This study demonstrates that primary HIV-1, HIV-2, and SIVmac nef
alleles modulate MHC-II and Ii cell surface expression, indicating that the ability of Nef to impair MHC-II-mediated antigen presentation and, hence, CD4+
-T-helper-cell responses are conserved among different groups of primate lentiviruses. Usually, virus-specific T helper responses are low or absent in most hosts infected with pathogenic HIV-1 or SIV (19
) but strong in HIV-1-infected individuals harboring nef
-defective forms of the virus and in monkeys experimentally infected with attenuated SIVmac239 variants from which nef
has been deleted (15
). However, it remains to be clarified whether these differences result from the lack of Nef function or are just due to the lower viral loads and asymptomatic status of hosts infected with attenuated virus variants. Vigorous and sustained CD4+
-T-helper responses have been observed in HIV-1-infected long-term nonprogressors (53
). To further elucidate whether Nef specifically impairs CD4+
-T-helper responses in vivo, it would be interesting to compare the T-helper and CTL responses in macaques infected with either nef
-defective or vpx/vpr
-defective SIVmac variants showing similar degrees of attenuation (21
Surface expression of Ii was strongly increased in transfected cells expressing low to moderate levels of Nef and in HIV-1-infected cells. In contrast, down-modulation of mature MHC-II was only observed at high levels of Nef expression. Extending a previous study, we demonstrated that the levels of MHC-II on the surface of Nef-expressing HIV-1-infected HeLa CIITA cells are slightly reduced (62
). However, we did not observe a significant decrease in MHC-II surface levels on 221-B7 cells or human PBMC transduced with proviral HIV-1 constructs (Fig. ). It has been demonstrated recently that Nef down-modulates MHC-I cell surface expression much more efficiently on primary T cells compared to HeLa cells (34
). The influence of Nef on MHC-II surface expression might also be cell type dependent, and it will be important to investigate this in HIV-1-infected primary APCs.
The importance of both Nef functions for MHC-II antigen presentation and the pathogenicity of HIV-1 remains to be defined. However, since surface expression of Ii was usually dramatically increased and MHC-II associated with Ii cannot stimulate CD4+
T cells, it is likely that this Nef function interferes with MHC-II-restricted peptide presentation. Furthermore, Ii up-regulation was seen for nef
alleles derived from all progressing HIV-1-infected individuals studied except for two of four LTNPs only, suggesting that this Nef function might be relevant for progressive infection. nef
alleles from patients LTNP2 and 039nm94 did not up-regulate Ii; however, they maintained low viral loads and stable CD4+
-T-cell counts despite more than 15 and 12 years, respectively, of documented HIV-1 infection. LTNP2 is among seven individuals of 128 HIV-1-infected participants monitored by the New England Hemophilia Center of the University of Massachusetts/Memorial Health Care System who met the criteria for long-term nonprogression (24
). Notably, LTNP2 was the only individual with nonprogressive infection in this cohort in whom no unusual, difficult-to-revert polymorphisms in HIV-1, which might attenuate viral replication, could be detected (2
). Furthermore, this individual's class I HLA alleles (HLA-B49 and HLA-A29) are typically associated with more rapid progression to AIDS (24
). Clearly, a larger number of LTNPs must be analyzed, but the preliminary results suggest that the inability of Nef to up-regulate Ii might contribute to the lack of disease progression in LTNP2. nef
alleles derived from another nonprogressor, LTNP4, could modulate MHC-II and Ii surface expression (Fig. ), but neither down-modulated CD4 or enhanced viral replication (8
). Thus, in addition to grossly defective nef
genes, more-subtle differences in Nef function might contribute to nonprogressive HIV-1 infection.
Nef affects multiple aspects of the interactions between CD4+
T cells and MHC-II-expressing APCs (5
). The concerted modulation of CD4, CD28, Ii, MHC-II, and/or CD3 cell surface expression should impair both the adhesion between T cells and APCs, as well as the duration and strength of the antigen-specific signal in the T cell. Recent studies suggest that Nef prevents antigen-specific T-cell activation while activating downstream effectors in signaling pathways that mediate cellular activation and lead to efficient virus production (30
). SIVmac variants containing mutations that selectively disrupt specific in vitro functions of Nef are useful in studying the relative contribution of different Nef activities to viral replication and pathogenesis in vivo (30
). However, we have not been able to identify motifs in SIV Nef exclusively required for down-modulation of MHC-II or up-regulation of Ii. Thus, it will not be easy to determine to what extent these Nef functions contribute to viral pathogenesis.
We demonstrated previously that nef
alleles obtained during late stages of HIV-1 infection did not down-modulate MHC-I efficiently, but they were highly active in stimulating viral replication and down-regulating CD4 (8
). The progressor consensus nef
alleles (Pcon, Pex, and PexP) and the late stage P2-93 or SP7-93 nef
genes were less active in up-regulating Ii than the NPcon, NPex, P2-87, and SP7-88 nef allele
s (Fig. ). However, the average functional activity of Nef in modulating Ii and MHC-II surface expression did not change significantly during or after progression to AIDS in six progressing HIV-1-infected individuals. One possibility is that these Nef functions are relevant for efficient viral replication throughout the course of infection. However, it should be noted that mutations in the C-proximal flexible loop disrupted Ii up-regulation and impaired down-regulation of CD4 (Fig. ). Thus, whereas some mutations in Nef selectively affect either CD4 or Ii surface modulation, both functions are apparently mediated by overlapping domains. Accordingly, changes reducing the ability of Nef to modulate Ii or MHC-II surface expression might frequently also impair other Nef functions, which are important for efficient viral replication during late stages of HIV-1 infection. This might explain why a loss of Nef function in MHC-II down- and Ii up-regulation is not observed in most AIDS patients.
In agreement with Stumptner-Cuvelette and Benaroch (62
), we found that most, if not all, in vitro Nef functions investigated are genetically separable. The acidic domain of Nef was shown in both studies to be involved in down-modulation of MHC-II but dispensable for up-regulation of the Ii chain. Mutations in the C-proximal flexible loop consistently abolished the ability of Nef to modulate Ii surface expression but had little effect on down-regulation of MHC-II. Nef-induced down-regulation of CD4 and up-regulation of Ii chain required the presence of the dileucine and the five charged residues at positions 174, 175, and 177 to 179. However, they had opposite dependencies toward the dibasic motif (residues 105 and 106) and the diacidic motif (residues 154 and 155). These genetic differences probably reflect differences in the mechanisms underlying these effects. The dileucine motif of Nef has been implicated in the capacity of Nef to associate with clathrin adaptor complexes, including AP2, thus contributing to the Nef-induced accelerated endocytosis of CD4 (12
). However, the effect of Nef on Ii surface expression does not seem to result from AP2 titration by the viral protein because other AP2-dependent proteins such as the transferrin receptor are not up-regulated. Mutation of prolines 72 and 75 moderately affected MHC-I down-regulation and disrupted the ability of Nef to modulate MHC-II but not Ii surface expression (Fig. ). In comparison, mutation of prolines 75 and 78 totally impaired MHC-I down-regulation but had only a moderate effect on MHC II and Ii modulation (62
). Of note, it has recently been shown that of the four prolines at positions 69, 72, 75, and 78 in Nef, only position 78 is critical for MHC-I down-regulation (66
). Taken together, these data suggest that proline 72 of Nef is involved in MHC-II down-modulation. However, an intact SH3-binding domain in Nef is not required for the modulation of Ii and MHC-I cell surface expression.
Overall, our results indicate that Nef-mediated up-regulation of Ii might play a relevant role in the pathogenesis of HIV-1. Other viruses might use a similar strategy to impair MHC-II antigen presentation (59
). However, much remains to be done to elucidate the mechanisms by which Nef modulates MHC-II and Ii cell surface expression and to clarify their roles in determining viral pathogenicity in vivo. It will be important to evaluate the effects of Nef on MHC-II antigen presentation in primary cells and to use Nef mutants, selectively impaired for modulation of CD3, CD4, CD28, Ii, and/or MHC-II surface expression, to clarify which activities are critical for the interaction between T cells and APCs. Such SIV and HIV-1 Nef mutants would also be useful for investigating the importance of these in vitro Nef activities to viral pathogenicity in the SIV/macaque and Nef-SHIV models (3
). Currently, we are analyzing nef
alleles derived from a larger panel of LTNPs to clarify whether long-term nonprogressive HIV-1 infection is frequently associated with impaired functional activity in Ii up-regulation or other Nef functions.