The ITAM in Nef influences acute pathogenesis of AIDS-inducing simian immunodeficiency viruses SIVsm and SIVagm without altering kinetics or extent of viremia

The role of the immunoreceptor tyrosine-based activation motif (ITAM) that is unique to the Nef protein of the acutely pathogenic simian immunodeficiency virus SIVsmPBj was studied in the context of two AIDS-inducing simian immunodeficiency virus molecular clones. NefY(+) variants of SIVagm9063-2 and SIVsmE543-3 replicated in and induced proliferation of unstimulated pig-tailed macaque PBMC. The pathogenesis of the NefY(+) and NefY(-) clones of SIVagm9063-2, SIVsmE543-3, and PBj6.6 were evaluated by intravenous inoculation of pig-tailed macaques (Macaca nemestrina). Introduction of the ITAM did not increase plasma viral RNA levels nor alter the kinetics of viremia compared with the NefY(-) versions of each clone. Clinical symptoms were not observed in animals inoculated with the NefY(-) variants. In contrast, characteristic PBj symptoms were observed in animals inoculated with any of the three NefY(+) clones. Blunting and fusion of intestinal villi and multifocal infiltration of mononuclear cells were observed in the gastrointestinal tracts of macaques inoculated with the NefY(+) versions. Lesions were associated with active viral replication, as demonstrated by simian immunodeficiency virus-specific in situ hybridization. However, only the macaque inoculated with wild-type NefY(+) SIVsmPBj developed fatal disease; lesions were more widespread and severe in this animal. A switch to macrophages as a viral reservoir and the presence of interleukin-6 in plasma was unique to the macaque infected with PBj6.6. Overall, these data suggest that the ITAM in SIV Nef alters the pathogenesis of simian immunodeficiency virus regardless of the viral background. The change in pathogenesis occurs without enhancement of viral replication. However, NefY(+) variants of SIVagm and SIVsm did not fully recapitulate the virulence of SIVsmPBj, implicating additional viral factors in this unique virus pathogenesis.     

Simian-Human Immunodeficiency Virus (SHIV) Containing the nef/Long Terminal Repeat Region of the Highly Virulent SIVsmmPBj14 Causes PBj-Like Activation of Cultured Resting Peripheral Blood Mononuclear Cells, but the Chimera Showed No Increase in Virulence

SIV_smmPBj14 is a highly pathogenic lentivirus which causes acute diarrhea, rash, massive lymphocyte proliferation predominantly in the gastrointestinal tract, and death within 7 to 14 days. In cell culture, the virus has mitogenic effects on resting macaque T lymphocytes. In contrast, SIV_mac239 causes AIDS in rhesus macaques, generally within 2 years after inoculation. In a previous study, replacement of amino acid residues 17 and 18 of the Nef protein of SIV_mac239 with the corresponding amino acid residues of the Nef protein of SIV_smmPBj14 yielded a PBj-like virus that caused extensive activation of resting T lymphocytes in cultures and acute PBj-like disease when inoculated into pig-tailed macaques. This study suggested that nef played a major role in both processes. In this study, we replaced the nef/long terminal repeat (LTR) region of a nonpathogenic simian-human immunodeficiency virus (SHIV), SHIV_PPc, with the corresponding region from SIV_smmPBj14 and examined the biological properties of the resultant virus. Like SIV_smmPBj14, SHIV_PPcPBjnef caused massive stimulation of resting peripheral blood mononuclear cells (PBMC), which then produced virus in the absence of extraneous interleukin 2. However, when inoculated into macaques, the virus failed to replicate productively or cause disease. Thus, while these results confirmed that the nef/LTR region of SIV_smmPBj14 played a major role in the activation of resting PBMC, duplication of the cellular activation process in macaques may require a further interaction between nef and the envelope glycoprotein of simian immunodeficiency virus because SHIV, containing the envelope of human immunodeficiency virus type 1, failed to cause activation in vivo.     

Functional analysis of the simian immunodeficiency virus Vpx protein: identification of packaging determinants and a novel nuclear targeting domain

The vpx gene products of human immunodeficiency virus type 2 (HIV-2) and of the closely related simian immunodeficiency viruses from sooty mangabeys (SIVsm) and macaques (SIVmac) comprise a 112-amino-acid virion-associated protein that is critical for efficient virus replication in nondividing cells such as macrophages. When expressed in the absence of other viral proteins, Vpx localizes to the nuclear membrane as well as to the nucleus; however, in the context of virus replication Vpx is packaged into virions via interaction with the p6 domain of the Gag precursor polyprotein (p55(gag)). To identify the domains essential for virion incorporation and nuclear localization, site-directed mutations were introduced into the vpx gene of SIVsmPBj1.9 and functionally analyzed. Our results show that (i) mutation of two highly conserved L74 and I75 residues impaired both virion incorporation and nuclear localization of Vpx; (ii) substitution of conserved H82, G86, C87, P103, and P106 residues impaired Vpx nuclear localization but not virion incorporation; (iii) mutations of conserved Y66, Y69, and Y71 residues impaired virion incorporation but not the translocation of Vpx to the nucleus; and (iv) a mutation at E30 (predicted to disrupt an N-terminal alpha-helix) had no effect on either virion incorporation or nuclear localization of Vpx. Importantly, mutations in Vpx which impaired nuclear localization also reduced virus replication in macaque macrophages, suggesting an important role of the carboxyl terminus of Vpx in nuclear translocation of the viral preintegration complex. Analyzing this domain in greater detail, we identified a 26-amino-acid (aa 60 to 85) fragment that was sufficient to mediate the transport of a heterologous protein (green fluorescent protein [GFP]) to the nucleus. Taken together, these results indicate that virion incorporation and nuclear localization are encoded by two partially overlapping domains in the C-terminus of Vpx (aa 60 to 112). The identification of a novel 26-amino-acid nuclear targeting domain provides a new tool to investigate the nuclear import of the HIV-2/SIV preintegration complex.     

The nef gene products of both simian and human immunodeficiency viruses enhance virus infectivity and are functionally interchangeable.

Adult rhesus macaques infected with nef-defective simian immunodeficiency virus (SIV) exhibit extremely low levels of steady-state virus replication, do not succumb to immunodeficiency disease, and are protected from experimental challenge with pathogenic isolates of SIV. Similarly, rare humans found to be infected with nef-defective human immunodeficiency virus type 1 (HIV-1) variants display exceptionally low viral burdens and do not show evidence of disease progression after many years of infection. HIV-1 Nef induces the rapid endocytosis and lysosomal degradation of cell surface CD4 and enhances virus infectivity in primary human T cells and macrophages. Although expression of SIV Nef also leads to down-modulation of cell surface CD4 levels, no evidence for SIV Nef-induced enhancement of virus infectivity was observed in earlier studies. Thus, it remains unclear whether fundamental differences exist between the activities of HIV-1 and SIV Nef. To establish more clearly whether the SIV and HIV-1 nef gene products are functionally analogous, we compared the replication kinetics and infectivity of variants of SIVmac239 that either do (SIVnef+) or do not (SIV delta nef) encode intact nef gene products. SIVnef+ replicates more rapidly than nef-defective viruses in both human and rhesus peripheral blood mononuclear cells (PBMCs). As previously described for HIV-1 Nef, SIV Nef also enhances virus infectivity within each cycle of virus replication. As a strategy for evaluating the in vivo contribution of HIV-1 nef alleles and long terminal repeat regulatory sequences to the pathogenesis of immunodeficiency disease, we constructed SIV-HIV chimeras in which the nef coding and U3 regulatory regions of SIVmac239 were replaced by the corresponding regions from HIV-1/R73 (SIVR7nef+). SIVR7nef+ displays enhanced infectivity and accelerated replication kinetics in primary human and rhesus PBMC infections compared to its nef-defective counterpart. Converse chimeras, containing SIV Nef in an HIV-1 background (R7SIVnef+) also exhibit greater infectivity than matched nef-defective viruses (R7SIV delta nef). These data indicate that SIV Nef, like that of HIV-1, does enhance virus replication in primary cells in tissue culture and that HIV-1 and SIV Nef are functionally interchangeable in the context of both HIV-1 and SIV.     

Multiple viral determinants contribute to pathogenicity of the acutely lethal simian immunodeficiency virus SIVsmmPBj variant.

Simian immunodeficiency virus (SIV) induces an immunodeficiency syndrome similar to human AIDS. Although the disease course of SIV-induced immunodeficiency is generally measured in months to years, a disease syndrome that results in death in 5 to 14 days has been described in pig-tailed macaques infected with the SIVsmmPBj (PBj) strain. The purpose of this study was to derive an acutely lethal PBj molecular clone in order to study viral genes involved in pathogenesis. Six infectious molecular clones were generated; acutely fatal disease was induced by experimental inoculation of pig-tailed macaques with virus stocks derived from either of two clones, PBj6.6 or PBj14.6. Molecular chimeras were constructed by exchange of regions of the genome of PBj6.6 and a nonlethal, related clone, SIVsmH4. Only a chimera expressing the PBj genome under the control of a SIVsmH4 long terminal repeat induced death soon after inoculation. These studies suggest that multiple viral genes of PBj are critical for development of acute disease. More specifically, the env gene but not the long terminal repeat PBj was required for acute disease induction; however env must act in concert with another gene(s) of the PBj genome.     

Limited Protection from a Pathogenic Chimeric Simian-Human Immunodeficiency Virus Challenge following Immunization with Attenuated Simian Immunodeficiency Virus

Two live attenuated single-deletion mutant simian immunodeficiency virus (SIV) constructs, SIV_239Δnef and SIV_PBj6.6Δnef, were tested for their abilities to stimulate protective immunity in macaques. During the immunization period the animals were examined for specific immune responses and virus growth. Each construct generated high levels of specific immunity in all of the immunized animals. The SIV_239Δnef construct was found to grow to high levels in all immunized animals, with some animals remaining positive for virus isolation and plasma RNA throughout the immunization period. The SIV_PBj6.6Δnef was effectively controlled by all of the immunized animals, with virus mostly isolated only during the first few months following immunization and plasma RNA never detected. Following an extended period of immunization of over 80 weeks, the animals were challenged with a pathogenic simian-human immunodeficiency virus (SHIV) isolate, SIV_89.6PD, by intravenous injection. All of the SIV_239Δnef-immunized animals became infected with the SHIV isolate; two of five animals eventually controlled the challenge and three of five animals, which failed to check the immunizing virus, progressed to disease state before the unvaccinated controls. One of five animals immunized with SIV_PBj6.6Δnef totally resisted infection by the challenge virus, while three others limited its growth and the remaining animal became persistently infected and eventually died of a pulmonary thrombus. These data indicate that vaccination with attenuated SIV can protect macaques from disease and in some cases from infection by a divergent SHIV. However, if animals are unable to control the immunizing virus, potential damage that can accelerate the disease course of a pathogenic challenge virus may occur.     

Importance of the N-distal AP-2 binding element in Nef for simian immunodeficiency virus replication and pathogenicity in rhesus macaques

Point mutations in SIVmac239 Nef disrupting CD4 downmodulation and enhancement of virion infectivity attenuate viral replication in acutely infected rhesus macaques, but changes selected later in infection fully restore Nef function (A. J. Iafrate et al., J. Virol. 74:9836-9844, 2000). To further evaluate the relevance of these Nef functions for viral persistence and disease progression, we analyzed an SIVmac239 Nef mutant containing a deletion of amino acids Q64 to N67 (delta64-67Nef). This mutation inactivates the N-distal AP-2 clathrin adaptor binding element and disrupts the abilities of Nef to downregulate CD4, CD28 and CXCR4 and to stimulate viral replication in vitro. However, it does not impair the downmodulation of CD3 and class I major histocompatibility complex (MHC-I) or MHC-II and the upregulation of the MHC-II-associated invariant chain, and it has only a moderate effect on the enhancement of virion infectivity. Replication of the delta64-67Nef variant in acutely infected macaques was intermediate between grossly nef-deleted and wild-type SIVmac239. Subsequently, three of six macaques developed moderate to high viral loads and developed disease, whereas the remaining animals efficiently controlled SIV replication and showed a more attenuated clinical course of infection. Sequence analysis revealed that the deletion in nef was not repaired in any of these animals. However, some changes that slightly enhanced the ability of Nef to downmodulate CD4 and moderately increased Nef-mediated enhancement of viral replication and infectivity in vitro were observed in macaques developing high viral loads. Our results imply that both the Nef functions that were disrupted by the delta64-67 mutation and the activities that remained intact contribute to viral pathogenicity.     

The Proteolytic Cleavage of Human Immunodeficiency Virus Type 1 Nef Does Not Correlate with Its Ability To Stimulate Virion Infectivity

The Nef protein of human immunodeficiency virus type 1 (HIV-1) promotes virion infectivity through mechanisms that are yet ill defined. Some Nef is incorporated into particles, where it is cleaved by the viral protease between amino acids 57 and 58. The functional significance of this event, which liberates the C-terminal core domain of the protein from its membrane-associated N terminus, is unknown. To address this question, we examined the modalities of Nef virion association and processing. We found that although significant levels of Nef were detected in HIV-1 virions partly in a cleaved form, cell-specific variations existed in the efficiency of Nef proteolytic processing. The virion association of Nef was strongly enhanced by myristoylation but did not require other HIV-1-specific proteins, since Nef was efficiently incorporated into and cleaved inside murine leukemia virus particles. Substituting alanine for tryptophan⁵⁷ decreased the efficiency of Nef processing, while mutating leucine⁵⁸ had little effect. In contrast, replacing both of these residues simultaneously almost completely prevented this process. However, when the resulting mutants were compared with a wild-type control in viral infectivity assays, no correlation was found between the levels of cleavage and the ability to stimulate virion infectivity. Furthermore, simian immunodeficiency virus Nef, which lacks the sequence recognized by the protease and as a consequence is not cleaved despite its incorporation into virions, could stimulate the infectivity of a nef-defective HIV-1 variant as efficiently as HIV-1 Nef. On these bases, we conclude that the proteolytic processing of Nef is not required for the ability of this protein to enhance virion infectivity.     

Increased APOBEC3G and APOBEC3F expression is associated with low viral load and prolonged survival in simian immunodeficiency virus infected rhesus monkeys

Background

The non-pathogenic course of SIV infection in its natural host is characterized by robust viral replication in the absence of chronic immune activation and T cell proliferation. In contrast, acutely lethal enteropathic SIVsmm strain PBj induces a strong immune activation and causes a severe acute and lethal disease in pig-tailed macaques after cross-species transmission. One important pathogenicity factor of the PBj virus is the PBj-Nef protein, which contains a conserved diacidic motif and, unusually, an immunoreceptor tyrosine-based activation motif (ITAM).

Results

Mutation of the diacidic motif in the Nef protein of the SIVsmmPBj abolishes the acute phenotype of this virus. In vitro, wild-type and mutant PBj (PBj-Nef202/203GG) viruses replicated to similar levels in macaque PBMCs, but PBj-Nef202/203GG no longer triggers ERK mitogen-activated protein (MAP) kinase pathway including an alteration of a Nef-associated Raf-1/ERK-2 multiprotein signaling complex. Moreover, stimulation of IL-2 and down-modulation of CD4 and CD28 were impaired in the mutant virus. Pig-tailed macaques infected with PBj-Nef202/203GG did not show enteropathic complications and lethality as observed with wild-type PBj virus, despite efficient replication of both viruses in vivo. Furthermore, PBj-Nef202/203GG infected animals revealed reduced T-cell activation in periphery lymphoid organs and no detectable induction of IL-2 and IL-6.

Conclusions

In sum, we report here that mutation of the diacidic motif in the PBj-Nef protein abolishes disease progression in pig-tailed macaques despite efficient replication. These data suggest that alterations in the ability of a lentivirus to promote T cell activation and proliferation can have a dramatic impact on its pathogenic potential.     

Nef from Human Immunodeficiency Virus Type 1F12 Inhibits Viral Production and Infectivity

Human immunodeficiency virus type 1(F12) (HIV-1(F12)) interferes with the replication of other strains of HIV. Its accessory protein, Nef, is sufficient for this phenotype, where the production and infectivity of HIV are impaired significantly. The analysis of three rare mutations in this Nef protein revealed that these effects could be separated genetically. Moreover, the defect in virus production correlated with the lack of processing of the p55(Gag) precursor in the presence of Nef from HIV-1(F12). Importantly, the introduction of one of these mutations (E177G) into Nef from HIV-1(NL4-3) also created a dominant-negative Nef protein. Effects of Nef from HIV-1(F12) on virus production and Gag processing correlated with its altered subcellular distribution. Moreover, the association with two new cellular proteins with molecular masses of 74 and 75 kDa, which do not interact with other Nef proteins, correlated with the decreased virion infectivity. The identification of a dominant-negative protein for the production and infectivity of HIV suggests that Nef plays an active role at this stage of the viral replicative cycle.     

Comprehensive analysis of nef functions selected in simian immunodeficiency virus-infected macaques

A variety of simian immunodeficiency virus (SIVmac) nef mutants have been investigated to clarify which in vitro Nef functions contribute to efficient viral replication and pathogenicity in rhesus macaques. Most of these nef alleles, however, were only functionally characterized for their ability to down-modulate CD4 and class I major histocompatibility complex (MHC-I) cell surface expression and to enhance SIV replication and infectivity. To obtain information on the in vivo relevance of more recently established Nef functions, we examined the ability of a large panel of constructed SIVmac Nef mutants and of variants that emerged in infected macaques to down-regulate CD3, CD28, and MHC-II and to up-regulate the MHC-II-associated invariant chain (Ii). We found that all these four Nef functions were restored in SIV-infected macaques. In most cases, however, the initial mutations and the changes selected in vivo affected several in vitro Nef functions. For example, truncated Nef proteins that emerged in animals infected with SIVmac239 containing a 152-bp deletion in nef efficiently modulated both CD3 and Ii surface expression. Overall, our results suggest that the effect of Nef on each of the six cellular receptors investigated contributes to viral fitness in the infected host but also indicate that modulation of CD3, MHC-I, MHC-II, or Ii surface expression alone is insufficient for SIV virulence.     

Infective Virus Substructure from Vesicular Stomatitis Virus

Treatment of suspensions of vesicular stomatitis virus with Tween-ether results in a rapid and considerable loss of infectivity (ca. 4 logs in 2 min), but the residual infectivity is comparatively stable to further treatment with ether. The infectivity remaining after the short exposure to Tween-ether is not due to virus for the following reasons. (i) It is much less infective for tissue cultures than for mice, whereas the intact virion is equally infective for both hosts. (ii) The residual infectivity is much less stable than virus infectivity in both sucrose and tartrate gradients. (iii) Virus immune serum does not neutralize its activity. (iv) The infectivity is associated with material which sediments further in sucrose gradients and has a greater buoyant density in tartrate gradients than the virion. Experiments with (32)P-labeled virion showed that the infective substructure contains ribonucleic acid with the same sedimentation characteristics as that extracted from the virion. Electron microscopy shows that the infective component has the same overall bullet-like structure as the virion but lacks the outer envelope and fringe structure.     

Impairment of hepatitis B virus virion secretion by single-amino-acid substitutions in the small envelope protein and rescue by a novel glycosylation site

Mutations in the S region of the hepatitis B virus (HBV) envelope gene are associated with immune escape, occult infection, and resistance to therapy. We previously identified naturally occurring mutations in the S gene that alter HBV virion secretion. Here we used transcomplementation assay to confirm that the I110M, G119E, and R169P mutations in the S domain of viral envelope proteins impair virion secretion and that an M133T mutation rescues virion secretion of the I110M and G119E mutants. The G119E mutation impaired detection of secreted hepatitis B surface antigen (HBsAg), suggesting immune escape. The R169P mutant protein is defective in HBsAg secretion as well and has a dominant negative effect when it is coexpressed with wild-type envelope proteins. Although the S domain is present in all three envelope proteins, the I110M, G119E, and R169P mutations impair virion secretion through the small envelope protein. Conversely, coexpression of just the small envelope protein of the M133T mutant could rescue virion secretion. The M133T mutation could also overcome the secretion defect caused by the G145R immune-escape mutation or mutation at N146, the site of N-linked glycosylation. In fact, the M133T mutation creates a novel N-linked glycosylation site ((131)NST(133)). Destroying this site by N131Q/T mutation or preventing glycosylation by tunicamycin treatment of transfected cells abrogated the effect of the M133T mutation. Our findings demonstrate that N-linked glycosylation of HBV envelope proteins is critical for virion secretion and that the secretion defect caused by mutations in the S protein can be rescued by an extra glycosylation site.     

Disrupting surfaces of nef required for downregulation of CD4 and for enhancement of virion infectivity attenuates simian immunodeficiency virus replication in vivo

The multifunctional simian and human immunodeficiency virus (SIV and HIV) Nef proteins are important for virulence. We studied the importance of selected Nef functions using an SIV Nef with mutations in two regions that are required for CD4 downregulation. This Nef mutant is defective for downregulating CD4 and, in addition, for enhancing SIV infectivity and induction of SIV replication from infected quiescent peripheral blood mononuclear cells, but not for other known functions, including downregulation of class I major histocompatibility complex (MHC) cell surface expression. Replication of SIV containing this Nef variant in rhesus monkeys was attenuated early during infection. Subsequent increases in viral load coincided with selection of reversions and second-site compensatory changes in Nef. Our results indicate that the surfaces of Nef that mediate CD4 downregulation and the enhancement of virion infectivity are critical for SIV replication in vivo. Furthermore, these findings indicate that class I MHC downregulation by Nef is not sufficient for SIV virulence early in infection.     

Virion Incorporation of Human Immunodeficiency Virus Type 1 Nef Is Mediated by a Bipartite Membrane-Targeting Signal: Analysis of Its Role in Enhancement of Viral Infectivity

The nef gene of primate immunodeficiency viruses is essential for high-titer virus replication and AIDS pathogenesis in vivo. In tissue culture, Nef is not required for human immunodeficiency virus (HIV) infection but enhances viral infectivity. We and others have shown that Nef is incorporated into HIV-1 particles and cleaved by the viral proteinase. To determine the signal for Nef incorporation and to analyze whether virion-associated Nef is responsible for enhancement of infectivity, we generated a panel of nef mutants and analyzed them for virion incorporation of Nef and for their relative infectivities. We report that N-terminal truncations of Nef abolished its incorporation into HIV particles. Incorporation was reconstituted by targeting the respective proteins to the plasma membrane by using a heterologous signal. Mutational analysis revealed that both myristoylation and an N-terminal cluster of basic amino acids were required for virion incorporation and for plasma membrane targeting of Nef. Grafting the N-terminal anchor domain of Nef onto the green fluorescent protein led to membrane targeting and virion incorporation of the resulting fusion protein. These results indicate that Nef incorporation into HIV-1 particles is mediated by plasma membrane targeting via an N-terminal bipartite signal which is reminiscent of a Src homology region 4. Virion incorporation of Nef correlated with enhanced infectivity of the respective viruses in a single-round replication assay. However, the phenotypes of HIV mutants with reduced Nef incorporation only partly correlated with their ability to replicate in primary lymphocytes, indicating that additional or different mechanisms may be involved in this system.     

Human immunodeficiency virus type 1 particles pseudotyped with envelope proteins that fuse at low pH no longer require Nef for optimal infectivity

We have investigated the effects of Nef on infectivity in the context of various viral envelope proteins. These experiments were performed with a minimal vector system where Nef is the only accessory protein present. Our results support the hypothesis that the route of entry influences the ability of Nef to enhance human immunodeficiency virus (HIV) infectivity. We show that HIV particles pseudotyped with Ebola virus glycoprotein or vesicular stomatitis virus glycoprotein (VSV-G), which fuse at low pH, do not require Nef for optimal infectivity. In contrast, Nef significantly enhances the infectivity of virus particles that contain envelope proteins that fuse at neutral pH (CCR5-dependent HIV Env, CXCR4-dependent HIV Env, or amphotropic murine leukemia virus Env). In addition, our results demonstrate that virus particles containing mixed CXCR4-dependent HIV and VSV-G envelope proteins show a conditional requirement for Nef for optimal infectivity, depending on which protein is allowed to facilitate entry.     

Nef enhances human immunodeficiency virus type 1 infectivity and replication independently of viral coreceptor tropism

We investigated the infectivities and replicative capacities of a large panel of variants of the molecular human immunodeficiency virus type 1 (HIV-1) NL4-3 clone that differ exclusively in the V3 region of the viral envelope glycoprotein and the nef gene. Our results demonstrate that Nef enhances virion infectivity and HIV-1 replication independently of the viral coreceptor tropism.     

T-cell receptor:CD3 down-regulation is a selected in vivo function of simian immunodeficiency virus Nef but is not sufficient for effective viral replication in rhesus macaques

We investigated the function of severely truncated simian immunodeficiency virus (SIV) Nef proteins (tNef) in vitro and in vivo. These variants emerged in rhesus monkeys infected with SIVmac239 containing a 152-bp deletion in the nef-unique region and have been suggested to enhance SIV virulence (E. T. Sawai, M. S. Hamza, M. Ye, K. E. Shaw, and P. A. Luciw, J. Virol. 74:2038-2045, 2000). We found that the tNef proteins were unable to down-regulate the cell surface expression of major histocompatibility complex class I proteins, CD4, and CD28 and neither stimulated SIV replication nor enhanced virion infectivity. The tNef proteins did efficiently down-regulate T-cell receptor (TCR):CD3 cell surface expression. Nevertheless, the SIVmac239 tnef variants were strongly attenuated in six infected juvenile rhesus macaques. Thus, while the ability of SIV Nef to down-modulate TCR:CD3 cell surface expression apparently confers a selective advantage in vivo, it is insufficient for efficient viral replication in infected macaques. Additional mutations elsewhere in SIVmac239 tnef genomes are required for a virulent phenotype.     

Efficient class I major histocompatibility complex down-regulation by simian immunodeficiency virus Nef is associated with a strong selective advantage in infected rhesus macaques

Substitution of Y223F disrupts the ability of simian immunodeficiency virus (SIV) Nef to down-modulate major histocompatibility complex (MHC) class I from the cell surface but has no effect on other Nef functions, such as down-regulation of CD4, CD28, and CD3 cell surface expression or stimulation of viral replication and enhancement of virion infectivity. Inoculation of three rhesus macaques with the SIVmac239 Y223F-Nef variant revealed that this point mutation consistently reverts and that Nef activity in MHC class I down-modulation is fully restored within 4 weeks after infection. Our results demonstrate a strong selective pressure for a tyrosine at amino acid position 223 in SIV Nef, and they constitute evidence that Nef-mediated MHC class I down-regulation provides a selective advantage for viral replication in vivo.