In vivo analysis of highly conserved Nef activities in HIV-1 replication and pathogenesis
© Watkins et al.; licensee BioMed Central Ltd. 2013
Received: 16 August 2013
Accepted: 23 October 2013
Published: 30 October 2013
The HIV-1 accessory protein, Nef, is decisive for progression to AIDS. In vitro characterization of the protein has described many Nef activities of unknown in vivo significance including CD4 downregulation and a number of activities that depend on Nef interacting with host SH3 domain proteins. Here, we use the BLT humanized mouse model of HIV-1 infection to assess their impact on viral replication and pathogenesis and the selection pressure to restore these activities using enforced in vivo evolution.
We followed the evolution of HIV-1LAI (LAI) with a frame-shifted nef (LAINeffs) during infection of BLT mice. LAINeffs was rapidly replaced in blood by virus with short deletions in nef that restored the open reading frame (LAINeffs∆-1 and LAINeffs∆-13). Subsequently, LAINeffs∆-1 was often replaced by wild type LAI. Unexpectedly, LAINeffs∆-1 and LAINeffs∆-13 Nefs were specifically defective for CD4 downregulation activity. Viruses with these mutant nefs were used to infect BLT mice. LAINeffs∆-1 and LAINeffs∆-13 exhibited three-fold reduced viral replication (compared to LAI) and a 50% reduction of systemic CD4+ T cells (>90% for LAI) demonstrating the importance of CD4 downregulation. These results also demonstrate that functions other than CD4 downregulation enhanced viral replication and pathogenesis of LAINeffs∆-1 and LAINeffs∆-13 compared to LAINeffs. To gain insight into the nature of these activities, we constructed the double mutant P72A/P75A. Multiple Nef activities can be negated by mutating the SH3 domain binding site (P72Q73V74P75L76R77) to P72A/P75A and this mutation does not affect CD4 downregulation. Virus with nef mutated to P72A/P75A closely resembled the wild-type virus in vivo as viral replication and pathogenesis was not significantly altered. Unlike LAINeffs described above, the P72A/P75A mutation had a very weak tendency to revert to wild type sequence.
The in vivo phenotype of Nef is significantly dependent on CD4 downregulation but minimally on the numerous Nef activities that require an intact SH3 domain binding motif. These results suggest that CD4 downregulation plus one or more unknown Nef activities contribute to enhanced viral replication and pathogenesis and are suitable targets for anti-HIV therapy. Enforced evolution studies in BLT mice will greatly facilitate identification of these critical activities.
KeywordsHIV-1 Nef Replication Pathogenesis BLT humanized mice Mutation
Patients infected with nef-defective HIV-1, have strongly attenuated viral replication and pathogenesis [1–4]. In vitro studies have defined numerous Nef activities but how this 206 amino acid protein has such a major effect on the outcome of HIV-1 infection in patients is unknown [5–9]. One view of Nef’s overall impact on HIV-1 infection is that there is a cumulative effect of multiple activities to achieve high viral loads resulting in the development of AIDS [10, 11]. In support of this view, a number of Nef activities have been found to be conserved in monkey, ape and human immunodeficiency viruses [12–17]. A difficulty with this interpretation is that there are so many Nef activities that the effect of any given activity on replication and pathogenesis would be small. Alternatively, one or a few Nef functionalities may be the major contributors to viral replication and pathogenesis. In this regard CD4 downregulation, a highly conserved Nef function, is of particular interest. Ex vivo studies with activated peripheral blood T cells and cultures of tonsil tissue support a dominant role for CD4 downregulation in establishing high rates of viral replication [18–20]. Another factor that may be critical is the SH3 domain binding site in Nef’s polyproline helix [21–23]. This ten amino acid segment (PVRPQVPLRP) is the most highly conserved stretch of amino acids in the protein . Evidence exists for SH3 domain binding site involvement with enhanced viral replication [21, 23, 25], cytotoxic effects [26–30], activation of Hck  and antagonism of host immune responses [32–36]. Nef structure/function studies have documented that the CD4 downregulation activity and the SH3 domain protein dependent activities are genetically distinct [21, 37, 38].
To gain greater understanding of the roles of Nef’s diverse activities during HIV-1 replication we have employed the BLT humanized mouse model. This model has stable reconstitution of a full spectrum of human immune cells and has been used to investigate a number of different aspects of HIV-1 infection [39–44]. With regard to Nef, we have previously compared the replicative properties of HIV-1LAI (LAI) and LAI with two large deletions in nef coding sequence (LAINefdd) in BLT humanzed mice . LAI exhibited high levels of viral replication and near total depletion of CD4+ T cells in blood and tissues, as well as, depletion of CD4+ CD8+ thymocytes from the human thymic organoid. LAINefdd had significantly reduced viral replication and dramatically reduced capacity for inducing CD4+ T cell and CD4+ CD8+ thymocyte loss . However, one important aspect of HIV-1 infection of BLT humanized mice that has not yet been investigated is the ability of nef to evolve during HIV-1 infection. In patients, HIV-1 nef extensively mutates resulting in tremendous sequence diversity but it has not been possible to clearly relate these changes to Nef activities or the pathogenic potential of the virus [24, 45–49]. Here, we investigate three critical features of Nef’s role during HIV-1 infection: 1) the ability of the virus to mutate nef sequences to gain enhanced replicative fitness, 2) the role of CD4 downregulation in viral replication and pathogenesis and 3) the importance of Nef’s interactions with host SH3 domain proteins in replication and pathogenesis. We find that Nef induced CD4 downregulation is highly significant for active viral replication and pathogenesis. In addition, there are unidentified function(s) that contribute to viral replication and/or CD4+ T cell depletion and are necessary for Nef’s full pathogenic potential. Importantly, this latter function or functions does not depend on interactions with host cell SH3 domain proteins.
The amino acid sequences of the restored nefs are reported in Figure 2B. The changes in Nef sequence resulting from the one base deletion (LAINeffs∆-1) and the thirteen base deletion (LAINeffs-13) were the replacement of three amino acids (DLE, 36–38) in wild type LAINef with four missense amino acids (SRPG) and the replacement of ten wild type amino acids (DLEKHGAITS, 36–45) with seven missense amino acids (SRPGKTC), respectively (Figure 2B). The sequencing data suggested that virus with fs∆-1 and fs∆-13 nefs had a strong replicative advantage over the nef-defective virus. However, the replacement of LAINeffs∆-1 with wild type (WT) virus in four mice further suggests a replicative advantage for wild type nef over revertant nefs. Based on these in vivo findings, we were interested in characterizing the in vitro activities of the ∆-1 and ∆-13 mutant Nefs.
In vitro functional analysis of nef mutants that evolved in vivo
Infection of BLT humanized mice with in vivo generated nefmutations
During infection with LAI, CD4+ T cell levels in blood were dramatically reduced (Figure 4B and D) while CD4+ T cells in the blood of uninfected mice were maintained at approximately 80% of total blood T cells (Figure 4B and D). For LAI, the average time to reduce CD4+ T cells to 50% of total blood T cells was 21.6 ± 2.4 days post infection (dpi, n = 7). For mice inoculated with LAINeffs∆-1 or LAINeffs∆-13, an intermediate loss of CD4+ T cells was evident (Figure 4B and D). The time for CD4+ T cells in blood to decline to 50% of total T cells was determined and compared to LAI (Figure 4B and D). As noted, LAI gave 21.6 ± 2.4 dpi (n = 7) which was significantly shorter than LAINeffs∆-1 at 65.1 ± 13.4 dpi (n = 4, p = 0.0106) and LAINeffs∆-13 at 52.5 ± 13.5 dpi (n = 4, p = 0.0294). LAINeffs∆-1 and LAINeffs∆-13 infected mice were not statistically different from each other. Together, the results from Figure 4 document an intermediate in vivo Nef phenotype for LAINeffs∆-1 and LAINeffs∆-13.
We have previously reported the phenotypes of LAI and LAI with a totally inactivated nef (LAINefdd, ). The observation that LAI expressing a Nef specifically defective for CD4 downregulation has an intermediate phenotype not expected based on previous reports [18, 19, 56]. In support of this conclusion, we also observed that a partial loss of CD4+ T cells from blood is established by six weeks. At this time point, the percent of CD4+ T cells in LAINeffs∆-1 and LAINeffs∆-13 infected mice were significantly lower than in Naïve mice but significantly higher than in LAI-infected mice (Figure 4B and D). For LAINeffs∆-1 inoculated mice, the percent CD4+ T cells of total T cells present in blood was 55.4 ± 3.3 (n = 4) compared to 77.5 ± 2.8 (n = 4) for Naïve (Figure 4B) with p = 0.0286. For LAINeffs∆-13 inoculated mice, the percentages were 47.0 ± 11.7 (n = 4) versus 77.5 ± 2.8 (n = 4) with p = 0.0286. Also at six weeks, LAINeffs∆-1 and LAINeffs∆-13 infected mice had higher percentages of CD4+ T cells than LAI infected mice (Figure 4B and D). Percent of CD4+ T cells for LAI was 12.5 ± 4.5 (n = 6), versus 55.4 ± 3.3 (n = 4, p = 0.0095) for LAINeffs∆-1. Percent of CD4+ T cells for LAI versus LAINeffs∆-13 was 12.5 ± 4.5 (n = 6) versus 47.0 ± 11.7 (n = 4, p = 0.0190).
At eight weeks, CD4+ T cells in blood of LAI infected mice are nearly depleted while Naïve mice maintained CD4+ T cells at approximately 80% of total CD4+ T cells (Figure 4B and D, [44, 57]). It was of interest to allow the LAINeffs∆-1 and LAINeffs∆-13 infections to continue past eight weeks to determine if these viruses would slowly deplete CD4+ T cells from blood. By 14 weeks, substantial levels of CD4+ T cells were still evident in blood for both viruses which emphasizes the persistence of the partial Nef phenotype in the absence of CD4 downregulation (Figure 4B and D).
Systemic loss of CD4+ T cells in BLT humanized mice infected with LAINeffs∆-1 and LAINeffs∆-13
We previously reported a devastating impact of LAI infection on CD4+ CD8+ thymocytes. However, LAI lacking a functional nef failed to reduce double positive thymocytes . In Figure 5B, drastic depletion of CD4+ CD8+ thymocytes was confirmed following inoculation with LAI. Intermediate losses were observed with LAINeffs∆-1 and LAINeffs∆-13 (Naive, 76.3 ± 3.0%; LAI, 1.7 ± 1.2%; LAINeffs∆-1, 35.0 ± 17.1%; LAINeffs∆-13, 29.3 ± 10.2%). On the basis of the above results, we conclude that the partial losses of LAINeffs∆-1 and LAINeffs∆-13 found for CD4+ T cells appeared to extend to CD4+ CD8+ thymocytes as well.
The mechanistic interpretation of the intermediate phenotype of the LAINeffs∆-1 and LAINeffs∆-13 viruses depends on the status of the sequence of nef. We sequenced nef in plasma virion RNA of LAINeffs∆-1 and LAINeffs∆-13 and found no reversions over the course of infection. Specifically, for LAINeffs∆-1, the four base insertion and the ∆-1 deletion remained intact. For LAINeffs∆-13, the four base insertion and the thirteen base deletion remained intact. There were no second site mutations present in nef either (not shown). The absence of wild type nef sequence from LAINeffs∆-1 and LAINeffs∆-13 infected BLT mice implies the stability of the phenotypic properties of these two nefs during infection. This failure of nefs from LAINeffs∆-1 and LAINeffs∆-13 to revert to wild type supports the hypothesis that the appearance of wild type nef sequence found in four of seven mice (Figure 2) infected with LAINeffs was the result of an exact four base deletion and not a two-step removal of the four base insertion plus a one base addition (Additional file 1). Therefore, our investigations of LAINeffs∆-1 and LAINeffs∆-13 demonstrate that LAIs stably lacking Nef’s CD4 downregulation activity have the in vivo phenotype of a reduced capacity for viral replication, for CD4+ T cell depletion and for CD4+ CD8+ thymocyte depletion relative to LAI .
LAI, LAINeffs∆-1 and LAINeffs∆-13 and systemic T cell activation
One explanation for the intermediate infection phenotypes of LAINeffs∆-1 and LAINeffs∆-13 would be an inability of these mutated HIV-1 to induce systemic T cell activation [58, 59]. It has been previously reported that naïve BLT mice have approximately 2% of CD8+ T cells that are CD38+ HLA-DR+ double positive in blood. Infection with LAI or LAINefdd elevates this fraction to approximately 8% [42, 44]. We observed similar effects of LAINeffs∆-1 and LAINeffs∆-13 infection on T cell activation. At six weeks post infection, LAINeffs∆-1 and LAINeffs∆-13 were determined to have 8.2 ± 3.5% (n = 4) and 6.1 ± 2.3% (n = 4) CD38+ HLA-DR+ double positive CD8+ T cells in blood, respectively. Thus, LAINeffs∆-1 and LAINeffs∆-13 exhibit the same enhancements of peripheral blood T cell activation as LAI and LAINefdd.
The role of SH3 domain dependent activities on LAI infection of BLT mice
Systemic depeltion of CD4+T cells and thymocytes by LAINefP72A/P75A
In vivoselection pressure to correct the P72A/P75A mutation is weak
Previously, we established that there are large phenotypic differences between infection of BLT mice with wild type LAI and the nef-defective LAINefdd in vivo. LAI replicates to high viral loads concomitantly with aggressive and systematic depletion of CD4+ T cells and CD4+ CD8+ thymocytes. LAINefdd exhibits 6–7 fold lower peak viral loads and has little to no capacity to deplete CD4+ T cells or thymocytes . These two large effects of Nef make it feasible to characterize the importance of Nef’s individual activities in BLT mice . Here, we have demonstrated a third important property of nef in the BLT mouse model- the ability to evolve and restore functionality. Viruses expressing Nef proteins have a decisive replicative advantage over the frame-shifted LAINeffs and replace the nef-defective virus within a few weeks. Hence, in seven mice, the input LAINeffs was lost after four weeks with either LAINeffs∆-1 (six mice) or LAINeffs∆-13 (one mouse) being the sole virus in peripheral blood. By eight weeks, four of seven mice further evolved to be predominantly wild type virus.
The strong in vivo selection of LAINeffs∆-1 and LAINeffs∆-13 over LAINeffs led us to characterize these in vivo selected mutant proteins in vitro. We discovered them to be stable but with a total loss of CD4 downregulation activity. Three other in vitro Nef activities, MHCI downregulation, PAK2 activation and enhancement of virion infectivity, remained intact. When BLT mice are infected with LAINeffs∆-1 and LAINeffs∆-13, we observed an approximate 3-fold reduction in peak viral load and a partial loss of CD4+ T cells and CD4+ CD8+ thymocytes relative to that observed for LAI. These observations suggest that the in vivo selection of the two viruses with mutant nefs relative to LAINeffs relied on activities beyond CD4 downregulation. Conversely, the partial reduction of Nef effectiveness observed for LAINeffs∆-1 and LAINeffs∆-13 demonstrates a significant role for CD4 downregulation.
Our data provided evidence that there is selective pressure for restoration of Nef activities other than CD4 downregulation. The identity of these activities is unknown. We considered likely candidates to be one or more of the SH3 domain binding site dependent activities. These activities include enhancement of virion infectivity [21, 25, 37], PAK2 activation [21–23], upregulation of FasL and PD1 [28, 29], activation of Hck , downregulation of MHCI [32–34] and Lck-dependent activation Ras-Erk signaling to promote the production of the T lymphocyte survival factor IL-2 [62, 63]. We mutated prolines 72 and 75 to alanine to prevent interactions between Nef and host cell SH3 domain proteins [21, 22]. This mutation did not exhibit a negative effect on Nef function in BLT mice. One explanation for this counter intuitive observation is that high levels of replication and rapid reduction in CD4+ T cell and CD4+ CD8+ thymocytes depend on only a few Nef activities.
Future studies with BLT mice will investigate Nef activities that are potentially responsible for the CD4 downregulation-independent aspects of Nef function in vivo. Possible activities include elevated secretion of exosomes, blocking the anti-viral effect of autophagy and inhibition of ASK1 [64–69]. Conversely, these studies may lead to the important result that known Nef activities may not account for a substantial portion of its impact on HIV-1 infection in vivo. In this regard, our mutational strategy of introducing palindromic insertions into Nef coding sequence can be extended to scan the protein for regions of special significance for viral replication and pathogenesis. The HIV-1/BLT mice infection model described here is a feasible experimental platform for resolving these questions.
CD4 downregulation activity accounts for approximately half of Nef’s capacity to enhance viral replication and deplete CD4+ T cells and CD4+ CD8+ thymocytes. This result is consistent with the high degree of conservation of the CD4 downregulation activity. Identities of the Nef activities that account for the remainder of Nef’s effects are unknown. We found these latter activities which are present in LAINeffs∆-1 and LAINeffs∆-13 provide the virus a strong selective advantage over LAINeffs that is fully defective for Nef expression. In addition, wild type virus, expressing a fully active Nef, out-competes virus expressing Nef specifically defective for CD4 downregulation. We tested the Nef activities dependent on the SH3 domain binding site because the corresponding amino acid sequence in the protein is highly conserved. However, the virus with nef mutated for SH3 domain binding was essentially wild type in its ability to enhance viral replication and deplete CD4+ T cells and CD4+ CD8+ thymocytes. Selective pressure for the mutant nef to revert to wild type was low. It is critical to determine which Nef activities or activities that do not depend on SH3 domain protein binding yet have major impacts on viral replication and pathogenesis.
Preparation of BLT humanized mice
BLT humanized mice were prepared as previously described [40–42, 44, 57, 71–77]. Briefly, thymus/liver implanted or NOD/SCID IL-2γ-/- mice (The Jackson Laboratories, Bar Harbor, ME) were transplanted with autologous human CD34+ cells isolated from fetal liver (Advanced Bioscience Resources, Alameda, CA). Human reconstitution in the peripheral blood of these mice was monitored periodically by flow cytometry (FACSCanto; BD Biosciences). Mice were maintained either at the Animal Resources Center, UT Southwestern Medical Center at Dallas (UTSWMC) or at the Division of Laboratory Animal Medicine, University of North Carolina at Chapel Hill (UNC-CH) in accordance with protocols approved by the UTSWMC or UNC-CH Institutional Animal Care and Use Committees.
To ensure genetic diversity, fifteen different tissue donors were used to generate five groups of mice used for the experiments presented in this manuscript. The overall level of engraftment for all the mice used in this manuscript was 60.9% ± 3.2% (n = 27). None of the groups (Naïve, LAI, LAINeffs∆-1, LAINeffs∆-13 and LAINefP72A/P75A) had significantly different engraftment levels compared to any of the other groups (p ≥ 0.1535). All groups had at least two different human genetic backgrounds included in the evaluation of infection. LAINeffs∆-1, LAINeffs∆-13 and LAINefP72A/P75A infected groups each shared a common donor with the LAI infected group.
Cell lines and culture conditions
HeLa Magi and TZM-bl cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Cellgro, Herndon, VA) supplemented with 10% fetal bovine serum (FBS; Cellgro), 100 IU/ml of penicillin, 100 μg/ml streptomycin, and 2 mM glutamine (Cellgro) in 10% CO2 at 37°C. Similarly, 293T cells were cultured under the same conditions as TZM-bl and HeLa Magi cells but in 5% CO2. The human CEM T cell line was cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (Hyclone), 50 IU of penicillin per ml, 50 μg streptomycin per ml, 2 mM L-glutamine and 1 mM sodium pyruvate in 10% CO2 at 37°C.
The proviral clone, pLAI (accession # K02013), was described by Peden et al. . pLAINeffs was constructed to be defective for nef by cutting with XhoI, filling in with Klenow and re-ligating. This leaves nef sequence intact but introduces a four-base frame-shift after nef codon 35 (Additional file 1). The one base deletion (8501) and thirteen base deletion (8511–8523) found in nef sequences from LAINeffs infected mice were inserted into pLAINef fs by site directed mutagenesis to produce pLAINeffs∆-1 and pLAINeffs∆-13, respectively.
Virus production, exposure of BLT mice to HIV-1LAI and HIV-1LAI with mutated nefs, tissue harvesting and flow cytometric analyses
Stocks of LAI, LAINeffs, LAINeffs∆-1, LAINeffs∆-13 and LAINefP72A/P75A were prepared and titered as we previously described [54, 79]. Briefly, proviral clones were transfected into 293T cells. Viral supernatant was collected 48 hours after transfection and diluted in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, 100 IU penicillin/ml, 100 μg/ml streptomycin, and 2 mM glutamine. TZM-bl cells were infected in 12-well tissue culture plates with 0.4 ml of virus at multiple dilutions in medium for two hours. Then, 1.0 ml of supplemented DMEM was added and the plates incubated overnight. Virus containing medium was removed the next day, replaced with fresh DMEM plus 10% fetal bovine serum and the incubation continued for 24 hours. The cells were fixed and stained with 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (40 hours after first exposure to virus). Individual blue cells were counted directly to determine infectious particles per ml (TCIU). Each titer of these viral stocks was performed in triplicate and at least two different titer determinations were performed for each virus preparation.
Intravenous exposure of BLT mice with infectious virus was conducted via tail vein injection with the indicated tissue culture infectious units (TCIU). Viral load in peripheral blood of infected mice was monitored longitudinally by quantitative real-time PCR using Taqman RNA to-CT™ 1-step kit from Applied Biosystems, USA [72, 73, 80]. The sequences of the forward and reverse primers and the Taqman probe for PCR were: 5′-CATGTTTTCAGCATTATCAGAAGGA-3′, 5′-TGCTTGATGTCCCCCCACT-3′, and 5′-FAM CCACCCCACAAGATTTAAACACCATGCTAA-Q-3′, respectively.
CD4+ and CD8+ T cell levels were monitored by flow cytometric analysis as previously described [40, 57, 76]. Immunophenotyping was performed on blood samples collected longitudinally and on mononuclear cells isolated from tissues at harvest. Whole peripheral blood (PB) from humanized mice was analyzed according to the BD Biosciences Lyse/Wash protocol (Cat. No. 349202) as we have previously described . Briefly, following antibody labeling of whole blood, red blood cells were lysed. The remaining cells were washed, fixed and the sample was analyzed by flow cytometry. Tissue mononuclear cell isolations and immunophenotyping analyses were also performed according to published methods [40, 57, 76]. Flow cytometric gating for CD4 and CD8 cell surface expression was performed as follows: (step 1) forward and side scatter properties were utilized to set a live cell gate; (step2) live cells were then analyzed for expression of the human pan-leukocyte marker CD45; (step 3) human leukocytes were then analyzed for hCD3 and (step 4) these T cells or thymocytes were analyzed for hCD4 and hCD8 expression.
The panel of antibodies for analysis of CD8+ T cells double positive for CD38+ and HLA-DR+ was CD8 FITC (SK1), HLA-DR, PE (TU36) or IgG2bκ PE, CD4 PerCP (SK3), CD3 PE-Cy7 (SK7), CD38 APC (HB7) or IgG1κ APC, and CD45 APC-Cy7 (2D1) (all purchased from BD Biosciences). Gating was performed as follows: (step 1) forward and side scatter properties were utilized to set a live cell gate; (step 2) live cells were then analyzed for expression of the human pan-leukocyte marker CD45; (step 3) human leukocytes were then analyzed for CD3; (step 4) T cells were analyzed for CD4 and/or CD8 expression; (step 5) activation of human CD8+ T cells was analyzed for HLA-DR and CD38 expression . Gates defining HLA-DR and CD38 expression were set with isotype-matched flourophore-conjugated antibodies.
Viral replication in vitro
The human T-cell line A3.01 (NIH AIDS Reagent Program) was used to propagate both wild-type and nef-mutant HIV-1LAI. Cells were infected with virus stocks at a multiplicity of infection (MOI) of 0.05 in complete RPMI (containing 10% fetal bovine serum (Hyclone), 50 IU of penicillin per ml, 50 μg streptomycin per ml, 2 mM L-glutamine, and 1 mM sodium pyruvate) plus 2 μg/ml polybrene at 37°C, 5% CO2 for 4 hours. The cells were washed extensively with PBS and cultured at 37°C, 5% CO2 in complete RPMI. Cell cultures were passaged twice weekly at which time a sample of the culture supernatant was collected for quantification of viral capsid protein by p24 gag ELISA (HIV-1 p24 Antigen Capture Assay (Advance Biosciences Library, Inc., #5421).
In vitroanalysis of Nef activities
The site directed mutations of nef in pLAINeffs∆-1, pLAINeffs∆-13 and pLAINefP72A/P75A were subcloned into pLXSN, a retroviral vector for transduction of CEM T cells and into pcDNA3.1 for transfection into 293T cells . Assays for CD4 downregulation, MHCI downregulation, and activation of PAK2 were described previously . Enhancement of virion infectivity was determined by single infection assays using HeLa-MAGI indicator cells with virus produced from proviral clones transfected into 293T cells [21, 82]. Protein expression was determined by Western Blot analysis with sheep anti-Nef antibody or mouse monoclonal anti-Nef [21, 83].
Sequence analysis of plasma virion RNA
Viral RNA was extracted from 20 μl of plasma from infected mice using the QIAamp Viral RNA Mini kit (Qiagen Sciences, USA). RNA was then reverse transcribed into cDNA, which was then subjected to nested PCR. The outer primers for nef amplification are 5′-AGCTTGCTCAATGCCACAGCC-3′ and 5′-GCTGCATATAAGCAGCTGCTTTTTG-3′. The inner primers are 5′-TAGAGCTATTCGCCACATACC-3′ and 5′-GCTTGCTACAAGGGACTTTCCGC-3′. Gel purified PCR products were sequenced and the sequences were aligned to HIVLAI sequences to determine if nucleotide changes had occurred.
Mann–Whitney tests were performed in Prism version 5 (Graph Pad). All data plotted as mean ± S.E.M.
This work was supported by grant AI33331 from the National Institute of Allergy and Infectious Diseases of the National Institutes of Health, USA and UNC CFAR P30 A1504410. RLW is supported by NIH Virology Training Grant 5T32A1007419.
- Calugi G, Montella F, Favalli C, Benedetto A: Entire genome of a strain of human immunodeficiency virus type 1 with a deletion of nef that was recovered 20 years after primary infection: large pool of proviruses with deletions of env. J Virol. 2006, 80: 11892-11896. 10.1128/JVI.00932-06.PubMed CentralView ArticlePubMedGoogle Scholar
- Gorry PR, McPhee DA, Verity E, Dyer WB, Wesselingh SL, Learmont J, Sullivan JS, Roche M, Zaunders JJ, Gabuzda D, et al: Pathogenicity and immunogenicity of attenuated, nef-deleted HIV-1 strains in vivo. Retrovirology. 2007, 4: 66-10.1186/1742-4690-4-66.PubMed CentralView ArticlePubMedGoogle Scholar
- Kirchhoff F, Greenough TC, Brettler DB, Sullivan JL, Desrosiers RC: Brief report: absence of intact nef sequences in a long-term survivor with nonprogressive HIV-1 infection. N Engl J Med. 1995, 332: 228-232. 10.1056/NEJM199501263320405.View ArticlePubMedGoogle Scholar
- Kondo M, Shima T, Nishizawa M, Sudo K, Iwamuro S, Okabe T, Takebe Y, Imai M: Identification of attenuated variants of HIV-1 circulating recombinant form 01_AE that are associated with slow disease progression due to gross genetic alterations in the nef/long terminal repeat sequences. J Infect Dis. 2005, 192: 56-61. 10.1086/430739.View ArticlePubMedGoogle Scholar
- Abraham L, Fackler OT: HIV-1 Nef: a multifaceted modulator of T cell receptor signaling. Cell Commun Signal. 2012, 10: 39-10.1186/1478-811X-10-39.PubMed CentralView ArticlePubMedGoogle Scholar
- Arhel NJ, Kirchhoff F: Implications of Nef: host cell interactions in viral persistence and progression to AIDS. Curr Top Microbiol Immunol. 2009, 339: 147-175.PubMedGoogle Scholar
- Foster JL, Denial SJ, Temple BR, Garcia JV: Mechanisms of HIV-1 Nef function and intracellular signaling. J Neuroimmune Pharmacol. 2011, 6: 230-246. 10.1007/s11481-011-9262-y.PubMed CentralView ArticlePubMedGoogle Scholar
- Kirchhoff F: Immune evasion and counteraction of restriction factors by HIV-1 and other primate lentiviruses. Cell Host Microbe. 2010, 8: 55-67. 10.1016/j.chom.2010.06.004.View ArticlePubMedGoogle Scholar
- Laguette N, Bregnard C, Benichou S, Basmaciogullari S: Human immunodeficiency virus (HIV) type-1, HIV-2 and simian immunodeficiency virus Nef proteins. Mol Aspects Med. 2010, 31: 418-433. 10.1016/j.mam.2010.05.003.View ArticlePubMedGoogle Scholar
- Kirchhoff F, Schindler M, Specht A, Arhel N, Munch J: Role of Nef in primate lentiviral immunopathogenesis. Cell Mol Life Sci. 2008, 65: 2621-2636. 10.1007/s00018-008-8094-2.View ArticlePubMedGoogle Scholar
- Mwimanzi P, Markle TJ, Ogata Y, Martin E, Tokunaga M, Mahiti M, Kuang XT, Walker BD, Brockman MA, Brumme ZL, Ueno T: Dynamic range of Nef functions in chronic HIV-1 infection. Virology. 2013, 439: 74-80. 10.1016/j.virol.2013.02.005.View ArticlePubMedGoogle Scholar
- Heigele A, Schindler M, Gnanadurai CW, Leonard JA, Collins KL, Kirchhoff F: Down-modulation of CD8alphabeta is a fundamental activity of primate lentiviral Nef proteins. J Virol. 2012, 86: 36-48. 10.1128/JVI.00717-11.PubMed CentralView ArticlePubMedGoogle Scholar
- Hrecka K, Swigut T, Schindler M, Kirchhoff F, Skowronski J: Nef proteins from diverse groups of primate lentiviruses downmodulate CXCR4 to inhibit migration to the chemokine stromal derived factor 1. J Virol. 2005, 79: 10650-10659. 10.1128/JVI.79.16.10650-10659.2005.PubMed CentralView ArticlePubMedGoogle Scholar
- Kirchhoff F, Schindler M, Bailer N, Renkema GH, Saksela K, Knoop V, Muller-Trutwin MC, Santiago ML, Bibollet-Ruche F, Dittmar MT, et al: Nef proteins from simian immunodeficiency virus-infected chimpanzees interact with p21-activated kinase 2 and modulate cell surface expression of various human receptors. J Virol. 2004, 78: 6864-6874. 10.1128/JVI.78.13.6864-6874.2004.PubMed CentralView ArticlePubMedGoogle Scholar
- Munch J, Rajan D, Schindler M, Specht A, Rucker E, Novembre FJ, Nerrienet E, Muller-Trutwin MC, Peeters M, Hahn BH, Kirchhoff F: Nef-mediated enhancement of virion infectivity and stimulation of viral replication are fundamental properties of primate lentiviruses. J Virol. 2007, 81: 13852-13864. 10.1128/JVI.00904-07.PubMed CentralView ArticlePubMedGoogle Scholar
- Schindler M, Wurfl S, Benaroch P, Greenough TC, Daniels R, Easterbrook P, Brenner M, Munch J, Kirchhoff F: Down-modulation of mature major histocompatibility complex class II and up-regulation of invariant chain cell surface expression are well-conserved functions of human and simian immunodeficiency virus nef alleles. J Virol. 2003, 77: 10548-10556. 10.1128/JVI.77.19.10548-10556.2003.PubMed CentralView ArticlePubMedGoogle Scholar
- Specht A, DeGottardi MQ, Schindler M, Hahn B, Evans DT, Kirchhoff F: Selective downmodulation of HLA-A and -B by Nef alleles from different groups of primate lentiviruses. Virology. 2008, 373: 229-237. 10.1016/j.virol.2007.11.019.View ArticlePubMedGoogle Scholar
- Fackler OT, Moris A, Tibroni N, Giese SI, Glass B, Schwartz O, Krausslich HG: Functional characterization of HIV-1 Nef mutants in the context of viral infection. Virology. 2006, 351: 322-339. 10.1016/j.virol.2006.03.044.View ArticlePubMedGoogle Scholar
- Glushakova S, Munch J, Carl S, Greenough TC, Sullivan JL, Margolis L, Kirchhoff F: CD4 down-modulation by human immunodeficiency virus type 1 Nef correlates with the efficiency of viral replication and with CD4(+) T-cell depletion in human lymphoid tissue ex vivo. J Virol. 2001, 75: 10113-10117. 10.1128/JVI.75.21.10113-10117.2001.PubMed CentralView ArticlePubMedGoogle Scholar
- Lundquist CA, Tobiume M, Zhou J, Unutmaz D, Aiken C: Nef-mediated downregulation of CD4 enhances human immunodeficiency virus type 1 replication in primary T lymphocytes. J Virol. 2002, 76: 4625-4633. 10.1128/JVI.76.9.4625-4633.2002.PubMed CentralView ArticlePubMedGoogle Scholar
- Kuo LS, Baugh LL, Denial SJ, Watkins RL, Liu M, Garcia JV, Foster JL: Overlapping effector interfaces define the multiple functions of the HIV-1 Nef polyproline helix. Retrovirology. 2012, 9: 47-10.1186/1742-4690-9-47.PubMed CentralView ArticlePubMedGoogle Scholar
- Manninen A, Hiipakka M, Vihinen M, Lu W, Mayer BJ, Saksela K: SH3-domain binding function of HIV-1 Nef is required for association with a PAK-related kinase. Virology. 1998, 250: 273-282. 10.1006/viro.1998.9381.View ArticlePubMedGoogle Scholar
- Olivieri KC, Mukerji J, Gabuzda D: Nef-mediated enhancement of cellular activation and human immunodeficiency virus type 1 replication in primary T cells is dependent on association with p21-activated kinase 2. Retrovirology. 2011, 8: 64-10.1186/1742-4690-8-64.PubMed CentralView ArticlePubMedGoogle Scholar
- O’Neill E, Kuo LS, Krisko JF, Tomchick DR, Garcia JV, Foster JL: Dynamic evolution of the human immunodeficiency virus type 1 pathogenic factor, Nef. J Virol. 2006, 80: 1311-1320. 10.1128/JVI.80.3.1311-1320.2006.PubMed CentralView ArticlePubMedGoogle Scholar
- Pizzato M, Helander A, Popova E, Calistri A, Zamborlini A, Palu G, Gottlinger HG: Dynamin 2 is required for the enhancement of HIV-1 infectivity by Nef. Proc Natl Acad Sci USA. 2007, 104: 6812-6817. 10.1073/pnas.0607622104.PubMed CentralView ArticlePubMedGoogle Scholar
- Baur AS, Sawai ET, Dazin P, Fantl WJ, Cheng-Mayer C, Peterlin BM: HIV-1 Nef leads to inhibition or activation of T cells depending on its intracellular localization. Immunity. 1994, 1: 373-384. 10.1016/1074-7613(94)90068-X.View ArticlePubMedGoogle Scholar
- Hanna Z, Weng X, Kay DG, Poudrier J, Lowell C, Jolicoeur P: The pathogenicity of human immunodeficiency virus (HIV) type 1 Nef in CD4C/HIV transgenic mice is abolished by mutation of its SH3-binding domain, and disease development is delayed in the absence of Hck. J Virol. 2001, 75: 9378-9392. 10.1128/JVI.75.19.9378-9392.2001.PubMed CentralView ArticlePubMedGoogle Scholar
- Muthumani K, Choo AY, Hwang DS, Premkumar A, Dayes NS, Harris C, Green DR, Wadsworth SA, Siekierka JJ, Weiner DB: HIV-1 Nef-induced FasL induction and bystander killing requires p38 MAPK activation. Blood. 2005, 106: 2059-2068. 10.1182/blood-2005-03-0932.PubMed CentralView ArticlePubMedGoogle Scholar
- Muthumani K, Choo AY, Shedlock DJ, Laddy DJ, Sundaram SG, Hirao L, Wu L, Thieu KP, Chung CW, Lankaraman KM, et al: Human immunodeficiency virus type 1 Nef induces programmed death 1 expression through a p38 mitogen-activated protein kinase-dependent mechanism. J Virol. 2008, 82: 11536-11544. 10.1128/JVI.00485-08.PubMed CentralView ArticlePubMedGoogle Scholar
- Stove V, Naessens E, Stove C, Swigut T, Plum J, Verhasselt B: Signaling but not trafficking function of HIV-1 protein Nef is essential for Nef-induced defects in human intrathymic T-cell development. Blood. 2003, 102: 2925-2932. 10.1182/blood-2003-03-0833.View ArticlePubMedGoogle Scholar
- Trible RP, Emert-Sedlak L, Smithgall TE: HIV-1 Nef selectively activates Src family kinases Hck, Lyn, and c-Src through direct SH3 domain interaction. J Biol Chem. 2006, 281: 27029-27038. 10.1074/jbc.M601128200.PubMed CentralView ArticlePubMedGoogle Scholar
- Blagoveshchenskaya AD, Thomas L, Feliciangeli SF, Hung CH, Thomas G: HIV-1 Nef downregulates MHC-I by a PACS-1- and PI3K-regulated ARF6 endocytic pathway. Cell. 2002, 111: 853-866. 10.1016/S0092-8674(02)01162-5.View ArticlePubMedGoogle Scholar
- Greenberg ME, Iafrate AJ, Skowronski J: The SH3 domain-binding surface and an acidic motif in HIV-1 Nef regulate trafficking of class I MHC complexes. Embo J. 1998, 17: 2777-2789. 10.1093/emboj/17.10.2777.PubMed CentralView ArticlePubMedGoogle Scholar
- Hung CH, Thomas L, Ruby CE, Atkins KM, Morris NP, Knight ZA, Scholz I, Barklis E, Weinberg AD, Shokat KM, Thomas G: HIV-1 Nef assembles a Src family kinase-ZAP-70/Syk-PI3K cascade to downregulate cell-surface MHC-I. Cell Host Microbe. 2007, 1: 121-133. 10.1016/j.chom.2007.03.004.View ArticlePubMedGoogle Scholar
- Jia X, Singh R, Homann S, Yang H, Guatelli J, Xiong Y: Structural basis of evasion of cellular adaptive immunity by HIV-1 Nef. Nat Struct Mol Biol. 2012, 19: 701-706. 10.1038/nsmb.2328.PubMed CentralView ArticlePubMedGoogle Scholar
- Xu W, Santini PA, Sullivan JS, He B, Shan M, Ball SC, Dyer WB, Ketas TJ, Chadburn A, Cohen-Gould L, et al: HIV-1 evades virus-specific IgG2 and IgA responses by targeting systemic and intestinal B cells via long-range intercellular conduits. Nat Immunol. 2009, 10: 1008-1017. 10.1038/ni.1753.PubMed CentralView ArticlePubMedGoogle Scholar
- Goldsmith MA, Warmerdam MT, Atchison RE, Miller MD, Greene WC: Dissociation of the CD4 downregulation and viral infectivity enhancement functions of human immunodeficiency virus type 1 Nef. J Virol. 1995, 69: 4112-4121.PubMed CentralPubMedGoogle Scholar
- Mangasarian A, Piguet V, Wang JK, Chen YL, Trono D: Nef-induced CD4 and major histocompatibility complex class I (MHC-I) down-regulation are governed by distinct determinants: N-terminal alpha helix and proline repeat of Nef selectively regulate MHC-I trafficking. J Virol. 1999, 73: 1964-1973.PubMed CentralPubMedGoogle Scholar
- Brainard DM, Seung E, Frahm N, Cariappa A, Bailey CC, Hart WK, Shin HS, Brooks SF, Knight HL, Eichbaum Q, et al: Induction of robust cellular and humoral virus-specific adaptive immune responses in human immunodeficiency virus-infected humanized BLT mice. J Virol. 2009, 83: 7305-7321. 10.1128/JVI.02207-08.PubMed CentralView ArticlePubMedGoogle Scholar
- Denton PW, Estes JD, Sun Z, Othieno FA, Wei BL, Wege AK, Powell DA, Payne D, Haase AT, Garcia JV: Antiretroviral pre-exposure prophylaxis prevents vaginal transmission of HIV-1 in humanized BLT mice. PLoS Med. 2008, 5: e16-10.1371/journal.pmed.0050016.PubMed CentralView ArticlePubMedGoogle Scholar
- Dudek TE, No DC, Seung E, Vrbanac VD, Fadda L, Bhoumik P, Boutwell CL, Power KA, Gladden AD, Battis L, et al: Rapid evolution of HIV-1 to functional CD8+ T cell responses in humanized BLT mice. Sci Transl Med. 2012, 4: 143ra198-View ArticleGoogle Scholar
- Long BR, Stoddart CA: Alpha interferon and HIV infection cause activation of human T cells in NSG-BLT mice. J Virol. 2012, 86: 3327-3336. 10.1128/JVI.06676-11.PubMed CentralView ArticlePubMedGoogle Scholar
- Murooka TT, Deruaz M, Marangoni F, Vrbanac VD, Seung E, von Andrian UH, Tager AM, Luster AD, Mempel TR: HIV-infected T cells are migratory vehicles for viral dissemination. Nature. 2012, 490: 283-287. 10.1038/nature11398.PubMed CentralView ArticlePubMedGoogle Scholar
- Zou W, Denton PW, Watkins RL, Krisko JF, Nochi T, Foster JL, Garcia JV: Nef functions in BLT mice to enhance HIV-1 replication and deplete CD4 + CD8+ thymocytes. Retrovirology. 2012, 9: 44-10.1186/1742-4690-9-44.PubMed CentralView ArticlePubMedGoogle Scholar
- Arganaraz ER, Schindler M, Kirchhoff F, Cortes MJ, Lama J: Enhanced CD4 down-modulation by late stage HIV-1 nef alleles is associated with increased Env incorporation and viral replication. J Biol Chem. 2003, 278: 33912-33919. 10.1074/jbc.M303679200.View ArticlePubMedGoogle Scholar
- Carl S, Greenough TC, Krumbiegel M, Greenberg M, Skowronski J, Sullivan JL, Kirchhoff F: Modulation of different human immunodeficiency virus type 1 Nef functions during progression to AIDS. J Virol. 2001, 75: 3657-3665. 10.1128/JVI.75.8.3657-3665.2001.PubMed CentralView ArticlePubMedGoogle Scholar
- Kirchhoff F, Easterbrook PJ, Douglas N, Troop M, Greenough TC, Weber J, Carl S, Sullivan JL, Daniels RS: Sequence variations in human immunodeficiency virus type 1 Nef are associated with different stages of disease. J Virol. 1999, 73: 5497-5508.PubMed CentralPubMedGoogle Scholar
- Lewis MJ, Balamurugan A, Ohno A, Kilpatrick S, Ng HL, Yang OO: Functional adaptation of Nef to the immune milieu of HIV-1 infection in vivo. J Immunol. 2008, 180: 4075-4081.View ArticlePubMedGoogle Scholar
- Michael NL, Chang G, d’Arcy LA, Tseng CJ, Birx DL, Sheppard HW: Functional characterization of human immunodeficiency virus type 1 nef genes in patients with divergent rates of disease progression. J Virol. 1995, 69: 6758-6769.PubMed CentralPubMedGoogle Scholar
- Chateau M, Swanson MD, Garcia JV: Inefficient vaginal transmission of tenofovir-resistant HIV-1. J Virol. 2012, 87: 1274-1277.View ArticlePubMedGoogle Scholar
- Krisko JF, Martinez-Torres F, Foster JL, Garcia JV: HIV restriction by APOBEC3 in humanized mice. PLoS Pathog. 2013, 9: e1003242-10.1371/journal.ppat.1003242.PubMed CentralView ArticlePubMedGoogle Scholar
- Garcia JV, Miller AD: Serine phosphorylation-independent downregulation of cell-surface CD4 by nef. Nature. 1991, 350: 508-511. 10.1038/350508a0.View ArticlePubMedGoogle Scholar
- Schwartz O, Marechal V, Le Gall S, Lemonnier F, Heard JM: Endocytosis of major histocompatibility complex class I molecules is induced by the HIV-1 Nef protein. Nat Med. 1996, 2: 338-342. 10.1038/nm0396-338.View ArticlePubMedGoogle Scholar
- Arora VK, Molina RP, Foster JL, Blakemore JL, Chernoff J, Fredericksen BL, Garcia JV: Lentivirus Nef specifically activates Pak2. J Virol. 2000, 74: 11081-11087. 10.1128/JVI.74.23.11081-11087.2000.PubMed CentralView ArticlePubMedGoogle Scholar
- Sawai ET, Baur A, Struble H, Peterlin BM, Levy JA, Cheng-Mayer C: Human immunodeficiency virus type 1 Nef associates with a cellular serine kinase in T lymphocytes. Proc Natl Acad Sci USA. 1994, 91: 1539-1543. 10.1073/pnas.91.4.1539.PubMed CentralView ArticlePubMedGoogle Scholar
- Lundquist CA, Zhou J, Aiken C: Nef stimulates human immunodeficiency virus type 1 replication in primary T cells by enhancing virion-associated gp120 levels: coreceptor-dependent requirement for Nef in viral replication. J Virol. 2004, 78: 6287-6296. 10.1128/JVI.78.12.6287-6296.2004.PubMed CentralView ArticlePubMedGoogle Scholar
- Sun Z, Denton PW, Estes JD, Othieno FA, Wei BL, Wege AK, Melkus MW, Padgett-Thomas A, Zupancic M, Haase AT, Garcia JV: Intrarectal transmission, systemic infection, and CD4+ T cell depletion in humanized mice infected with HIV-1. J Exp Med. 2007, 204: 705-714. 10.1084/jem.20062411.PubMed CentralView ArticlePubMedGoogle Scholar
- Schindler M, Munch J, Kutsch O, Li H, Santiago ML, Bibollet-Ruche F, Muller-Trutwin MC, Novembre FJ, Peeters M, Courgnaud V, et al: Nef-mediated suppression of T cell activation was lost in a lentiviral lineage that gave rise to HIV-1. Cell. 2006, 125: 1055-1067. 10.1016/j.cell.2006.04.033.View ArticlePubMedGoogle Scholar
- Schindler M, Schmokel J, Specht A, Li H, Munch J, Khalid M, Sodora DL, Hahn BH, Silvestri G, Kirchhoff F: Inefficient Nef-mediated downmodulation of CD3 and MHC-I correlates with loss of CD4 + T cells in natural SIV infection. PLoS Pathog. 2008, 4: e1000107-10.1371/journal.ppat.1000107.PubMed CentralView ArticlePubMedGoogle Scholar
- Casartelli N, Giolo G, Neri F, Haller C, Potesta M, Rossi P, Fackler OT, Doria M: The Pro78 residue regulates the capacity of the human immunodeficiency virus type 1 Nef protein to inhibit recycling of major histocompatibility complex class I molecules in an SH3-independent manner. J Gen Virol. 2006, 87: 2291-2296. 10.1099/vir.0.81775-0.View ArticlePubMedGoogle Scholar
- Hanna Z, Kay DG, Rebai N, Guimond A, Jothy S, Jolicoeur P: Nef harbors a major determinant of pathogenicity for an AIDS-like disease induced by HIV-1 in transgenic mice. Cell. 1998, 95: 163-175. 10.1016/S0092-8674(00)81748-1.View ArticlePubMedGoogle Scholar
- Kouwenhoven A, Minassian VD, Marsh JW: HIV-1 Nef mediates Pak phosphorylation of Mek1 Serine298 and elicits an active phospho-state of Pak2. Curr HIV Res. 2013, 11: 198-209. 10.2174/1570162X113119990039.View ArticlePubMedGoogle Scholar
- Pan X, Rudolph JM, Abraham L, Habermann A, Haller C, Krijnse-Locker J, Fackler OT: HIV-1 Nef compensates for disorganization of the immunological synapse by inducing trans-golgi network-associated Lck signaling. Blood. 2012, 119: 786-797. 10.1182/blood-2011-08-373209.View ArticlePubMedGoogle Scholar
- Ali SA, Huang MB, Campbell PE, Roth WW, Campbell T, Khan M, Newman G, Villinger F, Powell MD, Bond VC: Genetic characterization of HIV type 1 Nef-induced vesicle secretion. AIDS Res Hum Retroviruses. 2010, 26: 173-192. 10.1089/aid.2009.0068.PubMed CentralView ArticlePubMedGoogle Scholar
- Dinkins C, Arko-Mensah J, Deretic V: Autophagy and HIV. Semin Cell Dev Biol. 2010, 21: 712-718. 10.1016/j.semcdb.2010.04.004.PubMed CentralView ArticlePubMedGoogle Scholar
- Geleziunas R, Xu W, Takeda K, Ichijo H, Greene WC: HIV-1 Nef inhibits ASK1-dependent death signalling providing a potential mechanism for protecting the infected host cell. Nature. 2001, 410: 834-838. 10.1038/35071111.View ArticlePubMedGoogle Scholar
- Kyei GB, Dinkins C, Davis AS, Roberts E, Singh SB, Dong C, Wu L, Kominami E, Ueno T, Yamamoto A, et al: Autophagy pathway intersects with HIV-1 biosynthesis and regulates viral yields in macrophages. J Cell Biol. 2009, 186: 255-268. 10.1083/jcb.200903070.PubMed CentralView ArticlePubMedGoogle Scholar
- Lenassi M, Cagney G, Liao M, Vaupotic T, Bartholomeeusen K, Cheng Y, Krogan NJ, Plemenitas A, Peterlin BM: HIV Nef is secreted in exosomes and triggers apoptosis in bystander CD4+ T cells. Traffic. 2010, 11: 110-122. 10.1111/j.1600-0854.2009.01006.x.PubMed CentralView ArticlePubMedGoogle Scholar
- Shelton MN, Huang MB, Ali SA, Powell MD, Bond VC: Secretion modification region-derived peptide disrupts HIV-1 Nef’s interaction with mortalin and blocks virus and Nef exosome release. J Virol. 2012, 86: 406-419. 10.1128/JVI.05720-11.PubMed CentralView ArticlePubMedGoogle Scholar
- Baur AS: HIV-Nef and AIDS pathogenesis: are we barking up the wrong tree?. Trends Microbiol. 2011, 19: 435-440. 10.1016/j.tim.2011.06.002.View ArticlePubMedGoogle Scholar
- Denton PW, Krisko JF, Powell DA, Mathias M, Kwak YT, Martinez-Torres F, Zou W, Payne DA, Estes JD, Garcia JV: Systemic administration of antiretrovirals prior to exposure prevents rectal and intravenous HIV-1 transmission in humanized BLT mice. PLoS One. 2010, 5: e8829-10.1371/journal.pone.0008829.PubMed CentralView ArticlePubMedGoogle Scholar
- Denton PW, Olesen R, Choudhary SK, Archin NM, Wahl A, Swanson MD, Chateau M, Nochi T, Krisko JF, Spagnuolo RA, et al: Generation of HIV latency in humanized BLT mice. J Virol. 2012, 86: 630-634. 10.1128/JVI.06120-11.PubMed CentralView ArticlePubMedGoogle Scholar
- Denton PW, Othieno F, Martinez-Torres F, Zou W, Krisko JF, Fleming E, Zein S, Powell DA, Wahl A, Kwak YT, et al: One percent tenofovir applied topically to humanized BLT mice and used according to the CAPRISA 004 experimental design demonstrates partial protection from vaginal HIV infection, validating the BLT model for evaluation of new microbicide candidates. J Virol. 2011, 85: 7582-7593. 10.1128/JVI.00537-11.PubMed CentralView ArticlePubMedGoogle Scholar
- Kim SS, Peer D, Kumar P, Subramanya S, Wu H, Asthana D, Habiro K, Yang YG, Manjunath N, Shimaoka M, Shankar P: RNAi-mediated CCR5 silencing by LFA-1-targeted nanoparticles prevents HIV infection in BLT mice. Mol Ther. 2010, 18: 370-376. 10.1038/mt.2009.271.PubMed CentralView ArticlePubMedGoogle Scholar
- Lan P, Tonomura N, Shimizu A, Wang S, Yang YG: Reconstitution of a functional human immune system in immunodeficient mice through combined human fetal thymus/liver and CD34+ cell transplantation. Blood. 2006, 108: 487-492. 10.1182/blood-2005-11-4388.View ArticlePubMedGoogle Scholar
- Melkus MW, Estes JD, Padgett-Thomas A, Gatlin J, Denton PW, Othieno FA, Wege AK, Haase AT, Garcia JV: Humanized mice mount specific adaptive and innate immune responses to EBV and TSST-1. Nat Med. 2006, 12: 1316-1322. 10.1038/nm1431.View ArticlePubMedGoogle Scholar
- Rajesh D, Zhou Y, Jankowska-Gan E, Roenneburg DA, Dart ML, Torrealba J, Burlingham WJ: Th1 and Th17 immunocompetence in humanized NOD/SCID/IL2rgammanull mice. Hum Immunol. 2010, 71: 551-559. 10.1016/j.humimm.2010.02.019.PubMed CentralView ArticlePubMedGoogle Scholar
- Peden K, Emerman M, Montagnier L: Changes in growth properties on passage in tissue culture of viruses derived from infectious molecular clones of HIV-1LAI, HIV-1MAL, and HIV-1ELI. Virology. 1991, 185: 661-672. 10.1016/0042-6822(91)90537-L.View ArticlePubMedGoogle Scholar
- Wei BL, Denton PW, O’Neill E, Luo T, Foster JL, Garcia JV: Inhibition of lysosome and proteasome function enhances human immunodeficiency virus type 1 infection. J Virol. 2005, 79: 5705-5712. 10.1128/JVI.79.9.5705-5712.2005.PubMed CentralView ArticlePubMedGoogle Scholar
- Palmer S, Wiegand AP, Maldarelli F, Bazmi H, Mican JM, Polis M, Dewar RL, Planta A, Liu S, Metcalf JA, et al: New real-time reverse transcriptase-initiated PCR assay with single-copy sensitivity for human immunodeficiency virus type 1 RNA in plasma. J Clin Microbiol. 2003, 41: 4531-4536. 10.1128/JCM.41.10.4531-4536.2003.PubMed CentralView ArticlePubMedGoogle Scholar
- Denton PW, Garcia JV: Mucosal HIV-1 transmission and prevention strategies in BLT humanized mice. Trends Microbiol. 2012, 20: 268-274. 10.1016/j.tim.2012.03.007.PubMed CentralView ArticlePubMedGoogle Scholar
- Kimpton J, Emerman M: Detection of replication-competent and pseudotyped human immunodeficiency virus with a sensitive cell line on the basis of activation of an integrated beta-galactosidase gene. J Virol. 1992, 66: 2232-2239.PubMed CentralPubMedGoogle Scholar
- Chang AH, Hoxie JA, Cassol S, O’Shaughnessy M, Jirik F: Construction of single-chain antibodies that bind an overlapping epitope of HIV-1 Nef. FEBS Lett. 1998, 441: 307-312. 10.1016/S0014-5793(98)01569-5.View ArticlePubMedGoogle 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.