CA p24Gag-normalized purified viral cores revealed no significant differences in the count of HIV-1 RNA in the cores from Sup-T1 and both THP1-derived viruses (Figure 4D).

            Discussion

            The proteomic analysis of the cores isolated from HIV-1 virions assembled in T lymphocytes and activated (model of MDM) and non-activated monocytic cells revealed more than thirty cellular proteins which have been previously shown to be involved in different steps of HIV-1 replication and/or incorporated in HIV-1 virions (Table 3). Since HIV-1 is a retrovirus with a RNA genome of less than 10 kB and encodes only nine polypeptides, it engages numerous cellular factors and pathways at all stages of its life cycle. Some of the factors, especially proteins involved in RNA splicing and nuclear export, multi-vesicular bodies (MVB) and late endosomal pathway, as well as the proteins directly involved in the HIV-1 budding process, such as AIP1/Alix, may be incorporated into virions by being associated with the viral RNA and proteins, but do not play a visible role in subsequent stages of viral replication [4, 125, 130]. For the other factors, such as Hsp70, CLTC protein/clathrin and Rab GTP proteases, their important role in the molecular organization of mature virions and probably viral entry into the target cells has been proposed earlier [5, 11, 32, 33, 58]. Here, we focus on the proteins identified in the core structures of HIV-1 virions assembled in different cell types. These proteins can be potentially involved in post-entry stages of the viral replication.

            The HIV-1 morphogenesis is known to be different in T lymphocytes and myeloid cells. In T cells, the viral particle budding and assembly have been shown to take place directly at the plasma membrane (reviewed in [131]), whereas in macrophages, earlier studies detected assembling HIV-1 particles in the late endosomes [132] (reviewed in [131]) or in internally sequestered plasma membrane domains that contain late endosomal markers but are connected to the cell surface [133]. Recent reports revealed an extensive tubular network and large sheet-like structures which extended to the cell surface from vesicular compartments and contained HIV-1 virions, released into the extracellular media [134, 135]. Indeed, the cores of model MDM-derived virions contained twelve vesicular trafficking-associated proteins, whereas only six were detected in the T cell-derived viral cores. The larger proportion of cytoskeleton and cytoskeleton regulatory proteins in the cores of virions assembled in THP1 cells than in Sup-T1-derived cores may be dependent on the abundance of these proteins in producer cells. Uninfected THP1 cells contain larger amounts of actin and β tubulin than Sup-T1 (Figure 3A), suggesting that other cytoskeleton and associated cytoskeleton regulatory proteins may also be more abundant in these myeloid cells; hence, increasing the probability that the virus will hijack this subset of proteins. Our data suggest that the unique core-incorporated proteins, which are different in the viruses assembled in different cell types, are mostly indiscriminately hijacked during virion assembly and likely not important for subsequent infection.

            The group of forty two cellular proteins identified in the cores of virions produced by all types of cells contains at least thirteen proteins whose involvement in different stages of HIV-1 infection has been shown previously (Table 2 and 3). On the other hand, many proteins within this group have never been found to be implicated in any infection event. Meanwhile, incorporation of these proteins into the viral cores from different types of producer cells suggests that at least some of them may be important for successful infection. For instance, within the functional category of vesicular trafficking-associated proteins, the member of ESCRT pathway AIP1/ALIX detected in all our core preparations has been shown earlier to be interacting with HIV-1 Gag late domain and to be important for the release of viral particles [125, 136]. Clathrin has also been found to be abundant in HIV-1 viral particles and important for the correct assembly and maturation of viral particles through the regulation of proteolytic processing of virion components [5, 32]. Members of the RAB family of proteins were found to be important for different steps of HIV-1 particle assembly and probably RNA incorporation [112, 123, 124] (Table 3). Available data suggest that the proteins of this group are important factors of assembly and maturation of the viral particle and get into the viral cores in association with Gag and GagPol proteins or viral RNA. Some of the factors of vesicular trafficking may be potentially involved in the early stage of HIV-1 infection: the functional genomic screening of factors involved in HIV-1 infection showed that the vesicular RAB6A protein is important for the late phase of reverse transcription in infected cells [58].

            The cytoskeletal proteins were also abundant in all core preparations, however, the role of these proteins in HIV-1 virions is still questionable. The actin microfilaments form the plasma membrane cortex, and both actin and microtubular networks are involved in HIV-1 particle assembly [111, 112, 137], so that the proportion of these proteins in viral particles may be up to 15% of the molar level of Gag [94]. Actin is packaged in the virions probably in association with the NC domain of Gag [111, 137, 138]. Thus, actin and actin-associated proteins coronin, moesin, filamin, and FLII can get into the viral cores due to the actin interaction with Gag or GagPol. However, involvement of these virion-packaged molecules in post-assembly events of the virus life cycle has not been shown [12]. Our data indicate that the ratio of β tubulin between the viral cores reproduced the concentration ratio of this protein between the producer cells, suggesting the capturing, but not specific incorporation, of this cytoskeletal protein into assembling virions. On the contrary, the cytoskeleton regulation proteins were found to be mostly different in the viral cores from different cell types, which may reflect variability of the profiles of these proteins in virus-producing cells. Some of them, particularly Hsp27, a protein containing a nuclear localization signal [139] and found in the cores of virions from all cell types, can be potentially involved in post-entry steps of infection, although the role of this protein in viral replication remains unknown.

            The other category of proteins abundant in HIV-1 viral cores is the molecular chaperones. The profiles of these proteins are very similar in all analyzed samples. Indeed, Hsp27, Hsp40 (DnaJ) co-chaperone, Hsp70, Hsp90, numerous members of TCP1 (Hsp60) tetradecameric complex, as well as peptidylprolyl isomerase cyclophilin A were found in all core preparations (Table 2). Previously, these proteins were identified in purified samples of whole HIV-1 virions and Gag preparations [4, 10, 23]. Hsp70 was also found in HIV-2, SIVMAC and SIVAGM[11, 56]. Since the major function of these proteins is to regulate folding of newly synthesized polypeptides, facilitate intracellular protein transport and assemble multisubunit protein structures [140, 141], they likely play an important role in HIV-1 particle assembly, processing and folding of the viral proteins during virion core maturation and maintain structural integrity of the viral core and RTCs [11, 33, 49]. The early RTC functions, especially organization of reverse transcription, may also depend on the proper activity of incorporated chaperones.

            The RNA-binding proteins represent the most diverse group of cellular factors in viral cores. Although we identified 29 RNA-binding proteins in the core preparations, only four of them were found in all core samples (Table 2). These are (1) regulator of nonsense transcript stability (UPF1), (2) ATP-dependent RNA helicase A (RHA or DHX9), (3) small nuclear ribonucleoprotein component (Snrp116 or EFTUD2), and RNA helicase hBrr2 200 kDa (SNRNP200 or HELIC2). The role of the first two factors in HIV-1 infection was thoroughly characterized before. The UPF1 protein, RNA helicase from the SFI superfamily, involved in translation of Gag polypeptide, was found in virus-producing cells in association with HIV-1 ribonucleoprotein (RNP) along with Pr55Gag, viral RNA and cellular protein Staufen 1 [108]. Thus, UPF1 can be packaged in the virions in association with both Gag and the viral genomic RNA. RNA helicase A (RHA), a member of the DEAD family of proteins which are capable of unwinding the double-stranded RNA structure, was earlier found to be associated with HIV-1 Gag and incorporated into HIV-1 virions in an RNA-dependent manner. Packaging of this protein into HIV-1 virions was important for endogenous reverse transcription [10]. Jeang and Yedavalli suggested that RHA incorporated into HIV-1 viral cores might be important for the reverse transcription in RTCs [95]. A recent study that revealed an important role of this enzyme in the annealing of tRNALys3 primer [73], confirmed this suggestion. Two other proteins, SNRNP200 (member of the family of U5 DEXH-box RNA helicases) and Snrp116 (U5 snRNP specific protein, 116 kD), are both members of the U5 group of small nuclear RNA proteins, the spliceosome components, and have not been detected in HIV-1 virions before. Since these proteins are known as important components of splicing machinery required for a spliceosome catalytic activity [142, 143], they can be associated with HIV-1 pre-mRNA and remain associated with a mature viral RNA molecule.

            Interestingly, our analysis showed higher level of DHX9 (RHA) and SNRNP200 (HELIC2) in the cores of virions assembled in T cells, as compared with the viral cores from the monocyte and MDM models, which did not correlate with the abundance of these proteins in producer cells. Since we did not find significant differences in the RNA and CA protein count between the virions from analyzed cells, observed differences suggest that the mechanism of incorporation of these proteins into the virions (binding to viral RNA or interaction with Gag or/and GagPol) is more effective and likely selective in T lymphocytes, than in monocyte and MDM model cells. Because of importance of RNA helicase A for the reverse transcription in HIV-1 virions and RTCs, we expect that SNRNP200 protein may also be involved in cDNA synthesis or accumulation.

            Analysis of earlier published genome-wide screens performed by Warrilow and co-authors [22] to select the host factors potentially implicated in HIV reverse transcription showed that the proteins involved in DNA replication, transcription and repair, as well as proteins of the ubiquitin-proteasome pathway may also be important. Within the 17 DNA-binding proteins detected in our core preparations, only two, ATP-dependent DNA helicase II (XRCC5 or Ku80) and TATA binding protein interacting protein 49 kDa (RUVBL1 or Pontin52), were found in the core of all virions. Two other proteins, minichromosome maintenance complex component 5 (MCM5 or CDC46) and regulator of chromosome condensation protein 2 (RCC2), were identified only in Sup-T1 and activated THP1 cells (Table 2), although Western blot showed presence of MCM5 also in non-activated THP1. The protein RUVBL2 (Reptin52) was not identified by MS/MS in activated THP1, but the fact that in cells this DNA helicase is complexed with the closely related RUVBL1 protein in hetero-dodecamers [144] suggests incorporation of this protein in the virions from all studied cells. Our analysis showed that among DNA binding proteins present in the viral cores from different cell types only XRCC5 and especially MCM5 displayed an increased level of incorporation from the T cells, similar to RNA-binding DHX9 and SNRNP200, whereas core incorporation of RUVBL1 and RUVBL2 reproduced their level in virion-producing Sup-T1 and THP1 cells. The DNA helicase MCM5, a member of the MCM family of chromatin-binding proteins is involved in the initiation of DNA replication and was found to be upregulated during the transition from the G0 to G1/S phase of the cell cycle (RefSeq database). Interaction of this protein with HIV has not been shown before. Another DNA helicase, XRCC5 or Ku80, which is involved in repairing DNA double-strand breaks, was earlier found to be important for viral cDNA circularization, nuclear import and integration [145, 146]. However, this function was shown for the protein expressed in the infected target cells, but not for virion-incorporated Ku80. Packaging of both MCM5 and XRCC5 (Ku80) DNA helicases in all viral cores and their high levels in the cores of T lymphocyte-derived virions suggest that the core-incorporated molecules of these proteins can also be involved in processes associated with cDNA processing and/or integration during post-entry steps of infection, especially in T cells.

            Within the group of ubiquitin-proteasome pathway associated proteins a total of 9 proteins were detected; three of them were identified in viral cores from all producer cells (Table 2). Earlier, numerous 26 S proteasome-associated proteins were found in HIV-1 and SIV particles [4, 56]. Involvement of the ubiquitin-proteasome system in the budding of lentiviral particles was shown earlier (reviewed in [147]). Since all major domains of the membrane-associated HIV-1 Gag molecules have been shown to be ubiquitinated during virion budding [148], the ubiquitination factors could package into virions in association with Gag and then get into the viral cores. However, the role of virion-associated factors of the ubiquitin-proteasome system in the early steps of HIV-1 infection is unknown.

            Conclusions

            Taken together, results of our study indicate that the profile of host cell proteins packaged in the cores of HIV-1 virions depends on the type of producer cell. High abundance of certain proteins in the cell increases the probability of their capturing by the virions and hence their presence in the viral cores. However, certain members of functional groups of DNA- and RNA-binding proteins, molecular chaperones, cytoskeletal, vesicular trafficking-associated and ubiquitin-proteasome pathway-associated proteins were found in the cores of virions from all analyzed cells, suggesting that their incorporation is non-random and that they can be directly or indirectly involved in either the virus assembly/budding or early infection events. Our findings that the abundance of cellular proteins DHX9, (RHA) SNRNP200, MCM5, and XRCC5 (Ku80) within virus-producing cells did not correlate with the abundance seen in cores of produced virions, specifically their unexpected higher packaging in T cells, suggests that the incorporation of these factors in T lymphocytes is more efficient than in myeloid cells. These differences may be associated with variability of localization of these host proteins relative to the sites of virion assembly in different cell types and/or with different localization of virion assembly complexes. The host factors abundant in the viral cores may play a role in subsequent steps of HIV-1 infection, specifically in T cells. Further analysis of the role of these proteins in viral replication might reveal new mechanisms of the modulation of HIV infection by the host proteins and identify new targets for antiretroviral therapeutic interventions.

            Methods

            Cells and viruses

            The acute monocytic leukemia cell line THP1 (from S. Tsuchiya) and T lymphoblastoma Sup-T1 cells (from James Hoxie) were provided by the NIH AIDS Research & Reference Reagent Program. The human kidney fibroblasts 293 T/17 was purchased from ATCC (Manassas, VA). All cells were maintained at 37°C and 5% CO2 in 75 cm2 tissue culture flasks with RPMI-1640 culture media supplemented with 10% Fetal Bovine Serum, penicillin/streptomycin (100 μg/ml), and L-Glutamine.

            The stocks of the HIV-1 virus pseudotyped with amphotropic murine leukemia virus envelope glycoprotein (MLV Env) for synchronized infection of THP1 and Sup-T1 cells were prepared by transfection of 293 T/17 cells with HIV-1 NL4-3 provirus-encoding plasmid [149] and pcDNA-Env(MLV) plasmid (kindly provided by Nathaniel Landau) at a 4:1 ratio using Metafectene transfection reagent (Biontex, Planegg, Germany). The cells were transfected in 75 cm2 tissue culture flasks; the plasmid DNA containing media was changed at 5 h post-transfection. After overnight incubation with fresh RPMI-1640, the media was changed again and the cells were cultured in fresh media for an additional 48 h at 37°C and 5% CO2. Then, the supernatants were harvested, filtered through a 0.45-μm filter and stored on wet ice at 4°C for 1–4 days.

            Infection

            The viral suspensions were normalized according to their RT activity corresponding to 1 × 106 cpm per 1 × 106 cells, mixed with Polybrene (Sigma) to a final concentration of 8 μg/ml and used for infection of approximately 200 × 106 viable Sup-T1 or THP1 cells by spinoculation [150]. Infection was performed in 6-well plates (5 × 106 cells per well) by centrifugation at 1000xg for 2 h at 18°C. After a 2 h incubation at 37°C and 5% CO2, the cells were washed from the virus-containing media, re-suspended in RPMI-1640 (pre-warmed to 37°C) and seeded in a regular (Sup-T1 and non-activated THP1) or polylysine-treated (THP1 for activation) 75 cm2 tissue culture flasks at a concentration of 4 × 106 cells per ml (Sup-T1, non-activated THP1) or of 1 × 106 cells per ml (THP1 for activation). To get activated THP1 cells, the PMA and vitamin D3 solutions were added to cells to a final concentration of 100 nM. Then, the cells were incubated at 37°C and 5% CO2 for 72 h.

            Concentration of virus and “spin-thru” isolation of viral cores

            Virus-containing culture media from infected Sup-T1, activated and non-activated THP1 cells, as well as the media from the same types of non-infected cells (control) were harvested at 72 h after incubation with virus (or equivalent volume of the virus-negative culture media) and purified from cell debris by being centrifuged at 2,500 rpm and 4°C for 5 minutes and filtered through 0.45 μm syringe filters. Then, filtered samples were centrifuged at 100,000xg and 4°C for 3 h through 2 ml cushions of 30% sucrose in STE buffer (10 mM Tris–HCl [pH 7.4], 100 mM NaCl, and 1 mM EDTA) in a Beckman SW-41 rotor. The pellets were re-suspended in 300 μl of STE buffer and the viral cores were then isolated by “spin-thru” purification as described earlier [6366]. Briefly, 3.8 ml of a 30-50% linear density gradient of sucrose in STE buffer was overlaid with 1 ml of 15% sucrose containing 1% Triton X-100 and then covered with a 0.4-ml cushion of 7.5% sucrose in STE. The HIV-1 positive and negative samples, concentrated through 30% sucrose and resuspended in STE (0.3 ml) were carefully layered on top of the 7.5% sucrose layer and centrifuged in a Type 100 Ti rotor (Beckman Coulter) at 100,000xg and 4°C for 16–18 h. The pellets were re-suspended in 26 μl of STE buffer and replaced to polypropylene, non-siliconized Eppendorf microtubes; 4 μl aliquots were set aside for the p24CA ELISA assay. The CA p24Gag–normalized suspensions of HIV-1 cores and control suspensions were subjected to SDS-PAGE protein separation for subsequent LC-MS/MS analysis, Western blotting, or to In-solution protein digestion with trypsin for the LC-MS/MS analysis of unseparated protein samples.

            In order to test purity of the “spin-thru” isolated cores from undestroyed viral particles, 400 μl aliquots of the suspensions of viral cores and concentrated whole virions were separately subjected to centrifugation in a 30-70% sucrose gradient for 5 h at 125,000xg and 4°C in a SW-60Ti rotor (Beckman Coulter). Ten fractions of the gradient (each 400 μl) were then collected from the bottom of the tubes and densities were determined. All fractions were dialyzed versus 1 L of ice-cold PBS using Tube-O-DIALYZER 1 kDa MEDI Kit (G Biosciences, St. Louis, MO) according to the manufacturer’s protocol and then applied for p24 enzyme-linked immunosorbent assay using Alliance HIV-1 p24 ELISA Kit (PerkinElmer, Waltham, MA).

            Additionally, electron microscopy (EM) was applied to test purity of the viral and core preparations. For EM, the virions concentrated through 30% sucrose and “spin-thru”-purified core preparations were resuspended in 20 μl of STE buffer, incubated 20 minutes on formware carbon film-coated 100 square mesh nickel grids (Electron Microscopic Sciences) at room temperature, and then incubated with 4% glutaraldehyde fixing solution for 10 minutes. After five-time wash in molecular grade water (Mediatech, Manassas, VA), samples were stained with 2% uranyl acetate. For analysis of virion structure, the pellets of virions after centrifugation through 30% sucrose were washed twice with PBS and then fixed with 4% glutaraldehyde for 4 h. The preparations were further fixed with 2% OsO4 for 2 h, dehydrated with a graded series of ethanol dilutions ranging from 25% to 100% and then embedded in Araldite 502 resin. Ultrathin sections were contrasted with 2% uranyl acetate in methanol and 1% lead citrate. All preparations were examined on a JEOL JEM 1200 transmission electron microscope operating at 100 kV.

            Western blot analysis

            The aliquots of the lysates of HIV-1 infected Sup-T1 and THP1 cells, the virus samples and culture media from non-infected cells taken before and after the “spin-thru” isolation were subjected to SDS-PAGE, subsequently transferred to a PVDF membrane and then detected using anti-HIV-1 p24 (24–3) mouse monoclonal antibody and human HIV immunoglobulin (HIV-IgG) from NIH AIDS Research & Reference Reagent Program; anti-RNA Helicase A (ab70777) rabbit polyclonal antibody from Abcam; anti-CD45, clone F10-89-4 monoclonal antibody from Millipore (Temecula, CA); monoclonal anti-Actin clone AC-40 from Sigma; anti-β tubulin (D-10), anti-Reptin 52 (D-6), anti-Ku80 (B-1) and anti-MCM5 (G-1) mouse monoclonal antibodies from SantaCruz Biotechnology (Santa Cruz, CA); anti-HELIC2 (N-20) and anti-Pontin 52 (N-15) goat polyclonal antibodies also from Santa Criz. Specific bands were visualized by ECL (Thermo Scientific, Rockford, IL). Quantification of the Western blotting results was performed using ImageJ software.

            Gel separation of proteins, protein digestion and peptide extraction

            The volumes of viral core suspensions, each containing 400 ng of p24CA protein, and control suspensions taken in twofold excess were mixed with equal volumes of Laemmli Sample Buffer (BioRad, Hercules, CA) containing 5% β mercaptoethanol, heated in boiling water for 2 minutes and applied for SDS-PAGE protein separation. Separation of proteins was performed in 12.5% Tris–HCl Criterion Precast Gel (BioRad) at 100 V and 4°C for 2–2.5 h. The gel was stained in 0.1% (wt/v) Coomassie (BioRad) solution (40% methanol (v/v), 10% acetic acid (v/v) in water with 1 g/L of Brilliant Blue R-250) for 1 h at room temperature. After 7–8 washes in de-staining solution (contains the same components, as staining solution, except Brilliant Blue R-250) the gel was replaced to water, and each lane was sectioned into 10 contiguous pieces, which were subjected to the “in-gel” proteolysis according to the modified previously published protocol [151] Briefly, acetonitrile (ACN) dehydrated gel pieces were rehydrated in 10 mM DTT and incubated at 60°C for 1 h. After cooling at room temperature, the gel slices were incubated with 50 mM iodacetamide for 1 h at room temperature in the dark for alkylation of proteins. After the second dehydration, a 15 μl dose of Trypsin Gold (Promega, Madison, WI) solution (20 μg/ml) in 40 mM NH4HCO3/10% ACN was added to each of the gel pieces. After 1 h saturation at 4°C, the pieces were incubated at 37°C overnight. The resulted peptides were extracted three times: (1) with 25 mM of NH4HCO3:ACN (1:1); (2) 5% formic acid (FA); (3) 5% FA:ACN (1:1). After pooling all the extracts together, samples were purified through ZipTip pipette tips C18 (Millipore), eluted with 30 μl of 0.1% trifluoroacetic acid (TFA) in 80% ACN and subjected to HPLC separation and MS/MS analysis.

            For “in-solution” protein digestion, the suspensions of HIV-1 cores after “spin-thru” centrifugation were treated with 10 mM DTT (60°C for 1 h) and 150 mM iodacetamide (1 h at room temperature in the dark) in 20 ul of STE buffer. The protein samples were then mixed with 100 μl of 200 mM ammonium bicarbonate and treated with 200 ng of Trypsin Gold (Promega) at 37°C overnight. The resulted peptides were dried in SpeedVac, resuspended in water, purified through ZipTip pipette tips C18 as described above and then subjected to HPLC separation and MS/MS analysis.

            HPLC-MS/MS of tryptic digests and database search

            The peptides in each sample were separated by micro-capillary reversed-phase liquid chromatography (HPLC), coupled online to an ion trap mass spectrometer Thermo LTQ Orbitrap XL. The mass spectrometer was operated in a data-dependent MS/MS mode using a normalized collision-induced dissociation (CID) energy of 35%. The CID spectra were compared against those of the EMBL non-redundant protein database. Only peptides having cross-correlation (X corr) cutoffs of 2.6 for [M + 2 H]2+, 3.0 for [M + 3 H]3+ and higher charge state were considered. These SEQUEST criteria thresholds resulted in a 1-2% of False Descovery Rate. The proteome analysis of the spectra was made by Proteome Discoverer 1.2 software (Thermo Fisher Scientific). The protein profiles of the samples of viral cores were compared with identically prepared samples from non-infected cells. The sub-cellular localization and function of each filtered protein was determined using gene ontology (GO) information obtained from cross-referencing each protein’s Swiss-Prot accession number to the GO localization information available on the NCBI protein database (http://​www.​ncbi.​nlm.​nih.​gov/​sites/​entrez?​db=​Protein) and The Human Protein Atlas database (http://​www.​proteinatlas.​org). The involvement of the proteins in known cellular pathways associated with major biological processes such as cell cycle, intracytoplasmic transport, cytoplasm organization, nuclear transport, chromatin structure maintenance/regulation, RNA splicing and reorganization, transcription, apoptosis, proteasomal degradation, etc. were assessed using NCBI RefSeq database (http://​www.​ncbi.​nlm.​nih.​gov/​RefSeq/​) and DAVID Bioinformatics Resources 6.7 (NIAID NIH) (http://​david.​abcc.​ncifcrf.​gov).

            RNA purification and RT reaction

            RNA was purified from suspensions of “spin-thru” purified viral cores containing 250 ng of p24CA using TRI Reagent-LS (MRC, Cincinnati, OH) according to the manufacturer's protocol. A total of 0.5 μg of RNA from the RNA fraction was treated with 0.25 mg/ml DNase I RNase-free (Roche, Mannheim, Germany) for 60 minutes in the presence of 5 mM MgCl2, followed by the heat inactivation at 65°C for 15 minutes. A 250 ng aliquot of total RNA was used to generate cDNA with the GoScript Reverse Transcription System (Promega, Madison, WI) using oligo-dT reverse primers.

            DNA isolation and quantitative real-time PCR

            Lysates of HIV-1 infected (72 h p.i.) Sup-T1, activated and non-activated THP1 cells were normalized to the total protein count using DC Protein Assay (BioRad) following manufacturer’s protocol. The total DNA was isolated using an IsoQuick Nucleic Acid Extraction Kit (ISC BioExpress, Kaysville, UT) following manufacturer’s recommendations. After isolation, the cellular DNA samples were analyzed by quantitative TaqMan real-time PCR to quantify chrosomal DNA. Set of primers specific for the β-globin gene has been used: forward primer BGF1 (5’-CAACCTCAAACAGACACCATGG-3’), reverse primer BGR1 (5’-TCCACGTTCACCTTGCCC-3’), and probe BGX1 (5’-FAM-CTCCTGAGGAGAAGTCTGCCGTTACTGCC-TAMRA-3’). The 2 μl aliquots of RT reaction mixtures of the RNA samples from isolated viral cores (see above) were diluted to 10-fold and 100-fold and subjected to quantitative real-time PCR analysis with the set of primers specific for late HIV-1 reverse transcription product as described earlier [152]. The primers FOR-LATE (5’-TGTGTGCCCGTCTGTTGTGT-3’), REV-LATE (5’-GAGTCCTGCGTCGAGAGATC-3’), and probe Lt-LTR-Prb (5’-FAM-CAGTGGCGCCCGAACAGGGA-TAMRA-3’) recognized the positive-strand DNA, specific for the U5-Ψ LTR region. PCR reactions were performed with PerfeCTa qPCR FastMix, UNG (Quanta Biosciences, Gaithersburg, MD) using 300 nM of each primer and 200 nM of probe according to the manufacturer protocol. Serial dilutions of DNA from 8E5 cells (CEM cell line containing a single copy of HIV-1 LAV provirus per cell) were used as the quantitative standards. Real-time PCR reactions were carried out at least in triplicate using the PTC-200 Peltier Thermal Cycler with Chromo4 Continuous Fluorescence Detector (both from MJ Research) and Opticon Monitor 2.03 software.

            Declarations

            Acknowledgments

            The following reagents were obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH: Sup-T1 cells from Dr. James Hoxie, THP1 from Dr. S. Tsuchiya, 8E5 cells from Dr. Thomas Folks and anti-HIV-1 p24 mouse monoclonal antibody from Dr. Michael Malim. The HIV-1 proviral clone NL was a gift from Dr. Lee Ratner; pcDNA-Env(MLV) was kindly provided by Dr. Nathaniel Landau. Authors are grateful to Drs. Yetrib Hathout and Pawel Ciborowski for consultations on MS/MS data analysis, Drs. Fatah Kashanchi and David Jans for advice on the functional classification of HIV-associated proteins and to Dr. Tatyana Ammosova for advices on sample preparations for LC-MS/MS and Dr. Anastas Popratiloff for consultations and help with electron microscopy. This work was supported by the District of Columbia Developmental Center for AIDS Research (DC D-CFAR), an NIH-funded program 5P30AI087714-02 and the NIH grant from Research Centers in Minority Institutions (RCMI) Program of the Division of Research Infrastructure, National Center for Research Resources (RCMI-NIH 2G12RR003048). SS is a predoctoral student in the Microbiology and Immunology Program of the Institute for Biomedical Sciences at the George Washington University. This work is from a dissertation to be presented to the above program in partial fulfillment of the requirements for the Ph.D. degree.

            Authors’ Affiliations

            (1)
            Department of Microbiology, Immunology and Tropical Medicine, George Washington University School of Medicine and Health Sciences
            (2)
            Department of Medicine, Center for Sickle Cell Disease, Howard University College of Medicine
            (3)
            Howard University College of Medicine, RCMI Proteomics Core Facility

            References

            1. Bukrinskaya A, Brichacek B, Mann A, Stevenson M: Establishment of a functional human immunodeficiency virus type 1 (HIV-1) reverse transcription complex involves the cytoskeleton. J Exp Med 1998, 188:2113–2125.PubMedView Article
            2. VerPlank L, Bouamr F, LaGrassa TJ, Agresta B, Kikonyogo A, Leis J, Carter CA: Tsg101, a homologue of ubiquitin-conjugating (E2) enzymes, binds the L domain in HIV type 1 Pr55(Gag). Proc Natl Acad Sci USA 2001, 98:7724–7729.PubMedView Article
            3. Martin-Serrano J, Zang T, Bieniasz PD: HIV-1 and Ebola virus encode small peptide motifs that recruit Tsg101 to sites of particle assembly to facilitate egress. Nat Med 2001, 7:1313–1319.PubMedView Article
            4. Chertova E, Chertov O, Coren LV, Roser JD, Trubey CM, Bess JW, Sowder RC, Barsov E, Hood BL, Fisher RJ, et al.: Proteomic and biochemical analysis of purified human immunodeficiency virus type 1 produced from infected monocyte-derived macrophages. J Virol 2006, 80:9039–9052.PubMedView Article
            5. Zhang F, Zang T, Wilson SJ, Johnson MC, Bieniasz PD: Clathrin facilitates the morphogenesis of retrovirus particles. PLoS Pathog 2011, 7:e1002119.PubMedView Article
            6. Lawn SD, Butera ST: Incorporation of HLA-DR into the envelope of human immunodeficiency virus type 1 in vivo: correlation with stage of disease and presence of opportunistic infection. J Virol 2000, 74:10256–10259.PubMedView Article
            7. Tremblay MJ, Fortin JF, Cantin R: The acquisition of host-encoded proteins by nascent HIV-1. Immunol Today 1998, 19:346–351.PubMedView Article
            8. Mouland AJ, Mercier J, Luo M, Bernier L, DesGroseillers L, Cohen EA: The double-stranded RNA-binding protein Staufen is incorporated in human immunodeficiency virus type 1: evidence for a role in genomic RNA encapsidation. J Virol 2000, 74:5441–5451.PubMedView Article
            9. Chatel-Chaix L, Abrahamyan L, Frechina C, Mouland AJ, DesGroseillers L: The host protein Staufen1 participates in human immunodeficiency virus type 1 assembly in live cells by influencing pr55Gag multimerization. J Virol 2007, 81:6216–6230.PubMedView Article
            10. Roy BB, Hu J, Guo X, Russell RS, Guo F, Kleiman L, Liang C: Association of RNA helicase a with human immunodeficiency virus type 1 particles. J Biol Chem 2006, 281:12625–12635.PubMedView Article
            11. Gurer C, Cimarelli A, Luban J: Specific incorporation of heat shock protein 70 family members into primate lentiviral virions. J Virol 2002, 76:4666–4670.PubMedView Article
            12. Ott DE: Cellular proteins detected in HIV-1. Rev Med Virol 2008, 18:159–175.PubMedView Article
            13. Bukrinsky MI, Sharova N, McDonald TL, Pushkarskaya T, Tarpley WG, Stevenson M: Association of integrase, matrix, and reverse transcriptase antigens of human immunodeficiency virus type 1 with viral nucleic acids following acute infection. Proc Natl Acad Sci USA 1993, 90:6125–6129.PubMedView Article
            14. Gallay P, Swingler S, Song J, Bushman F, Trono D: HIV nuclear import is governed by the phosphotyrosine-mediated binding of matrix to the core domain of integrase. Cell 1995, 83:569–576.PubMedView Article
            15. Connor RI, Chen BK, Choe S, Landau NR: Vpr is required for efficient replication of human immunodeficiency virus type-1 in mononuclear phagocytes. Virology 1995, 206:935–944.PubMedView Article
            16. Aiken C, Trono D: Nef stimulates human immunodeficiency virus type 1 proviral DNA synthesis. J Virol 1995, 69:5048–5056.PubMed
            17. Haffar OK, Popov S, Dubrovsky L, Agostini I, Tang H, Pushkarsky T, Nadler SG, Bukrinsky M: Two nuclear localization signals in the HIV-1 matrix protein regulate nuclear import of the HIV-1 pre-integration complex. J Mol Biol 2000, 299:359–368.PubMedView Article
            18. McDonald D, Vodicka MA, Lucero G, Svitkina TM, Borisy GG, Emerman M, Hope TJ: Visualization of the intracellular behavior of HIV in living cells. J Cell Biol 2002, 159:441–452.PubMedView Article
            19. Yamashita M, Emerman M: Capsid is a dominant determinant of retrovirus infectivity in nondividing cells. J Virol 2004, 78:5670–5678.PubMedView Article
            20. Nisole S, Saib A: Early steps of retrovirus replicative cycle. Retrovirology 2004, 1:9.PubMedView Article
            21. Levin A, Loyter A, Bukrinsky M: Strategies to inhibit viral protein nuclear import: HIV-1 as a target. Biochim Biophys Acta 2011, 1813:1646–1653.PubMedView Article
            22. Warrilow D, Tachedjian G, Harrich D: Maturation of the HIV reverse transcription complex: putting the jigsaw together. Rev Med Virol 2009, 19:324–337.PubMedView Article
            23. Luban J, Bossolt KL, Franke EK, Kalpana GV, Goff SP: Human immunodeficiency virus type 1 Gag protein binds to cyclophilins A and B. Cell 1993, 73:1067–1078.PubMedView Article
            24. Cartier C, Deckert M, Grangeasse C, Trauger R, Jensen F, Bernard A, Cozzone A, Desgranges C, Boyer V: Association of ERK2 mitogen-activated protein kinase with human immunodeficiency virus particles. J Virol 1997, 71:4832–4837.PubMed
            25. Jacque JM, Mann A, Enslen H, Sharova N, Brichacek B, Davis RJ, Stevenson M: Modulation of HIV-1 infectivity by MAPK, a virion-associated kinase. EMBO J 1998, 17:2607–2618.PubMedView Article
            26. Campbell EM, Hope TJ: Live cell imaging of the HIV-1 life cycle. Trends Microbiol 2008, 16:580–587.PubMedView Article
            27. Hulme AE, Perez O, Hope TJ: Complementary assays reveal a relationship between HIV-1 uncoating and reverse transcription. Proc Natl Acad Sci USA 2011, 108:9975–9980.PubMedView Article
            28. Arhel NJ, Souquere-Besse S, Munier S, Souque P, Guadagnini S, Rutherford S, Prevost MC, Allen TD, Charneau P: HIV-1 DNA Flap formation promotes uncoating of the pre-integration complex at the nuclear pore. EMBO J 2007, 26:3025–3037.PubMedView Article
            29. Dismuke DJ, Aiken C: Evidence for a functional link between uncoating of the human immunodeficiency virus type 1 core and nuclear import of the viral preintegration complex. J Virol 2006, 80:3712–3720.PubMedView Article
            30. Lee K, Ambrose Z, Martin TD, Oztop I, Mulky A, Julias JG, Vandegraaff N, Baumann JG, Wang R, Yuen W, et al.: Flexible use of nuclear import pathways by HIV-1. Cell Host Microbe 2010, 7:221–233.PubMedView Article
            31. Schaller T, Ocwieja KE, Rasaiyaah J, Price AJ, Brady TL, Roth SL, Hue S, Fletcher AJ, Lee K, KewalRamani VN, et al.: HIV-1 capsid-cyclophilin interactions determine nuclear import pathway, integration targeting and replication efficiency. PLoS Pathog 2011, 7:e1002439.PubMedView Article
            32. Popov S, Strack B, Sanchez-Merino V, Popova E, Rosin H, Gottlinger HG: Human immunodeficiency virus type 1 and related primate lentiviruses engage clathrin through Gag-Pol or Gag. J Virol 2011, 85:3792–3801.PubMedView Article
            33. Gurer C, Hoglund A, Hoglund S, Luban J: ATPgammaS disrupts human immunodeficiency virus type 1 virion core integrity. J Virol 2005, 79:5557–5567.PubMedView Article
            34. Davis DA, Brown CA, Newcomb FM, Boja ES, Fales HM, Kaufman J, Stahl SJ, Wingfield P, Yarchoan R: Reversible oxidative modification as a mechanism for regulating retroviral protease dimerization and activation. J Virol 2003, 77:3319–3325.PubMedView Article
            35. Chatel-Chaix L, Boulay K, Mouland AJ, Desgroseillers L: The host protein Staufen1 interacts with the Pr55Gag zinc fingers and regulates HIV-1 assembly via its N-terminus. Retrovirology 2008, 5:41.PubMedView Article
            36. Guo F, Cen S, Niu M, Javanbakht H, Kleiman L: Specific inhibition of the synthesis of human lysyl-tRNA synthetase results in decreases in tRNA(Lys) incorporation, tRNA(3)(Lys) annealing to viral RNA, and viral infectivity in human immunodeficiency virus type 1. J Virol 2003, 77:9817–9822.PubMedView Article
            37. Selig L, Benichou S, Rogel ME, Wu LI, Vodicka MA, Sire J, Benarous R, Emerman M: Uracil DNA glycosylase specifically interacts with Vpr of both human immunodeficiency virus type 1 and simian immunodeficiency virus of sooty mangabeys, but binding does not correlate with cell cycle arrest. J Virol 1997, 71:4842–4846.PubMed
            38. Willetts KE, Rey F, Agostini I, Navarro JM, Baudat Y, Vigne R, Sire J: DNA repair enzyme uracil DNA glycosylase is specifically incorporated into human immunodeficiency virus type 1 viral particles through a Vpr-independent mechanism. J Virol 1999, 73:1682–1688.PubMed
            39. Mansky LM, Preveral S, Selig L, Benarous R, Benichou S: The interaction of vpr with uracil DNA glycosylase modulates the human immunodeficiency virus type 1 In vivo mutation rate. J Virol 2000, 74:7039–7047.PubMedView Article
            40. Chen R, Le Rouzic E, Kearney JA, Mansky LM, Benichou S: Vpr-mediated incorporation of UNG2 into HIV-1 particles is required to modulate the virus mutation rate and for replication in macrophages. J Biol Chem 2004, 279:28419–28425.PubMedView Article
            41. Harris RS, Bishop KN, Sheehy AM, Craig HM, Petersen-Mahrt SK, Watt IN, Neuberger MS, Malim MH: DNA deamination mediates innate immunity to retroviral infection. Cell 2003, 113:803–809.PubMedView Article
            42. Kaiser SM, Emerman M: Uracil DNA glycosylase is dispensable for human immunodeficiency virus type 1 replication and does not contribute to the antiviral effects of the cytidine deaminase Apobec3G. J Virol 2006, 80:875–882.PubMedView Article
            43. Guenzel CA, Herate C, Le Rouzic E, Maidou-Peindara P, Sadler HA, Rouyez MC, Mansky LM, Benichou S: Recruitment of the nuclear form of uracil DNA glycosylase into virus particles participates in the full infectivity of HIV-1. J Virol 2012, 86:2533–2544.PubMedView Article
            44. Yung E, Sorin M, Wang EJ, Perumal S, Ott D, Kalpana GV: Specificity of interaction of INI1/hSNF5 with retroviral integrases and its functional significance. J Virol 2004, 78:2222–2231.PubMedView Article
            45. Sorin M, Yung E, Wu X, Kalpana GV: HIV-1 replication in cell lines harboring INI1/hSNF5 mutations. Retrovirology 2006, 3:56.PubMedView Article
            46. Sorin M, Cano J, Das S, Mathew S, Wu X, Davies KP, Shi X, Cheng SW, Ott D, Kalpana GV: Recruitment of a SAP18-HDAC1 complex into HIV-1 virions and its requirement for viral replication. PLoS Pathog 2009, 5:e1000463.PubMedView Article
            47. Lesbats P, Botbol Y, Chevereau G, Vaillant C, Calmels C, Arneodo A, Andreola ML, Lavigne M, Parissi V: Functional coupling between HIV-1 integrase and the SWI/SNF chromatin remodeling complex for efficient in vitro integration into stable nucleosomes. PLoS Pathog 2011, 7:e1001280.PubMedView Article
            48. Luban J: Cyclophilin A, TRIM5, and resistance to human immunodeficiency virus type 1 infection. J Virol 2007, 81:1054–1061.PubMedView Article
            49. Briones MS, Dobard CW, Chow SA: Role of human immunodeficiency virus type 1 integrase in uncoating of the viral core. J Virol 2010, 84:5181–5190.PubMedView Article
            50. Sheehy AM, Gaddis NC, Malim MH: The antiretroviral enzyme APOBEC3G is degraded by the proteasome in response to HIV-1 Vif. Nat Med 2003, 9:1404–1407.PubMedView Article
            51. Zheng YH, Irwin D, Kurosu T, Tokunaga K, Sata T, Peterlin BM: Human APOBEC3F is another host factor that blocks human immunodeficiency virus type 1 replication. J Virol 2004, 78:6073–6076.PubMedView Article
            52. Zennou V, Perez-Caballero D, Gottlinger H, Bieniasz PD: APOBEC3G incorporation into human immunodeficiency virus type 1 particles. J Virol 2004, 78:12058–12061.PubMedView Article
            53. Malim MH: APOBEC proteins and intrinsic resistance to HIV-1 infection. Philos Trans R Soc Lond B Biol Sci 2009, 364:675–687.PubMedView Article
            54. Arhel N: Revisiting HIV-1 uncoating. Retrovirology 2010, 7:96.PubMedView Article
            55. Segura MM, Garnier A, Di Falco MR, Whissell G, Meneses-Acosta A, Arcand N, Kamen A: Identification of host proteins associated with retroviral vector particles by proteomic analysis of highly purified vector preparations. J Virol 2008, 82:1107–1117.PubMedView Article
            56. Stephenson ST, Bostik P, Song B, Rajan D, Bhimani S, Rehulka P, Mayne AE, Ansari AA: Distinct host cell proteins incorporated by SIV replicating in CD4+ T cells from natural disease resistant versus non-natural disease susceptible hosts. Retrovirology 2010, 7:107.PubMedView Article
            57. Konig R, Zhou Y, Elleder D, Diamond TL, Bonamy GM, Irelan JT, Chiang CY, Tu BP, De Jesus PD, Lilley CE, et al.: Global analysis of host-pathogen interactions that regulate early-stage HIV-1 replication. Cell 2008, 135:49–60.PubMedView Article
            58. Brass AL, Dykxhoorn DM, Benita Y, Yan N, Engelman A, Xavier RJ, Lieberman J, Elledge SJ: Identification of host proteins required for HIV infection through a functional genomic screen. Science 2008, 319:921–926.PubMedView Article
            59. Zhou H, Xu M, Huang Q, Gates AT, Zhang XD, Castle JC, Stec E, Ferrer M, Strulovici B, Hazuda DJ, Espeseth AS: Genome-scale RNAi screen for host factors required for HIV replication. Cell Host Microbe 2008, 4:495–504.PubMedView Article
            60. Travers SA, Tully DC, McCormack GP, Fares MA: A study of the coevolutionary patterns operating within the env gene of the HIV-1 group M subtypes. Mol Biol Evol 2007, 24:2787–2801.PubMedView Article
            61. Basheeruddin K, Rechtoris C, Mazzone T: Evaluation of the role of Ap1-like proteins in the enhanced apolipoprotein E gene transcription accompanying phorbol ester induced macrophage differentiation. Biochim Biophys Acta 1994, 1218:235–241.PubMedView Article
            62. Schwende H, Fitzke E, Ambs P, Dieter P: Differences in the state of differentiation of THP-1 cells induced by phorbol ester and 1,25-dihydroxyvitamin D3. J Leukoc Biol 1996, 59:555–561.PubMed
            63. Kewalramani VN, Emerman M: Vpx association with mature core structures of HIV-2. Virology 1996, 218:159–168.PubMedView Article
            64. Kotov A, Zhou J, Flicker P, Aiken C: Association of Nef with the human immunodeficiency virus type 1 core. J Virol 1999, 73:8824–8830.PubMed
            65. Aiken C: Cell-free assays for HIV-1 uncoating. Methods Mol Biol 2009, 485:41–53.PubMedView Article
            66. Shah VB, Aiken C: In vitro uncoating of HIV-1 cores. J Vis Exp 2011,(57):e3384.
            67. Yu X, Matsuda Z, Yu QC, Lee TH, Essex M: Vpx of simian immunodeficiency virus is localized primarily outside the virus core in mature virions. J Virol 1993, 67:4386–4390.PubMed
            68. Liu H, Wu X, Newman M, Shaw GM, Hahn BH, Kappes JC: The Vif protein of human and simian immunodeficiency viruses is packaged into virions and associates with viral core structures. J Virol 1995, 69:7630–7638.PubMed
            69. Gluschankof P, Mondor I, Gelderblom HR, Sattentau QJ: Cell membrane vesicles are a major contaminant of gradient-enriched human immunodeficiency virus type-1 preparations. Virology 1997, 230:125–133.PubMedView Article
            70. Trubey CM, Chertova E, Coren LV, Hilburn JM, Hixson CV, Nagashima K, Lifson JD, Ott DE: Quantitation of HLA class II protein incorporated into human immunodeficiency type 1 virions purified by anti-CD45 immunoaffinity depletion of microvesicles. J Virol 2003, 77:12699–12709.PubMedView Article
            71. Abdelhaleem MM, Hameed S, Klassen D, Greenberg AH: Leukophysin: an RNA helicase A-related molecule identified in cytotoxic T cell granules and vesicles. J Immunol 1996, 156:2026–2035.PubMed
            72. Bolinger C, Sharma A, Singh D, Yu L, Boris-Lawrie K: RNA helicase A modulates translation of HIV-1 and infectivity of progeny virions. Nucleic Acids Res 2010, 38:1686–1696.PubMedView Article
            73. Xing L, Liang C, Kleiman L: Coordinate roles of Gag and RNA helicase A in promoting the annealing of formula to HIV-1 RNA. J Virol 2011, 85:1847–1860.PubMedView Article
            74. Bushman FD, Malani N, Fernandes J, D'Orso I, Cagney G, Diamond TL, Zhou H, Hazuda DJ, Espeseth AS, Konig R, et al.: Host cell factors in HIV replication: meta-analysis of genome-wide studies. PLoS Pathog 2009, 5:e1000437.PubMedView Article
            75. Jager S, Cimermancic P, Gulbahce N, Johnson JR, McGovern KE, Clarke SC, Shales M, Mercenne G, Pache L, Li K, et al.: Global landscape of HIV-human protein complexes. Nature 2012, 481:365–370.
            76. Rhee SS, Hunter E: Structural role of the matrix protein of type D retroviruses in gag polyprotein stability and capsid assembly. J Virol 1990, 64:4383–4389.PubMed
            77. Rhee SS, Hunter E: Amino acid substitutions within the matrix protein of type D retroviruses affect assembly, transport and membrane association of a capsid. EMBO J 1991, 10:535–546.PubMed
            78. Bukrinsky MI, Haggerty S, Dempsey MP, Sharova N, Adzhubel A, Spitz L, Lewis P, Goldfarb D, Emerman M, Stevenson M: A nuclear localization signal within HIV-1 matrix protein that governs infection of non-dividing cells. Nature 1993, 365:666–669.PubMedView Article
            79. Mannioui A, Nelson E, Schiffer C, Felix N, Le Rouzic E, Benichou S, Gluckman JC, Canque B: Human immunodeficiency virus type 1 KK26–27 matrix mutants display impaired infectivity, circularization and integration but not nuclear import. Virology 2005, 339:21–30.PubMedView Article
            80. Yu X, Yuan X, Matsuda Z, Lee TH, Essex M: The matrix protein of human immunodeficiency virus type 1 is required for incorporation of viral envelope protein into mature virions. J Virol 1992, 66:4966–4971.PubMed
            81. Dorfman T, Mammano F, Haseltine WA, Gottlinger HG: Role of the matrix protein in the virion association of the human immunodeficiency virus type 1 envelope glycoprotein. J Virol 1994, 68:1689–1696.PubMed
            82. Checkley MA, Luttge BG, Freed EO: HIV-1 envelope glycoprotein biosynthesis, trafficking, and incorporation. J Mol Biol 2011, 410:582–608.PubMedView Article
            83. Qi M, Aiken C: Selective restriction of Nef-defective human immunodeficiency virus type 1 by a proteasome-dependent mechanism. J Virol 2007, 81:1534–1536.PubMedView Article
            84. Qi M, Aiken C: Nef enhances HIV-1 infectivity via association with the virus assembly complex. Virology 2008, 373:287–297.PubMedView Article
            85. Syed F, McCrae MA: Interactions in vivo between the Vif protein of HIV-1 and the precursor (Pr55(GAG)) of the virion nucleocapsid proteins. Arch Virol 2009, 154:1797–1805.PubMedView Article
            86. Cohen EA, Dehni G, Sodroski JG, Haseltine WA: Human immunodeficiency virus vpr product is a virion-associated regulatory protein. J Virol 1990, 64:3097–3099.PubMed
            87. Lu YL, Spearman P, Ratner L: Human immunodeficiency virus type 1 viral protein R localization in infected cells and virions. J Virol 1993, 67:6542–6550.PubMed
            88. Paxton W, Connor RI, Landau NR: Incorporation of Vpr into human immunodeficiency virus type 1 virions: requirement for the p6 region of gag and mutational analysis. J Virol 1993, 67:7229–7237.PubMed
            89. Heinzinger NK, Bukinsky MI, Haggerty SA, Ragland AM, Kewalramani V, Lee MA, Gendelman HE, Ratner L, Stevenson M, Emerman M: The Vpr protein of human immunodeficiency virus type 1 influences nuclear localization of viral nucleic acids in nondividing host cells. Proc Natl Acad Sci USA 1994, 91:7311–7315.PubMedView Article
            90. Popov S, Rexach M, Zybarth G, Reiling N, Lee MA, Ratner L, Lane CM, Moore MS, Blobel G, Bukrinsky M: Viral protein R regulates nuclear import of the HIV-1 pre-integration complex. EMBO J 1998, 17:909–917.PubMedView Article
            91. Vodicka MA, Koepp DM, Silver PA, Emerman M: HIV-1 Vpr interacts with the nuclear transport pathway to promote macrophage infection. Genes Dev 1998, 12:175–185.PubMedView Article
            92. Le Douce V, Herbein G, Rohr O, Schwartz C: Molecular mechanisms of HIV-1 persistence in the monocyte-macrophage lineage. Retrovirology 2010, 7:32.PubMedView Article
            93. Martin-Serrano J, Neil SJ: Host factors involved in retroviral budding and release. Nat Rev Microbiol 2011, 9:519–531.PubMedView Article
            94. Ott DE, Coren LV, Kane BP, Busch LK, Johnson DG, Sowder RC, Chertova EN, Arthur LO, Henderson LE: Cytoskeletal proteins inside human immunodeficiency virus type 1 virions. J Virol 1996, 70:7734–7743.PubMed
            95. Jeang KT, Yedavalli V: Role of RNA helicases in HIV-1 replication. Nucleic Acids Res 2006, 34:4198–4205.PubMedView Article
            96. Chai W, Ford LP, Lenertz L, Wright WE, Shay JW: Human Ku70/80 associates physically with telomerase through interaction with hTERT. J Biol Chem 2002, 277:47242–47247.PubMedView Article
            97. Pfingsten JS, Goodrich KJ, Taabazuing C, Ouenzar F, Chartrand P, Cech TR: Mutually exclusive binding of telomerase RNA and DNA by ku alters telomerase recruitment model. Cell 2012, 148:922–932.PubMedView Article
            98. Grigoletto A, Lestienne P, Rosenbaum J: The multifaceted proteins Reptin and Pontin as major players in cancer. Biochim Biophys Acta 2011, 1815:147–157.PubMed
            99. Humphries JD, Byron A, Bass MD, Craig SE, Pinney JW, Knight D, Humphries MJ: Proteomic analysis of integrin-associated complexes identifies RCC2 as a dual regulator of Rac1 and Arf6. Sci Signal 2009, 2:ra51.PubMedView Article
            100. Beddow AL, Richards SA, Orem NR, Macara IG: The Ran/TC4 GTPase-binding domain: identification by expression cloning and characterization of a conserved sequence motif. Proc Natl Acad Sci USA 1995, 92:3328–3332.PubMedView Article
            101. Kehlenbach RH, Dickmanns A, Kehlenbach A, Guan T, Gerace L: A role for RanBP1 in the release of CRM1 from the nuclear pore complex in a terminal step of nuclear export. J Cell Biol 1999, 145:645–657.PubMedView Article
            102. Ishaq M, Hu J, Wu X, Fu Q, Yang Y, Liu Q, Guo D: Knockdown of cellular RNA helicase DDX3 by short hairpin RNAs suppresses HIV-1 viral replication without inducing apoptosis. Mol Biotechnol 2008, 39:231–238.PubMedView Article
            103. Yedavalli VS, Neuveut C, Chi YH, Kleiman L, Jeang KT: Requirement of DDX3 DEAD box RNA helicase for HIV-1 Rev-RRE export function. Cell 2004, 119:381–392.PubMedView Article
            104. Li J, Tang H, Mullen TM, Westberg C, Reddy TR, Rose DW, Wong-Staal F: A role for RNA helicase A in post-transcriptional regulation of HIV type 1. Proc Natl Acad Sci USA 1999, 96:709–714.PubMedView Article
            105. Levesque K, Halvorsen M, Abrahamyan L, Chatel-Chaix L, Poupon V, Gordon H, DesGroseillers L, Gatignol A, Mouland AJ: Trafficking of HIV-1 RNA is mediated by heterogeneous nuclear ribonucleoprotein A2 expression and impacts on viral assembly. Traffic 2006, 7:1177–1193.PubMedView Article
            106. Okunola HL, Krainer AR: Cooperative-binding and splicing-repressive properties of hnRNP A1. Mol Cell Biol 2009, 29:5620–5631.PubMedView Article
            107. Marchand V, Santerre M, Aigueperse C, Fouillen L, Saliou JM, Van Dorsselaer A, Sanglier-Cianferani S, Branlant C, Motorin Y: Identification of protein partners of the human immunodeficiency virus 1 tat/rev exon 3 leads to the discovery of a new HIV-1 splicing regulator, protein hnRNP K. RNA Biol 2011, 8:325–342.PubMedView Article
            108. Ajamian L, Abrahamyan L, Milev M, Ivanov PV, Kulozik AE, Gehring NH, Mouland AJ: Unexpected roles for UPF1 in HIV-1 RNA metabolism and translation. RNA 2008, 14:914–927.PubMedView Article
            109. Liu B, Dai R, Tian CJ, Dawson L, Gorelick R, Yu XF: Interaction of the human immunodeficiency virus type 1 nucleocapsid with actin. J Virol 1999, 73:2901–2908.PubMed
            110. Arhel N, Genovesio A, Kim KA, Miko S, Perret E, Olivo-Marin JC, Shorte S, Charneau P: Quantitative four-dimensional tracking of cytoplasmic and nuclear HIV-1 complexes. Nat Methods 2006, 3:817–824.PubMedView Article
            111. Jolly C, Mitar I, Sattentau QJ: Requirement for an intact T-cell actin and tubulin cytoskeleton for efficient assembly and spread of human immunodeficiency virus type 1. J Virol 2007, 81:5547–5560.PubMedView Article
            112. Lehmann M, Milev MP, Abrahamyan L, Yao XJ, Pante N, Mouland AJ: Intracellular transport of human immunodeficiency virus type 1 genomic RNA and viral production are dependent on dynein motor function and late endosome positioning. J Biol Chem 2009, 284:14572–14585.PubMedView Article
            113. Welch MD, Mullins RD: Cellular control of actin nucleation. Annu Rev Cell Dev Biol 2002, 18:247–288.PubMedView Article
            114. Komano J, Miyauchi K, Matsuda Z, Yamamoto N: Inhibiting the Arp2/3 complex limits infection of both intracellular mature vaccinia virus and primate lentiviruses. Mol Biol Cell 2004, 15:5197–5207.PubMedView Article
            115. Christ F, Thys W, De Rijck J, Gijsbers R, Albanese A, Arosio D, Emiliani S, Rain JC, Benarous R, Cereseto A, Debyser Z: Transportin-SR2 imports HIV into the nucleus. Curr Biol 2008, 18:1192–1202.PubMedView Article
            116. Krishnan L, Matreyek KA, Oztop I, Lee K, Tipper CH, Li X, Dar MJ, Kewalramani VN, Engelman A: The requirement for cellular transportin 3 (TNPO3 or TRN-SR2) during infection maps to human immunodeficiency virus type 1 capsid and not integrase. J Virol 2010, 84:397–406.PubMedView Article
            117. Logue EC, Taylor KT, Goff PH, Landau NR: The cargo-binding domain of transportin 3 is required for lentivirus nuclear import. J Virol 2011, 85:12950–12961.PubMedView Article
            118. Gallay P, Hope T, Chin D, Trono D: HIV-1 infection of nondividing cells through the recognition of integrase by the importin/karyopherin pathway. Proc Natl Acad Sci USA 1997, 94:9825–9830.PubMedView Article
            119. Armon-Omer A, Graessmann A, Loyter A: A synthetic peptide bearing the HIV-1 integrase 161–173 amino acid residues mediates active nuclear import and binding to importin alpha: characterization of a functional nuclear localization signal. J Mol Biol 2004, 336:1117–1128.PubMedView Article
            120. Bouyac-Bertoia M, Dvorin JD, Fouchier RA, Jenkins Y, Meyer BE, Wu LI, Emerman M, Malim MH: HIV-1 infection requires a functional integrase NLS. Mol Cell 2001, 7:1025–1035.PubMedView Article
            121. Hearps AC, Jans DA: HIV-1 integrase is capable of targeting DNA to the nucleus via an importin alpha/beta-dependent mechanism. Biochem J 2006, 398:475–484.PubMedView Article
            122. Askjaer P, Bachi A, Wilm M, Bischoff FR, Weeks DL, Ogniewski V, Ohno M, Niehrs C, Kjems J, Mattaj IW, Fornerod M: RanGTP-regulated interactions of CRM1 with nucleoporins and a shuttling DEAD-box helicase. Mol Cell Biol 1999, 19:6276–6285.PubMed
            123. Murray JL, Mavrakis M, McDonald NJ, Yilla M, Sheng J, Bellini WJ, Zhao L, Le Doux JM, Shaw MW, Luo CC, et al.: Rab9 GTPase is required for replication of human immunodeficiency virus type 1, filoviruses, and measles virus. J Virol 2005, 79:11742–11751.PubMedView Article
            124. Stutz F, Izaurralde E, Mattaj IW, Rosbash M: A role for nucleoporin FG repeat domains in export of human immunodeficiency virus type 1 Rev protein and RNA from the nucleus. Mol Cell Biol 1996, 16:7144–7150.PubMed
            125. Strack B, Calistri A, Craig S, Popova E, Gottlinger HG: AIP1/ALIX is a binding partner for HIV-1 p6 and EIAV p9 functioning in virus budding. Cell 2003, 114:689–699.PubMedView Article
            126. Fukushi M, Dixon J, Kimura T, Tsurutani N, Dixon MJ, Yamamoto N: Identification and cloning of a novel cellular protein Naf1, Nef-associated factor 1, that increases cell surface CD4 expression. FEBS Lett 1999, 442:83–88.PubMedView Article
            127. Gurer C, Berthoux L, Luban J: Covalent modification of human immunodeficiency virus type 1 p6 by SUMO-1. J Virol 2005, 79:910–917.PubMedView Article
            128. Agostini I, Popov S, Li J, Dubrovsky L, Hao T, Bukrinsky M: Heat-shock protein 70 can replace viral protein R of HIV-1 during nuclear import of the viral preintegration complex. Exp Cell Res 2000, 259:398–403.PubMedView Article
            129. Iordanskiy S, Zhao Y, DiMarzio P, Agostini I, Dubrovsky L, Bukrinsky M: Heat-shock protein 70 exerts opposing effects on Vpr-dependent and Vpr-independent HIV-1 replication in macrophages. Blood 2004, 104:1867–1872.PubMedView Article
            130. von Schwedler UK, Stuchell M, Muller B, Ward DM, Chung HY, Morita E, Wang HE, Davis T, He GP, Cimbora DM, et al.: The protein network of HIV budding. Cell 2003, 114:701–713.PubMedView Article
            131. Benaroch P, Billard E, Gaudin R, Schindler M, Jouve M: HIV-1 assembly in macrophages. Retrovirology 2010, 7:29.PubMedView Article
            132. Pelchen-Matthews A, Kramer B, Marsh M: Infectious HIV-1 assembles in late endosomes in primary macrophages. J Cell Biol 2003, 162:443–455.PubMedView Article
            133. Deneka M, Pelchen-Matthews A, Byland R, Ruiz-Mateos E, Marsh M: In macrophages, HIV-1 assembles into an intracellular plasma membrane domain containing the tetraspanins CD81, CD9, and CD53. J Cell Biol 2007, 177:329–341.PubMedView Article
            134. Gousset K, Ablan SD, Coren LV, Ono A, Soheilian F, Nagashima K, Ott DE, Freed EO: Real-time visualization of HIV-1 GAG trafficking in infected macrophages. PLoS Pathog 2008, 4:e1000015.PubMedView Article
            135. Bennett AE, Narayan K, Shi D, Hartnell LM, Gousset K, He H, Lowekamp BC, Yoo TS, Bliss D, Freed EO, Subramaniam S: Ion-abrasion scanning electron microscopy reveals surface-connected tubular conduits in HIV-infected macrophages. PLoS Pathog 2009, 5:e1000591.PubMedView Article
            136. Usami Y, Popov S, Gottlinger HG: Potent rescue of human immunodeficiency virus type 1 late domain mutants by ALIX/AIP1 depends on its CHMP4 binding site. J Virol 2007, 81:6614–6622.PubMedView Article
            137. Ott DE: Potential roles of cellular proteins in HIV-1. Rev Med Virol 2002, 12:359–374.PubMedView Article
            138. Sasaki H, Ozaki H, Karaki H, Nonomura Y: Actin filaments play an essential role for transport of nascent HIV-1 proteins in host cells. Biochem Biophys Res Commun 2004, 316:588–593.PubMedView Article
            139. Kostenko S, Moens U: Heat shock protein 27 phosphorylation: kinases, phosphatases, functions and pathology. Cell Mol Life Sci 2009, 66:3289–3307.PubMedView Article
            140. Burston SG, Clarke AR: Molecular chaperones: physical and mechanistic properties. Essays Biochem 1995, 29:125–136.PubMed
            141. Mayer MP: Gymnastics of molecular chaperones. Mol Cell 2010, 39:321–331.PubMedView Article
            142. Shi Y, Reddy B, Manley JL: PP1/PP2A phosphatases are required for the second step of Pre-mRNA splicing and target specific snRNP proteins. Mol Cell 2006, 23:819–829.PubMedView Article
            143. Weber G, Cristao VF, De LAF, Santos KF, Holton N, Rappsilber J, Beggs JD, Wahl MC: Mechanism for Aar2p function as a U5 snRNP assembly factor. Genes Dev 2011, 25:1601–1612.PubMedView Article
            144. Torreira E, Jha S, Lopez-Blanco JR, Arias-Palomo E, Chacon P, Canas C, Ayora S, Dutta A, Llorca O: Architecture of the pontin/reptin complex, essential in the assembly of several macromolecular complexes. Structure 2008, 16:1511–1520.PubMedView Article
            145. Li L, Olvera JM, Yoder KE, Mitchell RS, Butler SL, Lieber M, Martin SL, Bushman FD: Role of the non-homologous DNA end joining pathway in the early steps of retroviral infection. EMBO J 2001, 20:3272–3281.PubMedView Article
            146. Jeanson L, Subra F, Vaganay S, Hervy M, Marangoni E, Bourhis J, Mouscadet JF: Effect of Ku80 depletion on the preintegrative steps of HIV-1 replication in human cells. Virology 2002, 300:100–108.PubMedView Article
            147. Martin-Serrano J: The role of ubiquitin in retroviral egress. Traffic 2007, 8:1297–1303.PubMedView Article
            148. Jager S, Gottwein E, Krausslich HG: Ubiquitination of human immunodeficiency virus type 1 Gag is highly dependent on Gag membrane association. J Virol 2007, 81:9193–9201.PubMedView Article
            149. Westervelt P, Trowbridge DB, Epstein LG, Blumberg BM, Li Y, Hahn BH, Shaw GM, Price RW, Ratner L: Macrophage tropism determinants of human immunodeficiency virus type 1 in vivo. J Virol 1992, 66:2577–2582.PubMed
            150. O'Doherty U, Swiggard WJ, Malim MH: Human immunodeficiency virus type 1 spinoculation enhances infection through virus binding. J Virol 2000, 74:10074–10080.PubMedView Article
            151. Formolo CA, Mintz M, Takanohashi A, Brown KJ, Vanderver A, Halligan B, Hathout Y: Time series proteome profiling. Methods Mol Biol 2011, 694:365–377.PubMedView Article
            152. Butler SL, Hansen MS, Bushman FD: A quantitative assay for HIV DNA integration in vivo. Nat Med 2001, 7:631–634.PubMedView Article

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