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.
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.
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