- Open Access
Intracellular HIV-1 Gag localization is impaired by mutations in the nucleocapsid zinc fingers
- Boyan Grigorov†1,
- Didier Décimo†1,
- Fatima Smagulova2,
- Christine Péchoux1,
- Marylène Mougel2,
- Delphine Muriaux1Email author and
- Jean-Luc Darlix1
© Grigorov et al; licensee BioMed Central Ltd. 2007
- Received: 04 May 2007
- Accepted: 03 August 2007
- Published: 03 August 2007
The HIV-1 nucleocapsid protein (NC) is formed of two CCHC zinc fingers flanked by highly basic regions. HIV-1 NC plays key roles in virus structure and replication via its nucleic acid binding and chaperoning properties. In fact, NC controls proviral DNA synthesis by reverse transcriptase (RT), gRNA dimerization and packaging, and virion assembly.
We previously reported a role for the first NC zinc finger in virion structure and replication . To investigate the role of both NC zinc fingers in intracellular Gag trafficking, and in virion assembly, we generated series of NC zinc fingers mutations. Results show that all Zinc finger mutations have a negative impact on virion biogenesis and maturation and rendered defective the mutant viruses. The NC zinc finger mutations caused an intracellular accumulation of Gag, which was found either diffuse in the cytoplasm or at the plasma membrane but not associated with endosomal membranes as for wild type Gag. Evidences are also provided showing that the intracellular interactions between NC-mutated Gag and the gRNA were impaired.
These results show that Gag oligomerization mediated by gRNA-NC interactions is required for correct Gag trafficking, and assembly in HIV-1 producing cells and the release of infectious viruses.
- Zinc Finger
- Late Endosome
- HeLaP4 Cell
- Lane Versus
- Zinc Finger Mutation
The retroviral Gag polyprotein precursor is formed of three essential domains, namely the matrix (MA), the capsid (CA) and the nucleocapsid (NC), which upon protease mediated processing of Gag constitute the architecture of the infectious mature viral particle. The three Gag domains contain the critical determinants that orchestrate virus assembly in the infected cell, via membrane-MA, CA-CA and NC-gRNA interactions [2–8]. In the mature virus, the MA protein is located under the virion envelope, which derives from the infected cell membrane. In the case of HIV-1, MA is myristoylated and contains basic amino acids within its N-terminus required for Gag-membrane binding and determinants that specifically interact with the cellular adaptator proteins AP-3 and AP-2. These AP proteins contribute to the intracellular transport of Gag to endosomal compartments and retroviral budding [9–11]. The CA molecules form the outer shell of the viral core while NC molecules extensively coat and condense the gRNA in the interior of the virion core . HIV-1 NC contains two zinc fingers flanked by basic regions and is located at the C-terminus of Gag, followed by the p6 domain. This later p6 domain is required for particle budding during which the viral particles pinch-off from the cellular membrane (reviewed in . The p6 domain contains a Proline-rich and a di-Leucine domains, which are the target of the cellular proteins Tsg101 and Alix, respectively, involved in the cellular class E protein sorting pathway and the HIV-1 budding machinery [5, 12, 13].
HIV-1 NC has been extensively studied during the past 15 years and was shown to be implicated in virus structure, gRNA dimerization and proviral DNA synthesis [3, 4, 7]. The highly basic nature of NC makes it a partner of choice of RNA while the zinc fingers appear to provide specific recognition of the HIV-1 Psi packaging signal necessary for gRNA packaging . Furthermore, specific RNA-NC interactions promote Gag-Gag oligomerization which turns out to be a prerequisite for assembly and virus biogenesis [15–18]. Both NC zinc fingers and basic domains are essential for virus formation and infectivity [1, 16, 17, 19–21]. Mutations in NC basic residues cause defects in Gag-viral RNA interactions and thus in HIV-1 assembly and budding [15, 16, 22]. More recently, new insights into the role of NC in Gag assembly show that mutations and deletions in the basic residues of NC prevent Gag-Gag multimerization but not Gag association with cellular membranes .
In the present study, we explored the influence of the NC zinc fingers in HIV-1 assembly by analyzing intracellular Gag and gRNA localization, Gag/membrane association and virion morphogenesis.
HIV-1 pNL4-3 DNA was provided by the National Institute of Health, USA. The HIV-1 ΔZF1 and H23C Gag mutant DNA constructs were described elsewhere . The HIV-1 GagΔNC proviral DNA construct  was provided by A.Cimarelli. The HIV-1 ΔZF2 and H44C Gag mutants were obtained by site directed mutagenesis on the pNL4.3 HIV-1 molecular clone as described  using the following oligonucleotides 5'CCTGTCTCTCAGTACCGCCCTTTTTCCTAG3' and 5'CTTTCATTTGGCATCCTTCC3', respectively. The double ΔZF1ZF2 and H23C/H44C Gag mutants were obtained by cloning the ApaI-AgeI fragments of H44C and ΔZF2 into the H23C and ΔZF1 pNL4.3 mutant clone, respectively. The pcDNA3.1 plasmid (Clonetech) was used as a control DNA vector.
Mammalian cell culture, DNA transfection and virus production
The human 293T cell line, HeLa P4 cells expressing the CD4 receptor and the LacZ gene under the control of the HIV-1 LTR and HeLa cells used were grown in Dulbecco's modified essential medium (DMEM), all supplemented with 10% fetal calf serum and antibiotics. 293T were transfected using the calcium phosphate method . For immunofluorescence staining, HeLa cells were transfected with DNA using the Fugene® transfection method (Invitrogen). To analyse virus production, cells were washed with PBS and medium was changed 5 h post-transfection. Culture supernatants containing virus particles were harvested 24 hours later and clarified by filtration (0.45 μm, Nalgen). The cells were then washed and lysed with 0,5% Triton-PBS.
Virions were purified from filtered culture supernatants by pelleting them through a cushion of 20% sucrose in TNE (100 mM NaCl, 10 mM Tris HCl, pH 7.4 and 1 mM EDTA) at 35 K rpm for 1 h in a Beckman SW41 rotor.
CAp24 antigen ELISA
To measure viral production, a CAp24 ELISA test was used. Aliquots of the same volume of viral supernatants (free CAp24 + virion associated = S) and pellet virions by ultracentrifugation (V) were resuspended in cell media with 0.5% Triton, and administered on 96 well plates coated with 10 μg/ml anti-CAp24 antibodies (23A5G and 3D10G9B8, BioMérieux) and then blocked with 10% horse serum in PBS-0.05% Tween-20. A biotinylated anti-CAp24 antibody (bioMérieux) was added and the ELISA was revealed with streptavidin and orthophenylene-diamine (OPD)-H2O2 (Sigma). The plate was read on ELISA-reader at 490 and 630 nm.
HIV Infectivity assays
Virus infectivity was assessed on HeLaP4 cells as described in . The infectivity was determined by counting the number of blue cells.
Genomic RNA analysis by Dot-Blot
For viral RNA analysis, virus pellets were resuspended in TNE buffer and lysed in 1% SDS, 100 μg of proteinase K per ml. Nucleic acids were extracted twice with phenol-chloroform and ethanol precipitated. Pellets were resuspended in DNase buffer (40 mM Tris-HCl, pH 7.5, 6 mM MgCl2, 10 mM NaCl, 10 mM dithiotreitol, 200 U of RNasin per ml) and contaminant plasmid DNA was digested with RQ1 DNase (100 U/ml) for 20 min at 37°C. RNA was purified by phenol-chloroform extraction, ethanol precipitated and resuspended in water. Hybridization with a random 32P-labeled 5.3 kb SacI-SalI fragment of the pNL4-3 plasmid corresponding to gag and pol sequences and quantitative analyses were done as previously described .
Twenty-four hours post transfection, 293T cells were washed with PBS and removed from the plate in PBS-1 mM EDTA, pelleted by centrifugation at 600 × g, resuspended in 1 ml of a homogenization buffer containing 10 mM Tris-HCl, pH 7.5, 0.25 M sucrose, 1 mM EDTA and protease inhibitors (Complete Mini EDTA-free from Roche), and then fragmented using a glass homogenizer. Nuclei were eliminated by centrifugation at 600 × g for 10 min at 4°C. The resulting post-nuclear supernatant (PNS) was subjected to subcellular fractionation on OptiPrep® gradient for the separation of different membrane compartments as described elsewhere . Fractions were collected and proteins were analyzed by SDS-PAGE and immunoblotting.
Viral proteins were separated on 10% SDS-PAGE and detected by immunoblotting with a mouse anti-CAp24 (P3D10G9B8, BioMérieux), and the cellular protein in the gradient was detected with the mouse anti-Lamp2 (Santa Cruz Biotechnology Inc.). The corresponding immunoglobulins conjugated with horse radish peroxidase (HRP) (DakoCytomation) were used and the signal was detected using SuperSignal® West Pico Chemiluminescent Substrate (Pierce).
Fractions from Optiprep® gradients were resuspended in equal volumes of a lysis buffer containing 100 mM Tris-HCl, pH 7.4, 20 mM EDTA, 2% SDS, 200 mM NaCl and 200 μg/ml proteinase K and incubated at 37°C for 30 min. RNA was purified by phenol/chloroform extraction, and precipitated with ethanol. RNA samples were pelleted by centrifugation at 4°C, 14 000 rpm for 30 min, and resuspended in RNAse-free water. Contaminant DNA was eliminated by digestion with RQ1 DNAse. RNA aliquots were reverse transcribed using the Invitrogene RT assay. The RT reaction was followed by PCR of the cDNA using primers for the cPPT as follows: up cPPT- nt. 4775 GCGCGATCGATCCACAATTTTAAAAGAAAAGGGGGGATTG, and down cPPT- nt. 4907 GCGCGATCGATTGTAATAAACCCGAAAATTTTG. The PCR DNA product of 132 bp was separated on a 2% agarose gel and visualized by ethidium bromide staining. The gel images were quantified by Metamorph software and semi-quantitave analysis of the gRNA was evaluated.
Immunofluorescence staining and confocal microscopy imaging
293T cells grown on poly-lysine coated coverslips and HeLa cells were transfected and, 24 h later, fixed in 3% paraformaldehyde-PBS for 20 min. After fixation, cells were permeabilized using 0.2 % Triton, and then incubated in 1% BSA-PBS with primary antibodies: mouse anti-CAp24 (BioMérieux or NIH), rabbit anti-MAp17 (NIH, USA), mouse anti-Lamp1 and anti-Lamp3 (Santa Cruz Biotechnology Inc.). The corresponding fluorescent Alexa® 488 and 546-conjugated secondary antibodies were used (Molecular probes). Coverslips were washed and mounted on microscope slides with Mowiol (Sigma). Images were acquired on Axioplan 2 Zeiss CLSM 510 confocal microscope with Argon 488/458, HeNe 543 lasers and plan apochromat 63× 1.4 oil objective, supplied with LSM 510 software. The percentage of colocalization (merge signals) was evaluated by the Metamorph software (UIC).
Fluorescent in situ hybridization (FISH)
Transfected 293T or HeLa cells were grown on poly-lysine treated coverslips and 24 h post-transfection were washed with PBS and fixed as described before  and stored at 4°C in 70% ethanol. Detection of the HIV-1 genomic RNA was performed by FISH  with a Cy3-conjugated oligonucleotide (GagHIVCy3), corresponding to position 1524 to 1563 of the HIV-1 sequence (MWG-Biotech). The probe was adjusted to 1 ng/ul in 66% formamide, 0.2× SSC, 2 μg/μl tRNA and 2 μg/μl of sheared salmon sperm DNA. After a denaturation step of 5 min at 95°c, the probe was mixed V/V with a solution containing 20% sulfate dextran, 4 × SSC, 0,04% RNase-free BSA and 4 mM Vanadyl-ribonucleoside complex, and applied to each coverslip. Hybridization was performed at 37°C overnight in a humid chamber. 24 hs later, the coverslips were washed twice with 50% formamide, 2× SSC at 37°c, followed by 3 washes with 50% formamide, 1× SSC at 37°c, and mounted on a slide in Vectashield with DAPI (Vector Laboratories Inc.). Image acquisition and analysis were performed in the Montpellier RIO Imaging microscopy facility. Images were taken with a Leica DMRA wide-field microscope and acquisition was performed with a Coolsnap HQ camera driven by Metamorph software.
Transmission electron microscopy
HeLaP4 cells expressing HIV-1 or either one of the NC zinc finger mutants were fixed in 2% glutaraldehyde, 0.1 M Sörensen phosphate buffer, pH 7.4 for 30 min at 4°C. Then, cells were washed 3 × 10 min with phosphate buffer containing 0.2 M sucrose and post-fixed in 1% OsO4 that was 1.5% with respect to potassium ferrocyanide for 1 hr at room temperature. Cells were dehydrated through graded ethanol and embedded in Epon 812. Thin sections were cut and picked up on 200 mesh copper grids, stained with uranyl acetate and counter-stained with lead citrate. Specimens were analyzed with a Philips CM120 electron microscope (CMEABG – Villeurbanne – France).
1- Mutations in NC zinc fingers impair virus production, maturation, gRNA packaging and infectivity
Properties of HIV-1 NC zinc fingers mutants produced by HeLa-P4 cells
total p24 release (S)
% of p24 in virions (V)
relative levels of gRNA in virions
37 ± 9
31.5 ± 4.5
10 ± 3
18.5 ± 9.5
3 ± 1
3.5 ± 1.5
43 ± 6
8.3 ± 0.5
5 ± 1
24.7 ± 12
17 ± 7
4 ± 0
23.7 ± 10
1 ± 0.1
3.5 ± 0.5
0.5 ± 0.1
Viral proteins present in cells and viral particles were analyzed by immunoblotting using anti-CAp24 (Fig. 1B). In wild-type virions, the vast majority of Gag has been processed (Fig. 1B, lane wt, V) while large amounts of unprocessed Gag and p41/p49 were found in the ZF mutant virions (Fig. 1B, lanes V). In comparison with the wild-type HIV-1, processing of Gag in viral particles was partially changed by mutating the NC zinc fingers, as evidenced by an accumulation of the MA-CA precursor (p41) and probably MA-CA-p2-NC (p49) (Fig. 1B, lanes V). The same defect in Gag processing was observed following mutating or deleting the first ZF . Processing of Gag was modified by the H23H44C mutations and ΔZF1ZF2 deletions, as indicated by the accumulation of partially cleaved Gag products, in particular p41 in virions (Fig. 1B, see arrows; compare lane "V" of these ZF mutants with lane "V" for the WT). In particular, these two NC mutants lack p49; thus the absence of p49 could be due to an undetectable level of protein or that mutations in both ZFs still permit maturation of p49. All the other NC mutants show p41 and p49 accumulation in the virions (Fig. 1B, lanes V), indicating a defect in maturation cleavage between NC-p1 or p1-p6 (that can be due to a conformational change of NC induced by ZF mutation, as previously reported . All NC mutants show intracellular Gag and p41/49 accumulation, except for limited amounts of CAp24/25, suggesting some defect in the budding process (Fig 1B, lanes Cell). These results suggest that ZF mutants have a negative impact on virus budding and Gag processing.
Although the viral production was low, enough virus could be recovered to monitor the relative level of genomic RNA (gRNA) in virions. The level of gRNA in virions was analyzed by dot blot hybridization (not shown) and, as reported in Table 1, none of the NC mutants harbored a wt gRNA level. In fact, mutations in the ZF reduced gRNA levels by 10 to 20 fold in comparison with wild-type HIV-1. Finally, the infectivity of HIV-1 ZF mutants was assessed and none of the mutants were infectious (Table 1).
2- Intracellular accumulation and localization of Gag proteins with NC ZF mutations
3- Influence of ZF mutations on intracellular genomic RNA localization
These results suggest that ZF-mutated Gag is poorly associated with the gRNA at the cell surface and that the ΔZF1ZF2 and H23H44C NC mutations alter intracellular Gag-gRNA interactions.
4- NC ZF mutations prevent intracellular Gag-RNA localization in late endosomes
In the case of the ΔZF1ZF2-NC mutant, the subcellular fractionation only reveals the mutated Gag and the viral RNA located at the bottom of the gradient (Fig. 5B, fractions 18–20), probably associated with active ribosomes. Semi-quantitative analysis of the gRNA level by RT-PCR show that 70% of the gRNA for ΔZF1ZF2-NC mutant and 15% for ΔNC-Gag mutant is remaining in comparison with wild-type gRNA (100% in the whole gradient), indicating that deletion of the NC domain results in gRNA instability, possibly due to impaired interactions between Gag and the gRNA. In addition, colocalization of the mutated Gag and the gRNA disappeared at the level of late endosomes and at the PM (Fig. 5B, fractions 7–10, and fraction 1, respectively). Abnormal processed mutated Gag was found in fractions 14–15 in comparison with wild-type Gag, suggesting a defect in Gag targeting and/or budding. As in HeLa cells, we observed by immunofluorescence microscopy of 293T cells that mutated Gag accumulated in endosomal membranes and in discrete domains at the PM (Fig. 5B, see IF). By FISH, the gRNA accumulated in the cytoplasm, and again the PM labeling was lost (Fig. 5B, see FISH). Similar results were obtained upon deletion of NC (Fig. 5C) since GagΔNC was found all over the gradient, in agreement with the immunofluorescence analysis where Gag was evenly distributed within the cell (Fig. 5C, see IF) as well as the gRNA (see FISH). Similar results were obtained with the H23H44C-Gag mutant (data not shown).
Taken together, these results indicate that the ZF mutations impair intracellular Gag/gRNA association, most probably due to an alteration of their interactions.
5- Impact of the NC zinc finger mutations on the structure of the viral particles as seen by electron microscopy
Quantitative analysis of virus core morphology of NC mutant HIV-1 particles produced by HeLaP4 cells
Dense cone shape core
Dense material in base of cone
Round centered core
Round displaced core
Tubular core structure
Two core structures or membrane*
No defined core
Diameter of particles
113 ± 4
134 ± 14
129 ± 14
96 ± 12
131 ± 12
101 ± 13
Taken together, these results show that HIV-1 NC zinc fingers play an important role in viral core structure and suggest that NC-NC and/or NC-gRNA interactions are essential for HIV-1 Gag assembly and particle release.
The retroviral Gag polyprotein orchestrates retrovirus assembly in the infected cell via two platforms, which are a cellular membrane and a RNA (reviewed in [2, 6]. The current view of the assembly process implies that the newly made Gag binds to the cellular membrane by the N-terminal myristoylated domain and stretches of basic residues of the matrix domain (reviewed in [31, 32], also involving inositol phosphates/Gag interactions [33–36]. At the same time, the NC domain selectively binds the gRNA via specific interactions with the packaging Psi signal , which in turn promote Gag oligomerization . Although, a leucine zipper motif could functionally, at least in part, replace NC to drive the assembly of a "minimal" Gag . We propose that the interactions between Gag-NC and the genomic Psi signal will ensure both the formation of Gag oligomers and the selective recruitment of the gRNA. Consistent with this view, mutations in the first NC zinc finger or the flanking basic residues result in a strong decrease of viral particle production and infectivity [1, 16, 17, 19, 38]. Thus, it has been proposed that the NC domain of Gag is required for the proper assembly and release of infectious virions.
To confirm the multiple roles played by NC in HIV-1 assembly, we have examined the role of the NC zinc fingers (ZF) in Gag trafficking. Taken together, our results show that both NC zinc fingers play critical roles in the ability of Gag to properly assemble and ultimately to bud. In fact, all HIV-1 ZF mutants examined so far produced particles at levels five to ten fold, or more, lower than that of wild-type HIV-1 and were not infectious (Table 1).
In model cell lines, the wild-type HIV-1 Gag was found to accumulate either at the PM or on intracellular tetraspanin-rich endosomal membranes as recently reported [8, 25, 30, 39–43]. Mutating the NC Zinc fingers caused Gag to accumulate within the cell, in a diffuse manner and at the PM (Fig. 2). Thus, mutating the NC ZF appears to prevent Gag targeting to and accumulation in endosomes (Fig. 3). This favors the view that endosomes are an important site for virus formation and release (Fig. 3), and also maturation since intracellular mature CAp24 was drastically reduced in the case of the ZF mutants (Fig. 1B). It also indicates that NC is not the major determinant for Gag targeting to the PM, such as the basic MA domain of Gag and phosphatidyl inositol phosphate lipids are [8, 30, 34, 44, 45]. However, NC contributes to the localization of Gag in late endosomes (Fig. 3 and 5).
As already stated, the other platform necessary for virus assembly is the gRNA since it directs the oligomerization of the newly made Gag upon binding [15, 46, 47]. As previously shown for mutations in the first ZF , we found that mutating the second ZF and both of them strongly impaired gRNA content in mutant virions as well as particle release (Table 1), and resulted in the production of replication defective viruses. Staufen, a cellular RNA-binding protein involved in RNA transport and metabolism, was reported to interact with both the gRNA and the NC domain of HIV-1 Gag, and to have a role in HIV-1 gRNA encapsidation and Gag assembly [48, 49]. Mapping the interaction domain between Staufen and Gag reveals the importance of the NC domain, in particular the second ZF [48, 49]. This could explain, at least in part, the role played by the second ZF on gRNA encapsidation if Staufen is responsible for gRNA trafficking to the assembly site and consequently RNA-dependent Gag oligomerization and assembly.
However, Ott and colleagues reported that the decrease in viral production due to NC deletion can be compensated by an RNA binding site in HIV-1 MA domain of Gag . It is very difficult to dissociate the role of NC in virus assembly from NC-RNA interactions, which are critical for the structure of the viral particle [22, 51], rather than assembly or budding [16, 17]. Our data favor a model in wich all events are linked and dependent upon Gag-RNA interactions via the NC domain, required to achieve a proper viral assembly, i.e. multimerization of Gag, Gag oligomer targeting and trafficking, and ultimately particle assembly, budding and release.
To better examine the role of Gag-RNA interactions in assembly, we examined gRNA localization by FISH (Fig. 4) and by subcellular fractionation (Fig 5). The gRNA was found in the nucleus, most probably due to provirus transcription, in the cytoplasm and at the PM of cells expressing wild-type HIV-1 (Fig. 4), in agreement with Berthold and Mandarelli . Wild-type Gag and the gRNA were also found at the level of late endosomes [25, 53, 54]. Mutating the NC ZF motifs drastically altered the cellular distribution of the gRNA, because it was found evenly distributed within the cell and no longer associated with the PM (Fig. 4, by FISH) or the late endosomes (Fig. 5, by gradient), while the NC-mutated Gag accumulated mainly at the PM (Fig. 3 and 5). Thus, specific Gag-gRNA interactions via the NC-ZF are most probably required for proper Gag trafficking through Gag-Gag multimer complexes. In agreement with this view, it was reported that Gag expressed from Psi(-) RNA diffuses throughout the cell and shows delayed cytoplasmic colocalisation with the gRNA . The authors propose that the packaging signal may coordinate capture of the genomic Psi(+) RNA by Gag, followed by assembly and transport to the budding site. This data indeed mirrors the results obtained with the NC mutants, in particular the Gag-ΔNC mutant, for which both the gRNA and mutated Gag were found diffuse throughout the cell and had probably lost Gag-gRNA association (as seen in Fig. 4 and 5). Taken together, these data strongly suggest that the specific Gag-gRNA interactions via the NC domain are necessary for proper Gag trafficking and assembly, for Gag oligomers to be targeted to either specific domains at the PM or to the late endosomes.
Finally, the structure of the HIV-1 virions carrying one or several mutations in the NC ZF was determined by electron microscopy (Fig. 6). Results show that the NC mutated virions have lost their conical core shape and are immature, which confirm the role of NC in HIV-1 virion structure [1, 16], as well as for other retroviruses, such as SIV  and MLV . Our results correlate with previous studies on the assembly of several retroviruses, namely HIV, MLV, RSV that have revealed a critical role of NC in virus assembly and release [1, 16, 17, 21, 56, 57].
Thanks are due to Fabienne Simian-Lermé and Claire Lionnet (PLATIM, ENS Lyon, France) for their helpful assistance in CSLM. We thank Pr Radka Argirova (National Institute of Infectious and Parasitic Diseases, Bulgaria) for the support of B.G. Work supported by INSERM, CNRS, ANRS, French Embassy in Bulgaria, FRM, the european TRIoH consortium, and by the Ministère de la Recherche (ACI) and SIDACTION (to MM). D.M. was supported by FRM, TRIoH and ANRS (French agency against AIDS) and FS by the CNRS.
- Tanchou V, Decimo D, Pechoux C, Lener D, Rogemond V, Berthoux L, Ottmann M, Darlix JL: Role of the N-terminal zinc finger of human immunodeficiency virus type 1 nucleocapsid protein in virus structure and replication. Journal of virology. 1998, 72 (5): 4442-4447.PubMed CentralPubMedGoogle Scholar
- Cimarelli A, Darlix JL: Assembling the human immunodeficiency virus type 1. Cell Mol Life Sci. 2002, 59 (7): 1166-1184.View ArticlePubMedGoogle Scholar
- Darlix JL, Cristofari G, Rau M, Pechoux C, Berthoux L, Roques B: Nucleocapsid protein of human immunodeficiency virus as a model protein with chaperoning functions and as a target for antiviral drugs. Advances in pharmacology (San Diego, Calif. 2000, 48: 345-372.View ArticleGoogle Scholar
- Darlix JL, Lapadat-Tapolsky M, de Rocquigny H, Roques BP: First glimpses at structure-function relationships of the nucleocapsid protein of retroviruses. Journal of molecular biology. 1995, 254 (4): 523-537.View ArticlePubMedGoogle Scholar
- Demirov DG, Freed EO: Retrovirus budding. Virus research. 2004, 106 (2): 87-102.View ArticlePubMedGoogle Scholar
- Muriaux D, Darlix JL, Cimarelli A: Targeting the assembly of the human immunodeficiency virus type I. Current pharmaceutical design. 2004, 10 (30): 3725-3739.View ArticlePubMedGoogle Scholar
- Rein A, Henderson LE, Levin JG: Nucleic-acid-chaperone activity of retroviral nucleocapsid proteins: significance for viral replication. Trends in biochemical sciences. 1998, 23 (8): 297-301.View ArticlePubMedGoogle Scholar
- Resh MD: Intracellular trafficking of HIV-1 Gag: how Gag interacts with cell membranes and makes viral particles. AIDS reviews. 2005, 7 (2): 84-91.PubMedGoogle Scholar
- Batonick M, Favre M, Boge M, Spearman P, Honing S, Thali M: Interaction of HIV-1 Gag with the clathrin-associated adaptor AP-2. Virology. 2005, 342 (2): 190-200.View ArticlePubMedGoogle Scholar
- Dong X, Li H, Derdowski A, Ding L, Burnett A, Chen X, Peters TR, Dermody TS, Woodruff E, Wang JJ, Spearman P: AP-3 directs the intracellular trafficking of HIV-1 Gag and plays a key role in particle assembly. Cell. 2005, 120 (5): 663-674.View ArticlePubMedGoogle Scholar
- Camus G, Segura-Morales C, Molle D, Lopez-Verges S, Begon-Pescia C, Cazevieille C, Schu P, Bertrand E, Berlioz-Torrent C, Basyuk E: The Clathrin Adaptor Complex AP-1 Binds HIV-1 and MLV Gag and Facilitates Their Budding. Mol Biol Cell. 2007Google Scholar
- 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 (6): 689-699.View ArticlePubMedGoogle Scholar
- von Schwedler UK, Stuchell M, Muller B, Ward DM, Chung HY, Morita E, Wang HE, Davis T, He GP, Cimbora DM, Scott A, Krausslich HG, Kaplan J, Morham SG, Sundquist WI: The protein network of HIV budding. Cell. 2003, 114 (6): 701-713.View ArticlePubMedGoogle Scholar
- De Guzman RN, Wu ZR, Stalling CC, Pappalardo L, Borer PN, Summers MF: Structure of the HIV-1 nucleocapsid protein bound to the SL3 psi-RNA recognition element. Science (New York, NY. 1998, 279 (5349): 384-388.View ArticleGoogle Scholar
- Burniston MT, Cimarelli A, Colgan J, Curtis SP, Luban J: Human immunodeficiency virus type 1 Gag polyprotein multimerization requires the nucleocapsid domain and RNA and is promoted by the capsid-dimer interface and the basic region of matrix protein. Journal of virology. 1999, 73 (10): 8527-8540.PubMed CentralPubMedGoogle Scholar
- Cimarelli A, Sandin S, Hoglund S, Luban J: Basic residues in human immunodeficiency virus type 1 nucleocapsid promote virion assembly via interaction with RNA. Journal of virology. 2000, 74 (7): 3046-3057.PubMed CentralView ArticlePubMedGoogle Scholar
- Muriaux D, Costes S, Nagashima K, Mirro J, Cho E, Lockett S, Rein A: Role of murine leukemia virus nucleocapsid protein in virus assembly. Journal of virology. 2004, 78 (22): 12378-12385.PubMed CentralView ArticlePubMedGoogle Scholar
- Muriaux D, Mirro J, Harvin D, Rein A: RNA is a structural element in retrovirus particles. Proceedings of the National Academy of Sciences of the United States of America. 2001, 98 (9): 5246-5251.PubMed CentralView ArticlePubMedGoogle Scholar
- Berthoux L, Pechoux C, Darlix JL: Multiple effects of an anti-human immunodeficiency virus nucleocapsid inhibitor on virus morphology and replication. Journal of virology. 1999, 73 (12): 10000-10009.PubMed CentralPubMedGoogle Scholar
- Buckman JS, Bosche WJ, Gorelick RJ: Human immunodeficiency virus type 1 nucleocapsid zn(2+) fingers are required for efficient reverse transcription, initial integration processes, and protection of newly synthesized viral DNA. Journal of virology. 2003, 77 (2): 1469-1480.PubMed CentralView ArticlePubMedGoogle Scholar
- Gorelick RJ, Gagliardi TD, Bosche WJ, Wiltrout TA, Coren LV, Chabot DJ, Lifson JD, Henderson LE, Arthur LO: Strict conservation of the retroviral nucleocapsid protein zinc finger is strongly influenced by its role in viral infection processes: characterization of HIV-1 particles containing mutant nucleocapsid zinc-coordinating sequences. Virology. 1999, 256 (1): 92-104.View ArticlePubMedGoogle Scholar
- Wang SW, Noonan K, Aldovini A: Nucleocapsid-RNA interactions are essential to structural stability but not to assembly of retroviruses. Journal of virology. 2004, 78 (2): 716-723.PubMed CentralView ArticlePubMedGoogle Scholar
- Ono A, Waheed AA, Joshi A, Freed EO: Association of human immunodeficiency virus type 1 gag with membrane does not require highly basic sequences in the nucleocapsid: use of a novel Gag multimerization assay. Journal of virology. 2005, 79 (22): 14131-14140.PubMed CentralView ArticlePubMedGoogle Scholar
- Cimarelli A, Luban J: Human immunodeficiency virus type 1 virion density is not determined by nucleocapsid basic residues. Journal of virology. 2000, 74 (15): 6734-6740.PubMed CentralView ArticlePubMedGoogle Scholar
- Grigorov B, Arcanger F, Roingeard P, Darlix JL, Muriaux D: Assembly of infectious HIV-1 in human epithelial and T-lymphoblastic cell lines. Journal of molecular biology. 2006, 359 (4): 848-862.View ArticlePubMedGoogle Scholar
- Smagulova F, Maurel S, Morichaud Z, Devaux C, Mougel M, Houzet L: The highly structured encapsidation signal of MuLV RNA is involved in the nuclear export of its unspliced RNA. Journal of molecular biology. 2005, 354 (5): 1118-1128.View ArticlePubMedGoogle Scholar
- Demene H, Dong CZ, Ottmann M, Rouyez MC, Jullian N, Morellet N, Mely Y, Darlix JL, Fournie-Zaluski MC, Saragosti S, et al: 1H NMR structure and biological studies of the His23-->Cys mutant nucleocapsid protein of HIV-1 indicate that the conformation of the first zinc finger is critical for virus infectivity. Biochemistry. 1994, 33 (39): 11707-11716.View ArticlePubMedGoogle Scholar
- Ottmann M, Gabus C, Darlix JL: The central globular domain of the nucleocapsid protein of human immunodeficiency virus type 1 is critical for virion structure and infectivity. Journal of virology. 1995, 69 (3): 1778-1784.PubMed CentralPubMedGoogle Scholar
- Hermida-Matsumoto L, Resh MD: Localization of human immunodeficiency virus type 1 Gag and Env at the plasma membrane by confocal imaging. Journal of virology. 2000, 74 (18): 8670-8679.PubMed CentralView ArticlePubMedGoogle Scholar
- Ono A, Freed EO: Cell-type-dependent targeting of human immunodeficiency virus type 1 assembly to the plasma membrane and the multivesicular body. Journal of virology. 2004, 78 (3): 1552-1563.PubMed CentralView ArticlePubMedGoogle Scholar
- Conte MR, Matthews S: Retroviral matrix proteins: a structural perspective. Virology. 1998, 246 (2): 191-198.View ArticlePubMedGoogle Scholar
- Resh MD: A myristoyl switch regulates membrane binding of HIV-1 Gag. Proceedings of the National Academy of Sciences of the United States of America. 2004, 101 (2): 417-418.PubMed CentralView ArticlePubMedGoogle Scholar
- Campbell S, Fisher RJ, Towler EM, Fox S, Issaq HJ, Wolfe T, Phillips LR, Rein A: Modulation of HIV-like particle assembly in vitro by inositol phosphates. Proceedings of the National Academy of Sciences of the United States of America. 2001, 98 (19): 10875-10879.PubMed CentralView ArticlePubMedGoogle Scholar
- Ono A, Ablan SD, Lockett SJ, Nagashima K, Freed EO: Phosphatidylinositol (4,5) bisphosphate regulates HIV-1 Gag targeting to the plasma membrane. Proceedings of the National Academy of Sciences of the United States of America. 2004, 101 (41): 14889-14894.PubMed CentralView ArticlePubMedGoogle Scholar
- Saad JS, Miller J, Tai J, Kim A, Ghanam RH, Summers MF: Structural basis for targeting HIV-1 Gag proteins to the plasma membrane for virus assembly. Proceedings of the National Academy of Sciences of the United States of America. 2006, 103 (30): 11364-11369.PubMed CentralView ArticlePubMedGoogle Scholar
- Datta SA, Zhao Z, Clark PK, Tarasov S, Alexandratos JN, Campbell SJ, Kvaratskhelia M, Lebowitz J, Rein A: Interactions between HIV-1 Gag molecules in solution: an inositol phosphate-mediated switch. Journal of molecular biology. 2007, 365 (3): 799-811.PubMed CentralView ArticlePubMedGoogle Scholar
- Accola MA, Strack B, Gottlinger HG: Efficient particle production by minimal Gag constructs which retain the carboxy-terminal domain of human immunodeficiency virus type 1 capsid-p2 and a late assembly domain. Journal of virology. 2000, 74 (12): 5395-5402.PubMed CentralView ArticlePubMedGoogle Scholar
- Gorelick RJ, Benveniste RE, Gagliardi TD, Wiltrout TA, Busch LK, Bosche WJ, Coren LV, Lifson JD, Bradley PJ, Henderson LE, Arthur LO: Nucleocapsid protein zinc-finger mutants of simian immunodeficiency virus strain mne produce virions that are replication defective in vitro and in vivo. Virology. 1999, 253 (2): 259-270.View ArticlePubMedGoogle Scholar
- Jouvenet N, Neil SJ, Bess C, Johnson MC, Virgen CA, Simon SM, Bieniasz PD: Plasma membrane is the site of productive HIV-1 particle assembly. PLoS biology. 2006, 4 (12): e435-PubMed CentralView ArticlePubMedGoogle Scholar
- Nydegger S, Foti M, Derdowski A, Spearman P, Thali M: HIV-1 egress is gated through late endosomal membranes. Traffic (Copenhagen, Denmark). 2003, 4 (12): 902-910.View ArticleGoogle Scholar
- Perlman M, Resh MD: Identification of an intracellular trafficking and assembly pathway for HIV-1 gag. Traffic (Copenhagen, Denmark). 2006, 7 (6): 731-745.View ArticleGoogle Scholar
- Rudner L, Nydegger S, Coren LV, Nagashima K, Thali M, Ott DE: Dynamic fluorescent imaging of human immunodeficiency virus type 1 gag in live cells by biarsenical labeling. Journal of virology. 2005, 79 (7): 4055-4065.PubMed CentralView ArticlePubMedGoogle Scholar
- Sherer NM, Lehmann MJ, Jimenez-Soto LF, Ingmundson A, Horner SM, Cicchetti G, Allen PG, Pypaert M, Cunningham JM, Mothes W: Visualization of retroviral replication in living cells reveals budding into multivesicular bodies. Traffic (Copenhagen, Denmark). 2003, 4 (11): 785-801.View ArticleGoogle Scholar
- Freed EO, Orenstein JM, Buckler-White AJ, Martin MA: Single amino acid changes in the human immunodeficiency virus type 1 matrix protein block virus particle production. Journal of virology. 1994, 68 (8): 5311-5320.PubMed CentralPubMedGoogle Scholar
- Zhou W, Parent LJ, Wills JW, Resh MD: Identification of a membrane-binding domain within the amino-terminal region of human immunodeficiency virus type 1 Gag protein which interacts with acidic phospholipids. Journal of virology. 1994, 68 (4): 2556-2569.PubMed CentralPubMedGoogle Scholar
- Campbell S, Rein A: In vitro assembly properties of human immunodeficiency virus type 1 Gag protein lacking the p6 domain. Journal of virology. 1999, 73 (3): 2270-2279.PubMed CentralPubMedGoogle Scholar
- Roldan A, Russell RS, Marchand B, Gotte M, Liang C, Wainberg MA: In vitro identification and characterization of an early complex linking HIV-1 genomic RNA recognition and Pr55Gag multimerization. The Journal of biological chemistry. 2004, 279 (38): 39886-39894.View ArticlePubMedGoogle Scholar
- 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. Journal of virology. 2007, 81 (12): 6216-6230.PubMed CentralView ArticlePubMedGoogle Scholar
- Chatel-Chaix L, Clement JF, Martel C, Beriault V, Gatignol A, DesGroseillers L, Mouland AJ: Identification of Staufen in the human immunodeficiency virus type 1 Gag ribonucleoprotein complex and a role in generating infectious viral particles. Molecular and cellular biology. 2004, 24 (7): 2637-2648.PubMed CentralView ArticlePubMedGoogle Scholar
- Ott DE, Coren LV, Gagliardi TD: Redundant roles for nucleocapsid and matrix RNA-binding sequences in human immunodeficiency virus type 1 assembly. Journal of virology. 2005, 79 (22): 13839-13847.PubMed CentralView ArticlePubMedGoogle Scholar
- Wang SW, Aldovini A: RNA incorporation is critical for retroviral particle integrity after cell membrane assembly of Gag complexes. Journal of virology. 2002, 76 (23): 11853-11865.PubMed CentralView ArticlePubMedGoogle Scholar
- Berthold E, Maldarelli F: cis-acting elements in human immunodeficiency virus type 1 RNAs direct viral transcripts to distinct intranuclear locations. Journal of virology. 1996, 70 (7): 4667-4682.PubMed CentralPubMedGoogle Scholar
- Houzet L, Gay B, Morichaud Z, Briant L, Mougel M: Intracellular assembly and budding of the Murine Leukemia Virus in infected cells. Retrovirology. 2006, 3: 12-PubMed CentralView ArticlePubMedGoogle Scholar
- Poole E, Strappe P, Mok HP, Hicks R, Lever AM: HIV-1 Gag-RNA interaction occurs at a perinuclear/centrosomal site; analysis by confocal microscopy and FRET. Traffic (Copenhagen, Denmark). 2005, 6 (9): 741-755.View ArticleGoogle Scholar
- Yovandich JL, Chertova EN, Kane BP, Gagliardi TD, Bess JW, Sowder RC, Henderson LE, Gorelick RJ: Alteration of zinc-binding residues of simian immunodeficiency virus p8(NC) results in subtle differences in gag processing and virion maturation associated with degradative loss of mutant NC. Journal of virology. 2001, 75 (1): 115-124.PubMed CentralView ArticlePubMedGoogle Scholar
- Dawson L, Yu XF: The role of nucleocapsid of HIV-1 in virus assembly. Virology. 1998, 251 (1): 141-157.View ArticlePubMedGoogle Scholar
- Lee EG, Linial ML: Deletion of a Cys-His motif from the Alpharetrovirus nucleocapsid domain reveals late domain mutant-like budding defects. Virology. 2006, 347 (1): 226-233.View ArticlePubMedGoogle Scholar
- Bampi C, Bibillo A, Wendeler M, Divita G, Gorelick RJ, Le Grice SF, Darlix JL: Nucleotide excision repair and template-independent addition by HIV-1 reverse transcriptase in the presence of nucleocapsid protein. The Journal of biological chemistry. 2006, 281 (17): 11736-11743.View ArticlePubMedGoogle Scholar
- Levin JG, Guo J, Rouzina I, Musier-Forsyth K: Nucleic acid chaperone activity of HIV-1 nucleocapsid protein: critical role in reverse transcription and molecular mechanism. Progress in nucleic acid research and molecular biology. 2005, 80: 217-286.View ArticlePubMedGoogle Scholar
- Hu WS, Rhodes T, Dang Q, Pathak V: Retroviral recombination: review of genetic analyses. Front Biosci. 2003, 8: 143-155.View ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.