Primary Sooty Mangabey Simian Immunodeficiency Vir
http://www.100md.com
病菌学杂志 2005年第16期
Abteilung Virologie, Universit?tsklinikum, 89081 Ulm, Germany
Institut für Zellul?re und Molekulare Botanik, Universit?t Bonn, 53115 Bonn, Germany
Yerkes National Primate Research Center, Emory University, 954 N. Gatewood Rd., Atlanta, Georgia 30322
ABSTRACT
The nef gene of the pathogenic simian immunodeficiency virus (SIV) mac239 clone has been well characterized. Little is known, however, about the function of nef alleles derived from naturally SIVsm-infected sooty mangabeys (Cercocebus atys) and from human immunodeficiency virus type 2 (HIV-2)-infected individuals. Addressing this, we demonstrate that, similarly to the SIVmac239 nef, primary SIVsm and HIV-2 nef alleles down-modulate cell surface expression of human CD4, CD28, CD3, and class I or II major histocompatibility complex (MHC-I or MHC-II, respectively) molecules, up-regulate surface expression of the invariant chain (Ii) associated with immature MHC-II, inhibit early T-cell activation events, and enhance virion infectivity. Both also stimulate viral replication, although HIV-2 nef alleles were less active in this assay than SIVsm nef alleles. Mutational analysis showed that a dileucine-based sorting motif in the C-proximal loop of SIV or HIV-2 Nef is critical for its effects on CD4, CD28, and Ii but dispensable for down-regulation of CD3, MHC-I, and MHC-II. The C terminus of SIV and HIV-2 Nef was exclusively required for down-modulation of MHC-I, further demonstrating that analogous functions are mediated by different domains in Nef proteins derived from different groups of primate lentiviruses. Our results demonstrate that none of the eight Nef functions investigated had been newly acquired after cross-species transmission of SIVsm from naturally infected mangabeys to humans or macaques. Notably, HIV-2 and SIVsm nef alleles efficiently down-modulate CD3 and C28 surface expression and inhibit T-cell activation more efficiently than HIV-1 nef alleles. These differences in Nef function might contribute to the relatively low levels of immune activation observed in HIV-2-infected human individuals.
INTRODUCTION
Both simian immunodeficiency virus (SIVmac) infection of captive rhesus macaques and human immunodeficiency virus type 2 (HIV-2) infection of humans originate from zoonotic transmission of SIVsm in naturally infected sooty mangabeys (16, 22). SIVsm usually causes a persistent but asymptomatic infection in its natural simian host (48, 54), although the development of AIDS in a sooty mangabey after an 18-year natural SIVsm infection has recently been reported (35). In comparison, HIV-2 is frequently pathogenic in humans, but infected individuals progress more slowly to AIDS than HIV-1-infected individuals (37, 44). Finally, infection of macaques with SIVmac typically results in rapid immunosuppression and represents one of the best animal models for AIDS in humans (reviewed in references 13 and 15). The well-characterized molecular SIVmac239 clone (44) has been proven particularly useful in studying the determinants of viral pathogenicity because it usually causes simian AIDS within 1 year after infection (28).
More than a decade ago, it was demonstrated that a large deletion in the wild-type SIVmac239 (239wt) nef gene results in low viral loads and a strongly attenuated clinical course of infection (29). Subsequent in vitro studies identified a variety of 239wt Nef activities, including down-regulation of cell surface expression of CD4 (4, 24), CD3 (3, 23), CD28 (60), major histocompatibility complex class I (MHC-I) (58), and MHC-II (51, 57), up-regulation of the invariant chain (Ii) associated with immature MHC-II complexes (51, 57), enhancement of virion infectivity (34), and stimulation of viral replication (1, 20, 36). Accumulating evidence indicates that many of these Nef functions are genetically separable and contribute to the maintenance of high viral loads and to disease progression in SIVmac-infected rhesus macaques (14, 26, 39, 40, 52).
The 239wt nef gene has been thoroughly characterized (29, 47, 52). Much less is known, however, about the function of primary SIVsm and HIV-2 nef alleles. As mentioned above, Nef is multifunctional and a major pathogenesis factor in SIVmac239-infected rhesus macaques (29). SIVsm strains also contain intact nef genes, but infected mangabeys usually do not develop AIDS despite high levels of viral replication (35, 48, 54). Immunodeficiency viruses are highly variable, and Nef function might have changed after cross-species transmission of SIVsm from mangabeys to macaques or humans. Therefore, we investigated the functional activity of primary SIVsm and HIV-2 nef alleles in eight previously established in vitro assays for Nef function. We found that SIVsm nef alleles down-modulate cell surface expression of human CD4, CD28, CD3, and class I or II MHC molecules, up-regulate surface expression of the Ii typically associated with immature class II MHC, and enhance viral replication. They were more active than HIV-1 nef alleles in down-regulating CD3 and CD28 from the cell surface and in inhibiting T-cell receptor (TCR) signaling. Compared to SIVsm, the activity of HIV-2 nef alleles was more variable and the ability to stimulate viral replication was significantly reduced. However, as a group the HIV-2 Nefs were capable of performing all eight functions investigated. Our findings indicate that no additional Nef functions were acquired after zoonotic transmission of SIVsm from sooty mangabeys to humans or macaques. Concordant with a previous report (61), we found that some HIV-2 nef genes are truncated or nonfunctional, suggesting that a lack of Nef function might contribute to the limited pathogenicity of HIV-2 in some infected individuals.
MATERIALS AND METHODS
Nef expression plasmids. The generation of bicistronic cytomegalovirus-based pCG vectors coexpressing green fluorescent protein (GFP) and the SIVmac239 or HIV-2 ROD or BEN nef alleles has been described previously (51). To generate the nef-defective control vector, 239wt nef was amplified with p239nef-Xba (5'-TTTTTCTAGAgTGGGTtGAGCTATTTCCtaGAGGCGGT) and p239MluI (5'-GTCCCTACGCGTCAGCGAGTTTCCTTCTTG-3') (mutated positions are lowercase; underlining represents the XbaI and MluI restriction sites), resulting in an M1V change and introducing two premature stop codons at positions 3 and 7 of the nef reading frame. The HIV-2 CBL23 nef (53) was PCR amplified using primers HIV-2Xba1 (5'-CGGTCTAGATGCAATATGGGTGCGAG-3') and pHIV-2MluI (5'-CCACGCGTTAACTAAATGGTATTCCT-3') (underlining represents XbaI and MluI restriction sites). The remaining HIV-2 nef genes were amplified from the CDC 310248, 310072, 310319, and 60415K strains (17, 42) obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Diseases, National Institutes of Health, from Mark Rayfield, Stephen Wiktor, Feng Gao, and Beatrice Hahn. Primers were pHIV-2Xba2 (5'-CGGTCTAGATGGGATCAGCTGGTTC-3') or pHIV-2Xba3 (5'-CGGTCTAGATGGGTGCGAGTGGAT-3') (for 60415K) paired with either p310248Mlu (5'-GTACGCGTCTAATCTGTAGGTATTCCTCTT-3'), p310319Mlu (5'-GTACGCGTCTAATCTGTTGGTATTCCCCTT-3'), p7924AMlu (5'-GTACGCGTTCAGTTAAATGGAATTCCCCTT-3'), or p60415KMlu (5'-GTACGCGTTTAATTAAATGGTATTCCT-3'). SIVsm nef alleles were amplified by nested PCR from the blood of three naturally infected sooty mangabeys, designated FWr, FYr, and FFm (Table 1). The inner primers pSIVsm(Xba) (5'-ACCTATCTAGAGCCCTTATGGGTGGCGTTACC-3') and SIVsm(MluI) (5'-GTCCCTACGCGTCAGCTTGTTTCCTTCTTG-3') introduced XbaI and MluI restriction sites (underlined) flanking the nef gene. These sites were used for cloning into the bicistronic pCG vector coexpressing Nef and GFP via an internal ribosome entry site element (21). Five to six individual nef clones per SIVsm or HIV-2 sample were sequenced, and one or two representative alleles were selected for functional analysis (Table 1). Notably, the SIVsm strains were never passaged on human cells. For Western blot analysis, we also amplified all nef open reading frames (ORFs) with primers resulting in fusion of the AU1 peptide tag to the C terminus of Nef. Site-directed mutagenesis of nef was performed by splice overlap extension PCR essentially as described previously (26, 39, 40). All PCR-derived inserts were sequenced to confirm that no undesired nucleotide changes were present.
Proviral constructs. A modified form of the Nef-SHIV-K6 clone (31) was used for the insertion of HIV-2 and SIVsm nef genes into the SIVmac239 genome. This chimeric SIVmac239 construct contains the HIV-1 K6 nef allele downstream of env and upstream of the polypurine tract and the core enhancer region of the SIVmac239 long terminal repeat. The original SIVmac239 nef initiation codon and a second ATG at codon 7 of the nef ORF are mutated without changing the coding sequence of the overlapping env gene (31). In addition, we eliminated the SacI restriction site at position 6015 of the proviral SIVmac239 sequence (M33262) without altering the predicted Vif sequence. All HIV-2 and SIVsm nef alleles were PCR amplified with 5' primers containing the SacI restriction site (underlined) located upstream of the SIVmac239 env termination codon (bold) and the first 18 nucleotides of the respective nef genes (e.g., pHIV-2Sac1, 5'-GGGCTTGAGCTCACTCTCTTGTAAGATGGGTGCGAGTGGATCC-3') together with a 3' primer introducing an MluI restriction site just downstream of nef. All PCR fragments were cloned into the proviral SIVmac239 vector using the SacI and MluI sites. Thus, the resulting proviral SIVmac239 clones do not contain overlapping env-nef sequences and differ exclusively by their Nef coding sequences.
Cell culture and transfection. HeLa CIITA and Jurkat T cells were cultured as described previously (7, 50, 51, 57). Transfection of Jurkat T cells was performed using the DMRIE-C reagent (Gibco BRL) following manufacturer's instructions. HeLa CIITA cells were transfected with Metafectene (Biontex). Briefly, 2.5 μg DNA in 100 μl optimized minimum essential medium (OMEM; Invitrogen) was mixed with 10 μl Metafectene in 90 μl OMEM and incubated for 30 min at room temperature. Subsequently, the mixture was added to 2 x 105 cells and incubated for 6 h at 37°C. Thereafter, the medium was changed, and cells were analyzed by fluorescence-activated cell sorter on the following day. Transfection efficiencies varied between 20% and 35%.
Western blotting. 293T cells were transfected with 5 μg pCG expression vectors coexpressing GFP and Nef using the calcium phosphate method as described previously (7). Two days posttransfection, cells were pelleted and lysates were generated and separated through 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Expression of Nef proteins in whole cellular lysates was analyzed by immunoblotting. Nef was detected with 1:500 diluted mouse anti-AU1 antibody (Ab) (Covance). ?-Actin and GFP were detected with 1:2,000 dilutions of rabbit anti-?-actin and rabbit anti-GFP Abs (Abcam). Western blotting was performed using KPL's protein detector kit as recommended by the manufacturer with 1:500 dilutions of alkaline phosphatase-coupled goat anti-mouse and goat anti-rabbit Abs (Jackson Immuno Research).
Flow cytometry. CD4, MHC-I, CD28, CD3, and GFP reporter molecules in Jurkat T cells transfected with a bicistronic vector coexpressing Nef and GFP (21) were measured as described previously (7, 52). Down-modulation of MHC-II and up-regulation of Ii were measured on transfected HeLa CIITA cells (50, 57). The following phycoerythrin-conjugated antibodies were used: anti-human CD4, anti-human CD3, and anti-LeuTM-28 (BD Biosciences); anti-CD74/R-PE M-B741 (Ancell); anti-HLA-ABC antigen/RPE (DAKO); mouse anti-human HLA-DR TUE36 (Caltag laboratories); and L243 (BD Biosciences). Nef-mediated down- or up-regulation of cellular receptors was quantitated as described previously (7, 52). To test the inhibitory effect on T-cell activation, Jurkat cells were transfected as described previously (7, 52), except that no phytohemagglutinin (PHA) was added to the culture medium. Approximately 24 h posttransfection, cells were stimulated using 0.3 μg/ml HIT3a monoclonal antibody (MAb) (BD Biosciences), specific for the subunit of CD3, or by the addition of 1 μg/ml PHA and cultured for an additional 15 h prior to flow cytometric analysis of CD69 expression (clone FN50; BD Biosciences).
Intracellular Nef staining. Jurkat T cells were permeabilized as described previously (51). Intracellular Nef expression was detected with an anti-AU1 Ab, and two-color flow cytometric analysis for Nef and GFP expression was performed as mentioned above.
Virus stocks, infectivity, and replication. For virus production, 293T cells were transfected by the calcium phosphate method with 10 μg of the proviral constructs as described previously (7, 40). The medium was changed after overnight incubation, and virus was harvested 24 h later. The p27 antigen concentrations were quantified by using an SIV enzyme-linked immunosorbent assay provided by the National Institutes of Health AIDS Reagent Program. Cells were infected with virus stocks containing normalized quantities of p27 antigen, and virus production was measured by reverse transcriptase assay at 2- to 3-day intervals as described previously (8, 26). The rhesus T-cell line 221-89 (1) was maintained in the presence of 100 U of interleukin-2 (IL-2)/ml (Boehringer) and 20% fetal calf serum. Infections were performed in the absence of exogenous IL-2 or in the presence of 50 U IL-2/ml and 5% fetal calf serum. Virus infectivity was determined using TZM-BL and P4-CCR5 cells as described previously (12, 46). Infectivity was measured on P4-CCR5 and TZM-BL cells using the ?-galactosidase screen from TROPIX as recommended by the manufacturer.
Statistical methods. The mean activities of HIV-2 and SIVsm nef alleles were compared by using Student's t test. Similar results were obtained with the Mann-Whitney test. The software package StatView version 4.0 (Abacus Concepts, Berkeley, CA) was used for all calculations.
Phylogenetic analysis. Sequences were derived from the Los Alamos sequence database (http://hiv-web.lanl.gov). Phylogenetic tree construction was done with the minimum evolution criterion as implemented in MEGA3 (33). Poisson corrected amino acid distances were used assuming uniform rates over sites. The following search settings were used: CNI level = 2 and max trees = 20. Bootstrap node support was calculated from 10,000 replicates.
Nucleotide sequence accession numbers. The GenBank accession numbers for the HIV-2 and SIVsm sequences are DQ092758 to DQ92767.
RESULTS
Nef sequence and phylogenetic analysis. To investigate whether Nef function might have changed after cross-species transmission of SIVsm from sooty mangabeys to the new macaque or human hosts, we analyzed nef alleles derived from three sooty mangabeys (designated FWr, FYr, and FFm) and seven HIV-2 strains (Table 1). For comparison, we also determined the activity of the SIVmac239 and HIV-1 NA7 nef alleles, which have been well characterized in previous studies (21, 25, 26, 29, 47, 52). The SIVsm nef alleles were amplified directly from uncultured blood samples of naturally infected sooty mangabeys. Three of the HIV-2 nef alleles were derived from the infectious molecular ROD10 (9, 41), BEN (30), and CBL23 (53) clones. The remaining nef genes were amplified from the HIV-2 CDC 310248, 310072, 310319, and 60415K strains (16, 42) obtained through the AIDS Reagent Program. These four HIV-2 strains have been obtained by coculture with PHA-stimulated normal peripheral blood mononuclear cells (PBMC) and were never passaged on transformed cell lines (16, 42). As summarized in Table 1, the HIV-2 strains originated from several African countries and were obtained at different clinical stages of infection.
Phylogenetic analysis revealed that the HIV-2/SIV Nef sequences formed five distinct clusters (Fig. 1). Three clusters included the various SIVsm and SIVmac Nef sequences, the fourth the HIV-2 group B strains as well as HIV-2 310248, and the fifth the HIV-2 group A ROD10, GH1, CBL23, BEN, and 60415K strains. Clustering of the 310248 Nef with that of HIV-2 group B strains was unexpected, because it has been previously demonstrated that the 310248 env sequence clusters with group A ROD10 and 60415K env genes (42). Thus, HIV-2 310248 might have arisen from recombination between a subtype A and B strain. Importantly, our analysis revealed that all sequences were animal or patient specific, confirming the authenticity of the Nef sequences and excluding cross-sample PCR contaminations.
Most SIVsm and HIV-2 nef genes predicted full-length proteins (Fig. 2). Exceptions were the six HIV-2 310072 nef ORFs analyzed, which all contained a premature stop at the 149th codon (Fig. 2 and data not shown). Several domains and protein interaction sites previously described to be relevant for HIV-1 function (reviewed in reference 18) were conserved in the remaining SIVsm and HIV-2 Nef sequences, including the N-terminal myristoylation signal, the acidic region, a diarginine motif, a C-proximal adaptor-protein interaction site, and a diacidic putative V1H binding site (Fig. 2). Concordant with their limited role for viral replication in vivo (8, 34), the N-proximal Y residues, proposed to represent endocytosis signals (6, 45), and the proline-rich region, which is involved in the interaction with cellular kinases (34), were more variable. All SIVsm FYr and HIV-2 60415K Nef sequences analyzed, for example, contain only a single proline residue at this location. In contrast, Y223, critical for MHC-I down-modulation by 239wt Nef (39, 58), was preserved at the corresponding position in all full-length SIVsm and HIV-2 Nef sequences. Residues P73A74 and D204, which are important for CD4 down-modulation and efficient replication of SIVmac239 in acutely infected macaques (26), were conserved among the HIV-2 group A and SIVsm Nef sequences. Unexpectedly, however, the HIV-2 group B 310072, 310248, and 310319 Nef sequences, which are closely related to one another and were all derived from Ivory Coast blood donors (Table 1), contained a deletion of 16 amino acid residues encompassing residues P73 and A74 (Fig. 2).
Modulation of cellular receptors by HIV-2 and SIVsm Nefs. Next, we investigated the activity of SIVsm and HIV-2 nef alleles in modulating various human cellular receptors. nef genes were cloned into a bicistronic vector coexpressing Nef and GFP for fluorescence-activated cell sorter analysis (21). Of the five to six individual nef clones per SIVsm or HIV-2 sample sequenced, one or two representative alleles were selected for functional studies (Fig. 2). Western blot analysis confirmed that all bicistronic vectors containing AU1-tagged nef open reading frames expressed proteins of the expected size (Fig. 3). Altogether, however, expression levels varied, and only small amounts of HIV-2 ROD10 Nef were detected in the extracts of transfected 293T cells (Fig. 3A). Next, we used permeabilized transfected Jurkat T cells to detect both Nef and GFP expression. Flow cytometric analysis showed that all HIV-2 and SIV nef alleles analyzed were expressed, albeit with differential efficiencies (Fig. 3B). Consistent with the results of the Western blot analysis (Fig. 3A), expression of the HIV-2 ROD Nef in Jurkat T cells was very inefficient (Fig. 3B).
As expected (52), the 239wt Nef modulated the surface expression of all six receptors investigated, whereas a construct containing inactivating point mutations in nef had no significant effects (Fig. 4, columns 1 and 2). The six SIVsm nef alleles analyzed showed comparable efficiencies in modulating cell surface expression of CD4, MHC-I, MHC-II, and Ii molecules but were less effective than 239wt nef in down-regulating CD3 and CD28 (Fig. 4, columns 3 to 5; Table 1). Similarly, the HIV-2 BEN and CBL23 nef alleles affected surface expression of six cellular receptors (Fig. 4, columns 6 and 7), whereas the 310319 Nef did not efficiently down-modulate CD28 (Fig. 4, panel 9C). As expected, the severely truncated HIV-2 310072 Nef was inactive in these assays (data not shown). The 310248 Nef was also generally inactive, although the full-length protein could be detected by Western blot analysis (Fig. 3). Tagged and untagged nef alleles did not differ significantly in the ability to modulate the surface expression of the six cellular factors analyzed (data not shown).
The bicistronic vector used for flow cytometric analysis allows us to readily quantitate the effect of Nef on the surface expression of cellular receptors. As described previously (7, 51, 52), we calculated n-fold up-regulation of Ii or down-modulation of the remaining surface receptors by dividing the mean fluorescence intensity obtained on Jurkat or HeLa CIITA cells coexpressing high levels of GFP and the different nef alleles by the mean fluorescence obtained for cells transfected with a control construct containing a disrupted nef gene. The same ranges of GFP expression (fluorescence intensities between 500 and 3,000) were used in all calculations. The activities of different nef alleles varied considerably, particularly within the HIV-2 group, e.g., CD4 down-modulation from 4.2- to 11.8-fold between the HIV-2 ROD10 and 60415K nef alleles. Furthermore, the HIV-2 60415K and 310319 nef alleles showed little activity in modulating CD28 and/or CD3 surface expression, although they were highly active in other aspects of Nef function (Table 1). On average, however, the ability to modulate CD4, CD28, MHC-I, MHC-II, and Ii surface expression did not differ significantly between the SIVsm and HIV-2 groups (Table 1). Notably, the five functional HIV-2 nef alleles were significantly more active than the six SIVsm nef alleles analyzed in down-regulating CD3 (P = 0.01) (Table 1). It is also noteworthy that the 239wt Nef was more active than all remaining SIVsm and HIV-2 Nefs analyzed in down-modulation of CD4, CD28, and CD3 molecules. Consistent with its inefficient expression (Fig. 3), the HIV-2 ROD10 nef was poorly active in several in vitro assays, including down-modulation of CD4, CD28, and MHC-II (Table 1). Taken together, our results demonstrate that the ability to modulate various human receptors involved in TCR signaling and MHC antigen presentation is conserved within the SIVsm/mac/HIV-2 group. With the exception of CD3 down-modulation, SIVsm Nefs manipulated cell surface expression of human receptors with the same efficiency as HIV-2 Nefs without any adaptive changes, suggesting that functional nef genes likely helped the virus to evade the human immune system immediately after cross-species transmission.
Interference of HIV-2 and SIVsm Nefs with T-cell activation. Next, we utilized a transient assay (25) to study the effect of various nef alleles on TCR-mediated T-cell activation. Stimulation of Jurkat T cells with PHA or CD3 MAb results in rapid induction of the early T-cell activation marker CD69 on the cell surface. Thus, induction of CD69 provides a convenient indicator of CD3 signaling and T-cell activation. Treatment of Jurkat T cells resulted in a readily detectable 5- to 10-fold increase in CD69 surface expression (Fig. 5A, compare panels 1 and 2). Transient transfection with bicistronic vectors allowed us to quantitate CD69 induction on cells expressing different levels of GFP and, indirectly, Nef. Cells transfected with a nef-defective control construct showed similar levels of CD69 induction at different levels of GFP expression (Fig. 5A, panel 3). In contrast, the increase in CD69 expression was blocked in Jurkat T cells expressing medium to high levels of the SIVmac239, SIVsm FFm1, and HIV-2 BEN Nef proteins (Fig. 5A, panels 4 to 6). In comparison, the HIV-1 NA7 Nef had little effect on CD69 induction (Fig. 5A, panel 7). Analysis of a larger set of nef alleles revealed that SIVmac, SIVsm, and HIV-2 Nefs efficiently inhibited both PHA- and anti-CD3 MAb-induced up-regulation of CD69 surface expression in Jurkat T cells in a dose-dependent manner, whereas HIV-1 Nefs showed little if any activity in blocking T-cell activation (Fig. 5B). The functional activities of these nef alleles in inhibiting PHA- and CD3-induced CD69 expression correlated significantly, suggesting that they might involve the same mechanism (P < 0.0001) (Fig. 5C). Consistent with their higher activity in CD3 down-modulation, most HIV-2 Nefs inhibited CD69 up-regulation more efficiently than SIVsm Nefs (Table 1). However, both HIV-2 and SIVsm Nefs interfered with early T-cell activation events much more severely than HIV-1 Nefs.
Mutational analysis of HIV-2 and SIVsm Nef proteins. It has been previously demonstrated that a dileucine motif in HIV-1 Nef is critical for down-modulation of CD4 and up-regulation of Ii but dispensable for down-regulation of MHC-I or -II (6, 10, 49, 57). We found that the dileucine-based sorting signal is also important for the ability of SIVsm and HIV-2 Nefs to down-modulate CD4 and to up-regulate Ii. Extending these previous studies, we investigated the functional activity of HIV-1 NA7, SIVmac239, HIV-2 BEN, and SIVsm FFm1 Nef proteins containing alanine (AXXXA) substitutions in the EXXXL motif, known to be important for adaptor protein interaction (10). These mutations also impaired down-modulation of CD28 (Fig. 6, row C). They had little if any disruptive effect, however, on down-regulation of CD3 and MHC-I or -II surface expression (Fig. 6, rows B, D, and E). Notably, the NA7 AXXXA Nef was entirely inactive in up-regulating Ii cell surface expression, whereas the mutated 239wt, BEN, and FFm1 Nefs showed some residual activity (Fig. 6, panel F, and data not shown). Thus, compared to HIV-1 Nef, additional regions in SIVmac and HIV-2 Nef might be involved in Ii up-regulation. These results suggest that the "dileucine" motif is of general importance for the ability of HIV-1, HIV-2, SIVsm, and SIVmac Nefs to modulate CD4 and CD28 but dispensable for the effect on CD3, MHC-I, and MHC-II surface expression.
It has been established that down-modulation of MHC-I is mediated by different domains in HIV-1 and SIVmac239 Nef (58). Three motifs in HIV-1 Nef have been implicated in MHC-I down-modulation, M20, EEEE65, and P72XXP75 (5). In contrast, the ability of the 239wt Nef to down-regulate MHC-I requires a unique C-terminal region, which is not found in HIV-1 Nef (58). Both the proline-rich and C-terminal regions are highly variable, and the C terminus of HIV-2 Nefs is truncated compared to SIVmac and SIVsm (Fig. 2). Several HIV-2 BEN Nef mutants were generated (Fig. 7A) to investigate whether its functional organization is reminiscent of that of HIV-1, having the same host, or of SIVmac239, showing much higher sequence similarity and belonging to the same phylogenetic group. As expected from previous studies (34, 52), alanine substitutions of the central proline residues did not impair 239wt Nef function (Fig. 7B, column 6). In comparison, mutation of P72XXP75 in the HIV-1 NA7 Nef reduced its activity in down-modulating MHC-I slightly from 7.0- to 4.7-fold and MHC-II down-regulation more strongly from 7.6- to 2.3-fold at high Nef expression levels (Fig. 7B, columns 2 and 3). A Y223F mutation in HIV-2 Nef selectively impaired its ability to down-modulate MHC-I, albeit less severely than the corresponding mutation in the 239wt Nef (Fig. 7B, panels 5D and 9D) (39, 52, 58). Mutation of the three central proline (AAA) residues in HIV-2 BEN Nef had little effect (Fig. 7B, column 10), whereas C-terminal truncation (G238) disrupted MHC-I down-modulation but not other functions (Fig. 7B, column 11). We also introduced four amino acid substitutions, T103R, R105Q, R108L, and E110P, into the HIV-2 BEN Nef to evaluate whether an HIV-1 PXXP consensus region (Fig. 7A) might allow MHC-I down-modulation in the absence of the C-terminal region. The HIV-1 PXXP mutant Nef was fully functional in all six assays (Fig. 7B, column 13). Similar to the effects on wild-type BEN and 239 Nef function, however, C-terminal truncation disrupted its effect on MHC-I (Fig. 7B, column 14). Further analysis revealed that C-terminal truncation of FFm1 Nef also impaired MHC-I down-modulation (data not shown). Our results are further evidence that different groups of primate immunodeficiency viruses utilize different Nef domains to perform analogous activities and demonstrate that the C-terminal region is required for MHC-I down-modulation by both SIV and HIV-2 Nef proteins.
Enhancement of viral infectivity and replication. Next, we investigated whether HIV-2 and SIVsm nef alleles also enhance viral infectivity. All nef ORFs were inserted downstream of the env gene of the proviral SIVmac239 clone as previously described for HIV-1 nef genes (31), except that the SacI site located at the 3' end of env was used for cloning (Fig. 8A). In these constructs, the original SIVmac239 nef initiation codon and a second ATG at codon 7 of the nef ORF are mutated without changing the predicted Env sequence (31). Sequence and functional analysis confirmed that the resulting proviral SIVmac239 constructs did not contain undesired changes in env and expressed the appropriate HIV-2 and SIVsm Nef proteins (data not shown). As a positive control, otherwise isogenic constructs containing the nef genes of 239wt, HIV-1 NL4-3, or the molecular SHIV-40K6 clone were generated. The 40K6 nef allele was derived from a Nef-SHIV-infected macaque with simian AIDS and is closely related to the HIV-1 group M subtype B Nef consensus sequence (31). As a negative control, we inserted a nef allele containing a premature stop codon at position 93 of the nef ORF (239nef). Compared to the parental SIVmac239 clone, the genomic size of these constructs is enlarged by about 170 nucleotides, and the nef ATG initiation codon is located at a different position because of the elimination of the env-nef overlap. On average, the infectivity of the modified SIVmac239 was approximately three- to fourfold reduced compared to that of the parental virus (Fig. 8B). Nevertheless, the construct containing an intact 239wt nef gene showed about 10-fold-higher activity than the otherwise isogenic nef-defective form (Fig. 8B). Thus, this experimental system allows us to study the effect of various nef alleles on SIVmac infectivity without the complications of overlapping env sequences. Functional assays revealed that the 239wt, HIV-1 K6 and NL4-3, SIVsm FWr1, FYr1, FFm1, and FFm2, and HIV-2 ROD10, BEN, and 310319 nef alleles efficiently enhanced viral infectivity (Fig. 8C). In comparison, the HIV-2 CBL23 and 60415K nef genes were only marginally active and the 310248 Nef was inactive in enhancing virion infectivity. These data demonstrate that some HIV-2 and SIVsm nef genes increase virion infectivity with high efficiency.
Finally, we investigated the functional activity of HIV-2 and SIVsm nef alleles in enhancing viral replication. All SIVmac239 nef recombinants replicated efficiently in CEMx174 cells (data not shown). The T-lymphoid cell line 221-89, derived from a rhesus monkey infected with herpesvirus saimiri (1), was utilized to evaluate the stimulatory effects of the various nef alleles on SIVmac replication. In our previous studies, the abilities of various nef alleles to stimulate SIV replication in 221-89 cells and in rhesus- or human-derived PBMC always correlated (8, 34, 40, 58), indicating that both experimental systems might measure analogous effects of Nef (1). However, the 221-89 assay is less variable, because SIV replication in PBMC is to some extent dependent on the blood donor. Intact 239wt and K6 nef alleles enhanced viral replication moderately in the presence of exogenous IL-2 (Fig. 9, left panels) and more strongly in the absence of IL-2 (Fig. 9, right panels). The four SIVsm nef alleles consistently stimulated SIVmac replication in 221-89 cells with high efficiency (representative example shown in Fig. 9A). With the exception of the 310248 Nef, the HIV-2 Nefs also enhanced viral replication (Fig. 9B). However, the ROD10, BEN, CBL23, and 60415K nef alleles were clearly less active than the four SIVsm nef alleles in enhancing viral replication in both the presence and absence of exogenous IL-2 (Fig. 9 and Table 1). This observation is consistent with previous studies showing that the efficiency of HIV-1 replication correlates with Nef activity in CD4 down-modulation but not enhancement of viral infectivity. On average, the HIV-2 nef alleles also enhanced viral replication in human PBMC less efficiently than SIVsm nef genes (data not shown). Because they were highly active in other functional aspects, it was unexpected that the HIV-2 nef alleles enhanced viral replication only slightly compared to the nef-defective controls. Our results demonstrate that primary SIVsm nef genes are able to stimulate viral replication in rhesus cells. Although our data are suggestive, a larger number of samples needs to be analyzed to elucidate whether HIV-2 Nefs are typically less active in enhancing viral replication than those derived from other groups of primate lentiviruses.
DISCUSSION
In the present study, we demonstrated that, similarly to SIVmac239 Nef (52), SIVsm nef alleles derived from naturally infected sooty mangabeys are able to modulate cell surface expression of human CD4, CD3, CD28, MHC-I, MHC-II, and Ii molecules and to enhance virion infectivity and replication. Thus, SIVsm Nefs derived from primary isolates can modulate cell surface expression of human receptors without adaptive changes following animal or cell culture passage. These results resemble those of our previous study showing that primary SIVcpz nef alleles also did not require any changes to be functionally active in human cells and likely enhanced viral replication and persistence immediately after cross-species transmission to the new human host (32). We found that most HIV-2 Nefs were also functional (Table 1). Thus, none of the eight Nef functions investigated have been newly acquired or lost after cross-species transmission from sooty mangabeys to humans or macaques. It is remarkable, however, that all HIV-2 and SIVsm nef alleles investigated were less active than the SIVmac239 Nef in down-modulating CD4, CD28, and CD3 cell surface expression. Taken together, these data show that primary SIVsm Nefs are able to perform a variety of functions that most likely contribute to immune evasion and efficient persistence in vivo (26, 39, 40, 52). Concordant with this, SIVsm infection of sooty mangabeys is characterized by high-level viremia and efficient viral replication (48, 54).
Although sooty mangabeys are not entirely resistant to SIV-induced disease (35), they are clearly less susceptible to the development of immunodeficiency than infected rhesus macaques, which often develop disease within 1 year after infection (28). In comparison, HIV-2 causes AIDS in humans with an average incubation period of about 14 to 16 years (37, 44). Our observation that SIVsm, SIVmac, and HIV-2 Nefs perform similar functions supports the hypothesis that host factors are important for the different clinical outcomes of infection of mangabeys, macaques, and humans. We also found, however, that all HIV-2 310072 nef alleles were prematurely truncated and that 310248 Nefs showed little functional activity. Unfortunately, these two HIV-2 strains were derived from a blood donor with unknown clinical status (42). Thus, it remains unclear whether the lack of HIV-2 Nef function was associated with attenuated viral replication in vivo. Notably, a high prevalence of defective nef genes in HIV-2 infection has been previously reported (61). A larger number of samples needs to be analyzed to draw definitive conclusions, but taken together these findings suggest that disrupted or nonfunctional nef genes contribute to the low level of viral replication and lack of disease progression in some HIV-2-infected individuals.
Recently, we performed a comprehensive analysis of HIV-1 and SIVcpz Nef function (32). A comparison of these previous results with those of the present study shows that nef alleles derived from the HIV-1/SIVcpz and HIV-2/SIVsm/mac groups show some remarkable functional differences. In contrast to HIV-2 and SIVsm or SIVmac Nefs, HIV-1 Nefs are generally inactive in down-regulating cell surface expression of CD3, a component of the T-cell receptor. Furthermore, HIV-2 and SIVsm Nefs are much more active than HIV-1 and SIVcpz Nefs in down-regulating CD28 from the cell surface and in inhibiting TCR-mediated T-cell activation. Recent studies indicate that chronic immune activation might play a major role in AIDS pathogenesis (11, 54). HIV-2 is less pathogenic than HIV-1, the major causative agent of AIDS (37, 44). Concordant with our data, it has been demonstrated that the levels of immune activation are relatively low in HIV-2 infection (38). Microarray studies in transfected Jurkat T cells have shown that HIV-1 Nef triggers a transcriptional program resembling TCR-mediated T-cell activation (56). It will be of interest to determine whether or not HIV-2 Nefs trigger similar T-cell signaling pathways. Analysis of Nef function clearly indicates that moderately pathogenic HIV-2 strains interfere with TCR signaling from the cell membrane more severely than highly virulent HIV-1 strains. Efficient inhibition of TCR signaling together with lower activity in triggering T-cell activation pathways might explain why HIV-2 causes weaker chronic immune activation and is less pathogenic than HIV-1. However, the determinants of HIV and SIV pathogenesis are complex, and other viral and host factors are clearly also important. For example, SIVmac239 expressing a nef allele that performs all of these functions is highly pathogenic in rhesus macaques (28, 29). It must be considered, however, that macaques are particularly susceptible to SIV-induced disease. It is noteworthy that the accessory vpr and vpx genes are not required for efficient viral spread and disease progression in this model (19), although Vpr might play a relevant role for AIDS pathogenesis in HIV-1-infected humans (50). SIVmac239 is nonpathogenic when reintroduced into sooty mangabeys (27). Macaques infected with SIVsm showed high levels of immune activation and developed CD4+-T-cell depletion, whereas mangabeys displayed little T-cell proliferation and remained healthy (55). Thus, the seemingly high virulence of SIVmac239 is not an inherent property of the virus itself but due to the unusually high susceptibility of macaques to SIV-induced disease. Mutations in Nef that increase its ability to cause T-cell activation enhance the virulence of SIVmac239 even further and result in an acute pathogenic phenotype (14). Most importantly, HIV-2, which is closely related to SIVmac and also originates from SIVsm-infected mangabeys (16), is clearly less virulent than HIV-1 in infected humans (37, 44). Thus, when compared in the same host, the SIVsm/mac/HIV-2 group is less pathogenic than the SIVcpz/HIV-1 group. Therefore, we feel that the observation that SIVmac239 is highly virulent in macaques does not contradict the possibility that down-modulation of CD3 and CD28 favors slow/nonprogressive infection.
Our results indicate that HIV-2 Nefs down-modulate CD3 and inhibit T-cell activation more effectively than SIVsm nef alleles but are less active in stimulating viral replication in 221-89 cells and in human PBMC. However, more samples need to be investigated to clarify how frequently HIV-2 nef genes are more active in inhibiting T-cell activation and less active in stimulating viral replication than those derived from other groups of primate lentiviruses. Furthermore, HIV-2 Nefs might be more active in stimulating viral replication in their usual genomic context. Our constructs have the advantage that nef function can be studied using the well-characterized proviral SIVmac239 clone without the complications of overlapping env sequences. However, the increased genomic size and the expression of Nef from a different location resulted in reduced viral infectivity and replication compared to the parental SIVmac239 clone. As discussed above, we feel that further studies on their ability to stimulate T-cell activation and viral replication might provide important new insights into the pathogenesis of HIV-1 and HIV-2 infection.
In addition to the functional differences of HIV-1/SIVcpz and HIV-2/SIVsm/mac Nefs in modulating CD3 and CD28 surface expression, it is noteworthy that some conserved Nef functions are mediated by different Nef domains. The C-terminal region of SIVmac239, SIVsm, and HIV-2 Nefs is highly variable and not found in HIV-1 Nef (Fig. 2). Our mutational analysis extends previous studies (39, 58) showing that this region is exclusively involved in MHC-I down-modulation by the mac239 Nef and demonstrates that it plays a similar role for HIV-2 Nef function. It has recently been shown that changes selected for in rhesus macaques infected with an SIVmac239 mutant containing deletions near the C terminus of Nef generate an additional PXXP motif, as present in most HIV-1 Nef sequences, and restore the ability to down-modulate MHC-I (59). In comparison, introduction of an HIV-1 PXXP consensus did not restore MHC-I down-modulation by the truncated BEN Nef. Thus, at least in the context of the BEN Nef, this proline motif is not sufficient to functionally compensate for the C-terminal region. Notably, additional variations changing amino acids in 239 Nef to those usually present in HIV-1 Nef were also observed in infected macaques (59) and might be required to restore this function. In contrast to the effect on MHC-I, down-modulation of CD4 requires similar functional domains, such as the EXXXLL motif, in the SIVcpz/HIV-1 and SIVsm/HIV-2 Nef proteins. These findings suggest that the nef gene of the unknown ancestor of these groups of primate lentiviruses might have already been able to perform this function. Some conserved domains in Nef are clearly critical for specific functions. Even within the same group of viruses, however, the requirement of specific residues seems to be often dependent on the specific nef allele investigated. For example, the PA motif located in the N-proximal adaptor protein interaction site in SIVmac239 Nef is required for CD4 down-modulation (26). These residues are missing in the HIV-2 group B 310319 Nef (Fig. 2), although it effectively down-regulates CD4 (Fig. 4, column 9). Thus, while the functional analysis of specific Nef mutants provides some important information on the functional relevance of specific sequence motifs, general conclusions must be made with great caution.
In contrast to the effect on CD4, down-regulation of MHC-I clearly requires distinct domains in HIV-1 and HIV-2 Nefs, suggesting that this function might have been acquired independently and later during the evolution of these primate lentiviruses. The observation that distinct domains mediate different Nef functions might be important for optimal viral spread in vivo, because it has been shown that various HIV-1 or SIVmac Nef activities can be modulated at different stages of disease progression (2, 7, 43). Our results also indicate that HIV-1 and HIV-2 Nefs use overlapping but distinct domains to up-regulate Ii. Mutations in the EXXXLL motif entirely disrupted HIV-1 Nef function but had less severe effects on SIVmac239 or HIV-2 Nefs. We are currently investigating whether additional functional domains are involved in up-regulation of Ii by SIVmac and HIV-2 Nef proteins.
Taken together, the results of the present study demonstrate that the ability of Nef to modulate cell surface expression of human CD4, CD3, CD28, MHC-I, MHC-II, and Ii and to enhance virion infectivity is conserved within the SIVsm/HIV-2/SIVmac group of primate lentiviruses. HIV-1 and SIVcpz Nefs are also able to perform a striking variety of functions. Nevertheless, compared to SIVsm and HIV-2 Nefs, they are generally unable to down-regulate CD3 and also have little effect on CD28 cell surface expression (32). It will be of interest to further evaluate whether these differences in Nef function contribute to the low pathogenicity of HIV-2 compared to HIV-1 in the human host.
ACKNOWLEDGMENTS
We thank Thomas Mertens for constant support and encouragement, Ingrid Bennett for critical reading of the manuscript, and Ainee McKnight for providing the HIV-2 CBL23 nef allele.
This work was supported by the Wilhelm-Sander Foundation, the Deutsche Forschungsgemeinschaft, and a grant from the Elitef?rderprogramm BW to J.M.
The first three authors are in alphabetical order and contributed equally to this work.
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Institut für Zellul?re und Molekulare Botanik, Universit?t Bonn, 53115 Bonn, Germany
Yerkes National Primate Research Center, Emory University, 954 N. Gatewood Rd., Atlanta, Georgia 30322
ABSTRACT
The nef gene of the pathogenic simian immunodeficiency virus (SIV) mac239 clone has been well characterized. Little is known, however, about the function of nef alleles derived from naturally SIVsm-infected sooty mangabeys (Cercocebus atys) and from human immunodeficiency virus type 2 (HIV-2)-infected individuals. Addressing this, we demonstrate that, similarly to the SIVmac239 nef, primary SIVsm and HIV-2 nef alleles down-modulate cell surface expression of human CD4, CD28, CD3, and class I or II major histocompatibility complex (MHC-I or MHC-II, respectively) molecules, up-regulate surface expression of the invariant chain (Ii) associated with immature MHC-II, inhibit early T-cell activation events, and enhance virion infectivity. Both also stimulate viral replication, although HIV-2 nef alleles were less active in this assay than SIVsm nef alleles. Mutational analysis showed that a dileucine-based sorting motif in the C-proximal loop of SIV or HIV-2 Nef is critical for its effects on CD4, CD28, and Ii but dispensable for down-regulation of CD3, MHC-I, and MHC-II. The C terminus of SIV and HIV-2 Nef was exclusively required for down-modulation of MHC-I, further demonstrating that analogous functions are mediated by different domains in Nef proteins derived from different groups of primate lentiviruses. Our results demonstrate that none of the eight Nef functions investigated had been newly acquired after cross-species transmission of SIVsm from naturally infected mangabeys to humans or macaques. Notably, HIV-2 and SIVsm nef alleles efficiently down-modulate CD3 and C28 surface expression and inhibit T-cell activation more efficiently than HIV-1 nef alleles. These differences in Nef function might contribute to the relatively low levels of immune activation observed in HIV-2-infected human individuals.
INTRODUCTION
Both simian immunodeficiency virus (SIVmac) infection of captive rhesus macaques and human immunodeficiency virus type 2 (HIV-2) infection of humans originate from zoonotic transmission of SIVsm in naturally infected sooty mangabeys (16, 22). SIVsm usually causes a persistent but asymptomatic infection in its natural simian host (48, 54), although the development of AIDS in a sooty mangabey after an 18-year natural SIVsm infection has recently been reported (35). In comparison, HIV-2 is frequently pathogenic in humans, but infected individuals progress more slowly to AIDS than HIV-1-infected individuals (37, 44). Finally, infection of macaques with SIVmac typically results in rapid immunosuppression and represents one of the best animal models for AIDS in humans (reviewed in references 13 and 15). The well-characterized molecular SIVmac239 clone (44) has been proven particularly useful in studying the determinants of viral pathogenicity because it usually causes simian AIDS within 1 year after infection (28).
More than a decade ago, it was demonstrated that a large deletion in the wild-type SIVmac239 (239wt) nef gene results in low viral loads and a strongly attenuated clinical course of infection (29). Subsequent in vitro studies identified a variety of 239wt Nef activities, including down-regulation of cell surface expression of CD4 (4, 24), CD3 (3, 23), CD28 (60), major histocompatibility complex class I (MHC-I) (58), and MHC-II (51, 57), up-regulation of the invariant chain (Ii) associated with immature MHC-II complexes (51, 57), enhancement of virion infectivity (34), and stimulation of viral replication (1, 20, 36). Accumulating evidence indicates that many of these Nef functions are genetically separable and contribute to the maintenance of high viral loads and to disease progression in SIVmac-infected rhesus macaques (14, 26, 39, 40, 52).
The 239wt nef gene has been thoroughly characterized (29, 47, 52). Much less is known, however, about the function of primary SIVsm and HIV-2 nef alleles. As mentioned above, Nef is multifunctional and a major pathogenesis factor in SIVmac239-infected rhesus macaques (29). SIVsm strains also contain intact nef genes, but infected mangabeys usually do not develop AIDS despite high levels of viral replication (35, 48, 54). Immunodeficiency viruses are highly variable, and Nef function might have changed after cross-species transmission of SIVsm from mangabeys to macaques or humans. Therefore, we investigated the functional activity of primary SIVsm and HIV-2 nef alleles in eight previously established in vitro assays for Nef function. We found that SIVsm nef alleles down-modulate cell surface expression of human CD4, CD28, CD3, and class I or II MHC molecules, up-regulate surface expression of the Ii typically associated with immature class II MHC, and enhance viral replication. They were more active than HIV-1 nef alleles in down-regulating CD3 and CD28 from the cell surface and in inhibiting T-cell receptor (TCR) signaling. Compared to SIVsm, the activity of HIV-2 nef alleles was more variable and the ability to stimulate viral replication was significantly reduced. However, as a group the HIV-2 Nefs were capable of performing all eight functions investigated. Our findings indicate that no additional Nef functions were acquired after zoonotic transmission of SIVsm from sooty mangabeys to humans or macaques. Concordant with a previous report (61), we found that some HIV-2 nef genes are truncated or nonfunctional, suggesting that a lack of Nef function might contribute to the limited pathogenicity of HIV-2 in some infected individuals.
MATERIALS AND METHODS
Nef expression plasmids. The generation of bicistronic cytomegalovirus-based pCG vectors coexpressing green fluorescent protein (GFP) and the SIVmac239 or HIV-2 ROD or BEN nef alleles has been described previously (51). To generate the nef-defective control vector, 239wt nef was amplified with p239nef-Xba (5'-TTTTTCTAGAgTGGGTtGAGCTATTTCCtaGAGGCGGT) and p239MluI (5'-GTCCCTACGCGTCAGCGAGTTTCCTTCTTG-3') (mutated positions are lowercase; underlining represents the XbaI and MluI restriction sites), resulting in an M1V change and introducing two premature stop codons at positions 3 and 7 of the nef reading frame. The HIV-2 CBL23 nef (53) was PCR amplified using primers HIV-2Xba1 (5'-CGGTCTAGATGCAATATGGGTGCGAG-3') and pHIV-2MluI (5'-CCACGCGTTAACTAAATGGTATTCCT-3') (underlining represents XbaI and MluI restriction sites). The remaining HIV-2 nef genes were amplified from the CDC 310248, 310072, 310319, and 60415K strains (17, 42) obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Diseases, National Institutes of Health, from Mark Rayfield, Stephen Wiktor, Feng Gao, and Beatrice Hahn. Primers were pHIV-2Xba2 (5'-CGGTCTAGATGGGATCAGCTGGTTC-3') or pHIV-2Xba3 (5'-CGGTCTAGATGGGTGCGAGTGGAT-3') (for 60415K) paired with either p310248Mlu (5'-GTACGCGTCTAATCTGTAGGTATTCCTCTT-3'), p310319Mlu (5'-GTACGCGTCTAATCTGTTGGTATTCCCCTT-3'), p7924AMlu (5'-GTACGCGTTCAGTTAAATGGAATTCCCCTT-3'), or p60415KMlu (5'-GTACGCGTTTAATTAAATGGTATTCCT-3'). SIVsm nef alleles were amplified by nested PCR from the blood of three naturally infected sooty mangabeys, designated FWr, FYr, and FFm (Table 1). The inner primers pSIVsm(Xba) (5'-ACCTATCTAGAGCCCTTATGGGTGGCGTTACC-3') and SIVsm(MluI) (5'-GTCCCTACGCGTCAGCTTGTTTCCTTCTTG-3') introduced XbaI and MluI restriction sites (underlined) flanking the nef gene. These sites were used for cloning into the bicistronic pCG vector coexpressing Nef and GFP via an internal ribosome entry site element (21). Five to six individual nef clones per SIVsm or HIV-2 sample were sequenced, and one or two representative alleles were selected for functional analysis (Table 1). Notably, the SIVsm strains were never passaged on human cells. For Western blot analysis, we also amplified all nef open reading frames (ORFs) with primers resulting in fusion of the AU1 peptide tag to the C terminus of Nef. Site-directed mutagenesis of nef was performed by splice overlap extension PCR essentially as described previously (26, 39, 40). All PCR-derived inserts were sequenced to confirm that no undesired nucleotide changes were present.
Proviral constructs. A modified form of the Nef-SHIV-K6 clone (31) was used for the insertion of HIV-2 and SIVsm nef genes into the SIVmac239 genome. This chimeric SIVmac239 construct contains the HIV-1 K6 nef allele downstream of env and upstream of the polypurine tract and the core enhancer region of the SIVmac239 long terminal repeat. The original SIVmac239 nef initiation codon and a second ATG at codon 7 of the nef ORF are mutated without changing the coding sequence of the overlapping env gene (31). In addition, we eliminated the SacI restriction site at position 6015 of the proviral SIVmac239 sequence (M33262) without altering the predicted Vif sequence. All HIV-2 and SIVsm nef alleles were PCR amplified with 5' primers containing the SacI restriction site (underlined) located upstream of the SIVmac239 env termination codon (bold) and the first 18 nucleotides of the respective nef genes (e.g., pHIV-2Sac1, 5'-GGGCTTGAGCTCACTCTCTTGTAAGATGGGTGCGAGTGGATCC-3') together with a 3' primer introducing an MluI restriction site just downstream of nef. All PCR fragments were cloned into the proviral SIVmac239 vector using the SacI and MluI sites. Thus, the resulting proviral SIVmac239 clones do not contain overlapping env-nef sequences and differ exclusively by their Nef coding sequences.
Cell culture and transfection. HeLa CIITA and Jurkat T cells were cultured as described previously (7, 50, 51, 57). Transfection of Jurkat T cells was performed using the DMRIE-C reagent (Gibco BRL) following manufacturer's instructions. HeLa CIITA cells were transfected with Metafectene (Biontex). Briefly, 2.5 μg DNA in 100 μl optimized minimum essential medium (OMEM; Invitrogen) was mixed with 10 μl Metafectene in 90 μl OMEM and incubated for 30 min at room temperature. Subsequently, the mixture was added to 2 x 105 cells and incubated for 6 h at 37°C. Thereafter, the medium was changed, and cells were analyzed by fluorescence-activated cell sorter on the following day. Transfection efficiencies varied between 20% and 35%.
Western blotting. 293T cells were transfected with 5 μg pCG expression vectors coexpressing GFP and Nef using the calcium phosphate method as described previously (7). Two days posttransfection, cells were pelleted and lysates were generated and separated through 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Expression of Nef proteins in whole cellular lysates was analyzed by immunoblotting. Nef was detected with 1:500 diluted mouse anti-AU1 antibody (Ab) (Covance). ?-Actin and GFP were detected with 1:2,000 dilutions of rabbit anti-?-actin and rabbit anti-GFP Abs (Abcam). Western blotting was performed using KPL's protein detector kit as recommended by the manufacturer with 1:500 dilutions of alkaline phosphatase-coupled goat anti-mouse and goat anti-rabbit Abs (Jackson Immuno Research).
Flow cytometry. CD4, MHC-I, CD28, CD3, and GFP reporter molecules in Jurkat T cells transfected with a bicistronic vector coexpressing Nef and GFP (21) were measured as described previously (7, 52). Down-modulation of MHC-II and up-regulation of Ii were measured on transfected HeLa CIITA cells (50, 57). The following phycoerythrin-conjugated antibodies were used: anti-human CD4, anti-human CD3, and anti-LeuTM-28 (BD Biosciences); anti-CD74/R-PE M-B741 (Ancell); anti-HLA-ABC antigen/RPE (DAKO); mouse anti-human HLA-DR TUE36 (Caltag laboratories); and L243 (BD Biosciences). Nef-mediated down- or up-regulation of cellular receptors was quantitated as described previously (7, 52). To test the inhibitory effect on T-cell activation, Jurkat cells were transfected as described previously (7, 52), except that no phytohemagglutinin (PHA) was added to the culture medium. Approximately 24 h posttransfection, cells were stimulated using 0.3 μg/ml HIT3a monoclonal antibody (MAb) (BD Biosciences), specific for the subunit of CD3, or by the addition of 1 μg/ml PHA and cultured for an additional 15 h prior to flow cytometric analysis of CD69 expression (clone FN50; BD Biosciences).
Intracellular Nef staining. Jurkat T cells were permeabilized as described previously (51). Intracellular Nef expression was detected with an anti-AU1 Ab, and two-color flow cytometric analysis for Nef and GFP expression was performed as mentioned above.
Virus stocks, infectivity, and replication. For virus production, 293T cells were transfected by the calcium phosphate method with 10 μg of the proviral constructs as described previously (7, 40). The medium was changed after overnight incubation, and virus was harvested 24 h later. The p27 antigen concentrations were quantified by using an SIV enzyme-linked immunosorbent assay provided by the National Institutes of Health AIDS Reagent Program. Cells were infected with virus stocks containing normalized quantities of p27 antigen, and virus production was measured by reverse transcriptase assay at 2- to 3-day intervals as described previously (8, 26). The rhesus T-cell line 221-89 (1) was maintained in the presence of 100 U of interleukin-2 (IL-2)/ml (Boehringer) and 20% fetal calf serum. Infections were performed in the absence of exogenous IL-2 or in the presence of 50 U IL-2/ml and 5% fetal calf serum. Virus infectivity was determined using TZM-BL and P4-CCR5 cells as described previously (12, 46). Infectivity was measured on P4-CCR5 and TZM-BL cells using the ?-galactosidase screen from TROPIX as recommended by the manufacturer.
Statistical methods. The mean activities of HIV-2 and SIVsm nef alleles were compared by using Student's t test. Similar results were obtained with the Mann-Whitney test. The software package StatView version 4.0 (Abacus Concepts, Berkeley, CA) was used for all calculations.
Phylogenetic analysis. Sequences were derived from the Los Alamos sequence database (http://hiv-web.lanl.gov). Phylogenetic tree construction was done with the minimum evolution criterion as implemented in MEGA3 (33). Poisson corrected amino acid distances were used assuming uniform rates over sites. The following search settings were used: CNI level = 2 and max trees = 20. Bootstrap node support was calculated from 10,000 replicates.
Nucleotide sequence accession numbers. The GenBank accession numbers for the HIV-2 and SIVsm sequences are DQ092758 to DQ92767.
RESULTS
Nef sequence and phylogenetic analysis. To investigate whether Nef function might have changed after cross-species transmission of SIVsm from sooty mangabeys to the new macaque or human hosts, we analyzed nef alleles derived from three sooty mangabeys (designated FWr, FYr, and FFm) and seven HIV-2 strains (Table 1). For comparison, we also determined the activity of the SIVmac239 and HIV-1 NA7 nef alleles, which have been well characterized in previous studies (21, 25, 26, 29, 47, 52). The SIVsm nef alleles were amplified directly from uncultured blood samples of naturally infected sooty mangabeys. Three of the HIV-2 nef alleles were derived from the infectious molecular ROD10 (9, 41), BEN (30), and CBL23 (53) clones. The remaining nef genes were amplified from the HIV-2 CDC 310248, 310072, 310319, and 60415K strains (16, 42) obtained through the AIDS Reagent Program. These four HIV-2 strains have been obtained by coculture with PHA-stimulated normal peripheral blood mononuclear cells (PBMC) and were never passaged on transformed cell lines (16, 42). As summarized in Table 1, the HIV-2 strains originated from several African countries and were obtained at different clinical stages of infection.
Phylogenetic analysis revealed that the HIV-2/SIV Nef sequences formed five distinct clusters (Fig. 1). Three clusters included the various SIVsm and SIVmac Nef sequences, the fourth the HIV-2 group B strains as well as HIV-2 310248, and the fifth the HIV-2 group A ROD10, GH1, CBL23, BEN, and 60415K strains. Clustering of the 310248 Nef with that of HIV-2 group B strains was unexpected, because it has been previously demonstrated that the 310248 env sequence clusters with group A ROD10 and 60415K env genes (42). Thus, HIV-2 310248 might have arisen from recombination between a subtype A and B strain. Importantly, our analysis revealed that all sequences were animal or patient specific, confirming the authenticity of the Nef sequences and excluding cross-sample PCR contaminations.
Most SIVsm and HIV-2 nef genes predicted full-length proteins (Fig. 2). Exceptions were the six HIV-2 310072 nef ORFs analyzed, which all contained a premature stop at the 149th codon (Fig. 2 and data not shown). Several domains and protein interaction sites previously described to be relevant for HIV-1 function (reviewed in reference 18) were conserved in the remaining SIVsm and HIV-2 Nef sequences, including the N-terminal myristoylation signal, the acidic region, a diarginine motif, a C-proximal adaptor-protein interaction site, and a diacidic putative V1H binding site (Fig. 2). Concordant with their limited role for viral replication in vivo (8, 34), the N-proximal Y residues, proposed to represent endocytosis signals (6, 45), and the proline-rich region, which is involved in the interaction with cellular kinases (34), were more variable. All SIVsm FYr and HIV-2 60415K Nef sequences analyzed, for example, contain only a single proline residue at this location. In contrast, Y223, critical for MHC-I down-modulation by 239wt Nef (39, 58), was preserved at the corresponding position in all full-length SIVsm and HIV-2 Nef sequences. Residues P73A74 and D204, which are important for CD4 down-modulation and efficient replication of SIVmac239 in acutely infected macaques (26), were conserved among the HIV-2 group A and SIVsm Nef sequences. Unexpectedly, however, the HIV-2 group B 310072, 310248, and 310319 Nef sequences, which are closely related to one another and were all derived from Ivory Coast blood donors (Table 1), contained a deletion of 16 amino acid residues encompassing residues P73 and A74 (Fig. 2).
Modulation of cellular receptors by HIV-2 and SIVsm Nefs. Next, we investigated the activity of SIVsm and HIV-2 nef alleles in modulating various human cellular receptors. nef genes were cloned into a bicistronic vector coexpressing Nef and GFP for fluorescence-activated cell sorter analysis (21). Of the five to six individual nef clones per SIVsm or HIV-2 sample sequenced, one or two representative alleles were selected for functional studies (Fig. 2). Western blot analysis confirmed that all bicistronic vectors containing AU1-tagged nef open reading frames expressed proteins of the expected size (Fig. 3). Altogether, however, expression levels varied, and only small amounts of HIV-2 ROD10 Nef were detected in the extracts of transfected 293T cells (Fig. 3A). Next, we used permeabilized transfected Jurkat T cells to detect both Nef and GFP expression. Flow cytometric analysis showed that all HIV-2 and SIV nef alleles analyzed were expressed, albeit with differential efficiencies (Fig. 3B). Consistent with the results of the Western blot analysis (Fig. 3A), expression of the HIV-2 ROD Nef in Jurkat T cells was very inefficient (Fig. 3B).
As expected (52), the 239wt Nef modulated the surface expression of all six receptors investigated, whereas a construct containing inactivating point mutations in nef had no significant effects (Fig. 4, columns 1 and 2). The six SIVsm nef alleles analyzed showed comparable efficiencies in modulating cell surface expression of CD4, MHC-I, MHC-II, and Ii molecules but were less effective than 239wt nef in down-regulating CD3 and CD28 (Fig. 4, columns 3 to 5; Table 1). Similarly, the HIV-2 BEN and CBL23 nef alleles affected surface expression of six cellular receptors (Fig. 4, columns 6 and 7), whereas the 310319 Nef did not efficiently down-modulate CD28 (Fig. 4, panel 9C). As expected, the severely truncated HIV-2 310072 Nef was inactive in these assays (data not shown). The 310248 Nef was also generally inactive, although the full-length protein could be detected by Western blot analysis (Fig. 3). Tagged and untagged nef alleles did not differ significantly in the ability to modulate the surface expression of the six cellular factors analyzed (data not shown).
The bicistronic vector used for flow cytometric analysis allows us to readily quantitate the effect of Nef on the surface expression of cellular receptors. As described previously (7, 51, 52), we calculated n-fold up-regulation of Ii or down-modulation of the remaining surface receptors by dividing the mean fluorescence intensity obtained on Jurkat or HeLa CIITA cells coexpressing high levels of GFP and the different nef alleles by the mean fluorescence obtained for cells transfected with a control construct containing a disrupted nef gene. The same ranges of GFP expression (fluorescence intensities between 500 and 3,000) were used in all calculations. The activities of different nef alleles varied considerably, particularly within the HIV-2 group, e.g., CD4 down-modulation from 4.2- to 11.8-fold between the HIV-2 ROD10 and 60415K nef alleles. Furthermore, the HIV-2 60415K and 310319 nef alleles showed little activity in modulating CD28 and/or CD3 surface expression, although they were highly active in other aspects of Nef function (Table 1). On average, however, the ability to modulate CD4, CD28, MHC-I, MHC-II, and Ii surface expression did not differ significantly between the SIVsm and HIV-2 groups (Table 1). Notably, the five functional HIV-2 nef alleles were significantly more active than the six SIVsm nef alleles analyzed in down-regulating CD3 (P = 0.01) (Table 1). It is also noteworthy that the 239wt Nef was more active than all remaining SIVsm and HIV-2 Nefs analyzed in down-modulation of CD4, CD28, and CD3 molecules. Consistent with its inefficient expression (Fig. 3), the HIV-2 ROD10 nef was poorly active in several in vitro assays, including down-modulation of CD4, CD28, and MHC-II (Table 1). Taken together, our results demonstrate that the ability to modulate various human receptors involved in TCR signaling and MHC antigen presentation is conserved within the SIVsm/mac/HIV-2 group. With the exception of CD3 down-modulation, SIVsm Nefs manipulated cell surface expression of human receptors with the same efficiency as HIV-2 Nefs without any adaptive changes, suggesting that functional nef genes likely helped the virus to evade the human immune system immediately after cross-species transmission.
Interference of HIV-2 and SIVsm Nefs with T-cell activation. Next, we utilized a transient assay (25) to study the effect of various nef alleles on TCR-mediated T-cell activation. Stimulation of Jurkat T cells with PHA or CD3 MAb results in rapid induction of the early T-cell activation marker CD69 on the cell surface. Thus, induction of CD69 provides a convenient indicator of CD3 signaling and T-cell activation. Treatment of Jurkat T cells resulted in a readily detectable 5- to 10-fold increase in CD69 surface expression (Fig. 5A, compare panels 1 and 2). Transient transfection with bicistronic vectors allowed us to quantitate CD69 induction on cells expressing different levels of GFP and, indirectly, Nef. Cells transfected with a nef-defective control construct showed similar levels of CD69 induction at different levels of GFP expression (Fig. 5A, panel 3). In contrast, the increase in CD69 expression was blocked in Jurkat T cells expressing medium to high levels of the SIVmac239, SIVsm FFm1, and HIV-2 BEN Nef proteins (Fig. 5A, panels 4 to 6). In comparison, the HIV-1 NA7 Nef had little effect on CD69 induction (Fig. 5A, panel 7). Analysis of a larger set of nef alleles revealed that SIVmac, SIVsm, and HIV-2 Nefs efficiently inhibited both PHA- and anti-CD3 MAb-induced up-regulation of CD69 surface expression in Jurkat T cells in a dose-dependent manner, whereas HIV-1 Nefs showed little if any activity in blocking T-cell activation (Fig. 5B). The functional activities of these nef alleles in inhibiting PHA- and CD3-induced CD69 expression correlated significantly, suggesting that they might involve the same mechanism (P < 0.0001) (Fig. 5C). Consistent with their higher activity in CD3 down-modulation, most HIV-2 Nefs inhibited CD69 up-regulation more efficiently than SIVsm Nefs (Table 1). However, both HIV-2 and SIVsm Nefs interfered with early T-cell activation events much more severely than HIV-1 Nefs.
Mutational analysis of HIV-2 and SIVsm Nef proteins. It has been previously demonstrated that a dileucine motif in HIV-1 Nef is critical for down-modulation of CD4 and up-regulation of Ii but dispensable for down-regulation of MHC-I or -II (6, 10, 49, 57). We found that the dileucine-based sorting signal is also important for the ability of SIVsm and HIV-2 Nefs to down-modulate CD4 and to up-regulate Ii. Extending these previous studies, we investigated the functional activity of HIV-1 NA7, SIVmac239, HIV-2 BEN, and SIVsm FFm1 Nef proteins containing alanine (AXXXA) substitutions in the EXXXL motif, known to be important for adaptor protein interaction (10). These mutations also impaired down-modulation of CD28 (Fig. 6, row C). They had little if any disruptive effect, however, on down-regulation of CD3 and MHC-I or -II surface expression (Fig. 6, rows B, D, and E). Notably, the NA7 AXXXA Nef was entirely inactive in up-regulating Ii cell surface expression, whereas the mutated 239wt, BEN, and FFm1 Nefs showed some residual activity (Fig. 6, panel F, and data not shown). Thus, compared to HIV-1 Nef, additional regions in SIVmac and HIV-2 Nef might be involved in Ii up-regulation. These results suggest that the "dileucine" motif is of general importance for the ability of HIV-1, HIV-2, SIVsm, and SIVmac Nefs to modulate CD4 and CD28 but dispensable for the effect on CD3, MHC-I, and MHC-II surface expression.
It has been established that down-modulation of MHC-I is mediated by different domains in HIV-1 and SIVmac239 Nef (58). Three motifs in HIV-1 Nef have been implicated in MHC-I down-modulation, M20, EEEE65, and P72XXP75 (5). In contrast, the ability of the 239wt Nef to down-regulate MHC-I requires a unique C-terminal region, which is not found in HIV-1 Nef (58). Both the proline-rich and C-terminal regions are highly variable, and the C terminus of HIV-2 Nefs is truncated compared to SIVmac and SIVsm (Fig. 2). Several HIV-2 BEN Nef mutants were generated (Fig. 7A) to investigate whether its functional organization is reminiscent of that of HIV-1, having the same host, or of SIVmac239, showing much higher sequence similarity and belonging to the same phylogenetic group. As expected from previous studies (34, 52), alanine substitutions of the central proline residues did not impair 239wt Nef function (Fig. 7B, column 6). In comparison, mutation of P72XXP75 in the HIV-1 NA7 Nef reduced its activity in down-modulating MHC-I slightly from 7.0- to 4.7-fold and MHC-II down-regulation more strongly from 7.6- to 2.3-fold at high Nef expression levels (Fig. 7B, columns 2 and 3). A Y223F mutation in HIV-2 Nef selectively impaired its ability to down-modulate MHC-I, albeit less severely than the corresponding mutation in the 239wt Nef (Fig. 7B, panels 5D and 9D) (39, 52, 58). Mutation of the three central proline (AAA) residues in HIV-2 BEN Nef had little effect (Fig. 7B, column 10), whereas C-terminal truncation (G238) disrupted MHC-I down-modulation but not other functions (Fig. 7B, column 11). We also introduced four amino acid substitutions, T103R, R105Q, R108L, and E110P, into the HIV-2 BEN Nef to evaluate whether an HIV-1 PXXP consensus region (Fig. 7A) might allow MHC-I down-modulation in the absence of the C-terminal region. The HIV-1 PXXP mutant Nef was fully functional in all six assays (Fig. 7B, column 13). Similar to the effects on wild-type BEN and 239 Nef function, however, C-terminal truncation disrupted its effect on MHC-I (Fig. 7B, column 14). Further analysis revealed that C-terminal truncation of FFm1 Nef also impaired MHC-I down-modulation (data not shown). Our results are further evidence that different groups of primate immunodeficiency viruses utilize different Nef domains to perform analogous activities and demonstrate that the C-terminal region is required for MHC-I down-modulation by both SIV and HIV-2 Nef proteins.
Enhancement of viral infectivity and replication. Next, we investigated whether HIV-2 and SIVsm nef alleles also enhance viral infectivity. All nef ORFs were inserted downstream of the env gene of the proviral SIVmac239 clone as previously described for HIV-1 nef genes (31), except that the SacI site located at the 3' end of env was used for cloning (Fig. 8A). In these constructs, the original SIVmac239 nef initiation codon and a second ATG at codon 7 of the nef ORF are mutated without changing the predicted Env sequence (31). Sequence and functional analysis confirmed that the resulting proviral SIVmac239 constructs did not contain undesired changes in env and expressed the appropriate HIV-2 and SIVsm Nef proteins (data not shown). As a positive control, otherwise isogenic constructs containing the nef genes of 239wt, HIV-1 NL4-3, or the molecular SHIV-40K6 clone were generated. The 40K6 nef allele was derived from a Nef-SHIV-infected macaque with simian AIDS and is closely related to the HIV-1 group M subtype B Nef consensus sequence (31). As a negative control, we inserted a nef allele containing a premature stop codon at position 93 of the nef ORF (239nef). Compared to the parental SIVmac239 clone, the genomic size of these constructs is enlarged by about 170 nucleotides, and the nef ATG initiation codon is located at a different position because of the elimination of the env-nef overlap. On average, the infectivity of the modified SIVmac239 was approximately three- to fourfold reduced compared to that of the parental virus (Fig. 8B). Nevertheless, the construct containing an intact 239wt nef gene showed about 10-fold-higher activity than the otherwise isogenic nef-defective form (Fig. 8B). Thus, this experimental system allows us to study the effect of various nef alleles on SIVmac infectivity without the complications of overlapping env sequences. Functional assays revealed that the 239wt, HIV-1 K6 and NL4-3, SIVsm FWr1, FYr1, FFm1, and FFm2, and HIV-2 ROD10, BEN, and 310319 nef alleles efficiently enhanced viral infectivity (Fig. 8C). In comparison, the HIV-2 CBL23 and 60415K nef genes were only marginally active and the 310248 Nef was inactive in enhancing virion infectivity. These data demonstrate that some HIV-2 and SIVsm nef genes increase virion infectivity with high efficiency.
Finally, we investigated the functional activity of HIV-2 and SIVsm nef alleles in enhancing viral replication. All SIVmac239 nef recombinants replicated efficiently in CEMx174 cells (data not shown). The T-lymphoid cell line 221-89, derived from a rhesus monkey infected with herpesvirus saimiri (1), was utilized to evaluate the stimulatory effects of the various nef alleles on SIVmac replication. In our previous studies, the abilities of various nef alleles to stimulate SIV replication in 221-89 cells and in rhesus- or human-derived PBMC always correlated (8, 34, 40, 58), indicating that both experimental systems might measure analogous effects of Nef (1). However, the 221-89 assay is less variable, because SIV replication in PBMC is to some extent dependent on the blood donor. Intact 239wt and K6 nef alleles enhanced viral replication moderately in the presence of exogenous IL-2 (Fig. 9, left panels) and more strongly in the absence of IL-2 (Fig. 9, right panels). The four SIVsm nef alleles consistently stimulated SIVmac replication in 221-89 cells with high efficiency (representative example shown in Fig. 9A). With the exception of the 310248 Nef, the HIV-2 Nefs also enhanced viral replication (Fig. 9B). However, the ROD10, BEN, CBL23, and 60415K nef alleles were clearly less active than the four SIVsm nef alleles in enhancing viral replication in both the presence and absence of exogenous IL-2 (Fig. 9 and Table 1). This observation is consistent with previous studies showing that the efficiency of HIV-1 replication correlates with Nef activity in CD4 down-modulation but not enhancement of viral infectivity. On average, the HIV-2 nef alleles also enhanced viral replication in human PBMC less efficiently than SIVsm nef genes (data not shown). Because they were highly active in other functional aspects, it was unexpected that the HIV-2 nef alleles enhanced viral replication only slightly compared to the nef-defective controls. Our results demonstrate that primary SIVsm nef genes are able to stimulate viral replication in rhesus cells. Although our data are suggestive, a larger number of samples needs to be analyzed to elucidate whether HIV-2 Nefs are typically less active in enhancing viral replication than those derived from other groups of primate lentiviruses.
DISCUSSION
In the present study, we demonstrated that, similarly to SIVmac239 Nef (52), SIVsm nef alleles derived from naturally infected sooty mangabeys are able to modulate cell surface expression of human CD4, CD3, CD28, MHC-I, MHC-II, and Ii molecules and to enhance virion infectivity and replication. Thus, SIVsm Nefs derived from primary isolates can modulate cell surface expression of human receptors without adaptive changes following animal or cell culture passage. These results resemble those of our previous study showing that primary SIVcpz nef alleles also did not require any changes to be functionally active in human cells and likely enhanced viral replication and persistence immediately after cross-species transmission to the new human host (32). We found that most HIV-2 Nefs were also functional (Table 1). Thus, none of the eight Nef functions investigated have been newly acquired or lost after cross-species transmission from sooty mangabeys to humans or macaques. It is remarkable, however, that all HIV-2 and SIVsm nef alleles investigated were less active than the SIVmac239 Nef in down-modulating CD4, CD28, and CD3 cell surface expression. Taken together, these data show that primary SIVsm Nefs are able to perform a variety of functions that most likely contribute to immune evasion and efficient persistence in vivo (26, 39, 40, 52). Concordant with this, SIVsm infection of sooty mangabeys is characterized by high-level viremia and efficient viral replication (48, 54).
Although sooty mangabeys are not entirely resistant to SIV-induced disease (35), they are clearly less susceptible to the development of immunodeficiency than infected rhesus macaques, which often develop disease within 1 year after infection (28). In comparison, HIV-2 causes AIDS in humans with an average incubation period of about 14 to 16 years (37, 44). Our observation that SIVsm, SIVmac, and HIV-2 Nefs perform similar functions supports the hypothesis that host factors are important for the different clinical outcomes of infection of mangabeys, macaques, and humans. We also found, however, that all HIV-2 310072 nef alleles were prematurely truncated and that 310248 Nefs showed little functional activity. Unfortunately, these two HIV-2 strains were derived from a blood donor with unknown clinical status (42). Thus, it remains unclear whether the lack of HIV-2 Nef function was associated with attenuated viral replication in vivo. Notably, a high prevalence of defective nef genes in HIV-2 infection has been previously reported (61). A larger number of samples needs to be analyzed to draw definitive conclusions, but taken together these findings suggest that disrupted or nonfunctional nef genes contribute to the low level of viral replication and lack of disease progression in some HIV-2-infected individuals.
Recently, we performed a comprehensive analysis of HIV-1 and SIVcpz Nef function (32). A comparison of these previous results with those of the present study shows that nef alleles derived from the HIV-1/SIVcpz and HIV-2/SIVsm/mac groups show some remarkable functional differences. In contrast to HIV-2 and SIVsm or SIVmac Nefs, HIV-1 Nefs are generally inactive in down-regulating cell surface expression of CD3, a component of the T-cell receptor. Furthermore, HIV-2 and SIVsm Nefs are much more active than HIV-1 and SIVcpz Nefs in down-regulating CD28 from the cell surface and in inhibiting TCR-mediated T-cell activation. Recent studies indicate that chronic immune activation might play a major role in AIDS pathogenesis (11, 54). HIV-2 is less pathogenic than HIV-1, the major causative agent of AIDS (37, 44). Concordant with our data, it has been demonstrated that the levels of immune activation are relatively low in HIV-2 infection (38). Microarray studies in transfected Jurkat T cells have shown that HIV-1 Nef triggers a transcriptional program resembling TCR-mediated T-cell activation (56). It will be of interest to determine whether or not HIV-2 Nefs trigger similar T-cell signaling pathways. Analysis of Nef function clearly indicates that moderately pathogenic HIV-2 strains interfere with TCR signaling from the cell membrane more severely than highly virulent HIV-1 strains. Efficient inhibition of TCR signaling together with lower activity in triggering T-cell activation pathways might explain why HIV-2 causes weaker chronic immune activation and is less pathogenic than HIV-1. However, the determinants of HIV and SIV pathogenesis are complex, and other viral and host factors are clearly also important. For example, SIVmac239 expressing a nef allele that performs all of these functions is highly pathogenic in rhesus macaques (28, 29). It must be considered, however, that macaques are particularly susceptible to SIV-induced disease. It is noteworthy that the accessory vpr and vpx genes are not required for efficient viral spread and disease progression in this model (19), although Vpr might play a relevant role for AIDS pathogenesis in HIV-1-infected humans (50). SIVmac239 is nonpathogenic when reintroduced into sooty mangabeys (27). Macaques infected with SIVsm showed high levels of immune activation and developed CD4+-T-cell depletion, whereas mangabeys displayed little T-cell proliferation and remained healthy (55). Thus, the seemingly high virulence of SIVmac239 is not an inherent property of the virus itself but due to the unusually high susceptibility of macaques to SIV-induced disease. Mutations in Nef that increase its ability to cause T-cell activation enhance the virulence of SIVmac239 even further and result in an acute pathogenic phenotype (14). Most importantly, HIV-2, which is closely related to SIVmac and also originates from SIVsm-infected mangabeys (16), is clearly less virulent than HIV-1 in infected humans (37, 44). Thus, when compared in the same host, the SIVsm/mac/HIV-2 group is less pathogenic than the SIVcpz/HIV-1 group. Therefore, we feel that the observation that SIVmac239 is highly virulent in macaques does not contradict the possibility that down-modulation of CD3 and CD28 favors slow/nonprogressive infection.
Our results indicate that HIV-2 Nefs down-modulate CD3 and inhibit T-cell activation more effectively than SIVsm nef alleles but are less active in stimulating viral replication in 221-89 cells and in human PBMC. However, more samples need to be investigated to clarify how frequently HIV-2 nef genes are more active in inhibiting T-cell activation and less active in stimulating viral replication than those derived from other groups of primate lentiviruses. Furthermore, HIV-2 Nefs might be more active in stimulating viral replication in their usual genomic context. Our constructs have the advantage that nef function can be studied using the well-characterized proviral SIVmac239 clone without the complications of overlapping env sequences. However, the increased genomic size and the expression of Nef from a different location resulted in reduced viral infectivity and replication compared to the parental SIVmac239 clone. As discussed above, we feel that further studies on their ability to stimulate T-cell activation and viral replication might provide important new insights into the pathogenesis of HIV-1 and HIV-2 infection.
In addition to the functional differences of HIV-1/SIVcpz and HIV-2/SIVsm/mac Nefs in modulating CD3 and CD28 surface expression, it is noteworthy that some conserved Nef functions are mediated by different Nef domains. The C-terminal region of SIVmac239, SIVsm, and HIV-2 Nefs is highly variable and not found in HIV-1 Nef (Fig. 2). Our mutational analysis extends previous studies (39, 58) showing that this region is exclusively involved in MHC-I down-modulation by the mac239 Nef and demonstrates that it plays a similar role for HIV-2 Nef function. It has recently been shown that changes selected for in rhesus macaques infected with an SIVmac239 mutant containing deletions near the C terminus of Nef generate an additional PXXP motif, as present in most HIV-1 Nef sequences, and restore the ability to down-modulate MHC-I (59). In comparison, introduction of an HIV-1 PXXP consensus did not restore MHC-I down-modulation by the truncated BEN Nef. Thus, at least in the context of the BEN Nef, this proline motif is not sufficient to functionally compensate for the C-terminal region. Notably, additional variations changing amino acids in 239 Nef to those usually present in HIV-1 Nef were also observed in infected macaques (59) and might be required to restore this function. In contrast to the effect on MHC-I, down-modulation of CD4 requires similar functional domains, such as the EXXXLL motif, in the SIVcpz/HIV-1 and SIVsm/HIV-2 Nef proteins. These findings suggest that the nef gene of the unknown ancestor of these groups of primate lentiviruses might have already been able to perform this function. Some conserved domains in Nef are clearly critical for specific functions. Even within the same group of viruses, however, the requirement of specific residues seems to be often dependent on the specific nef allele investigated. For example, the PA motif located in the N-proximal adaptor protein interaction site in SIVmac239 Nef is required for CD4 down-modulation (26). These residues are missing in the HIV-2 group B 310319 Nef (Fig. 2), although it effectively down-regulates CD4 (Fig. 4, column 9). Thus, while the functional analysis of specific Nef mutants provides some important information on the functional relevance of specific sequence motifs, general conclusions must be made with great caution.
In contrast to the effect on CD4, down-regulation of MHC-I clearly requires distinct domains in HIV-1 and HIV-2 Nefs, suggesting that this function might have been acquired independently and later during the evolution of these primate lentiviruses. The observation that distinct domains mediate different Nef functions might be important for optimal viral spread in vivo, because it has been shown that various HIV-1 or SIVmac Nef activities can be modulated at different stages of disease progression (2, 7, 43). Our results also indicate that HIV-1 and HIV-2 Nefs use overlapping but distinct domains to up-regulate Ii. Mutations in the EXXXLL motif entirely disrupted HIV-1 Nef function but had less severe effects on SIVmac239 or HIV-2 Nefs. We are currently investigating whether additional functional domains are involved in up-regulation of Ii by SIVmac and HIV-2 Nef proteins.
Taken together, the results of the present study demonstrate that the ability of Nef to modulate cell surface expression of human CD4, CD3, CD28, MHC-I, MHC-II, and Ii and to enhance virion infectivity is conserved within the SIVsm/HIV-2/SIVmac group of primate lentiviruses. HIV-1 and SIVcpz Nefs are also able to perform a striking variety of functions. Nevertheless, compared to SIVsm and HIV-2 Nefs, they are generally unable to down-regulate CD3 and also have little effect on CD28 cell surface expression (32). It will be of interest to further evaluate whether these differences in Nef function contribute to the low pathogenicity of HIV-2 compared to HIV-1 in the human host.
ACKNOWLEDGMENTS
We thank Thomas Mertens for constant support and encouragement, Ingrid Bennett for critical reading of the manuscript, and Ainee McKnight for providing the HIV-2 CBL23 nef allele.
This work was supported by the Wilhelm-Sander Foundation, the Deutsche Forschungsgemeinschaft, and a grant from the Elitef?rderprogramm BW to J.M.
The first three authors are in alphabetical order and contributed equally to this work.
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