Primary sooty mangabey simian immunodeficiency virus and human immunodeficiency virus type 2 nef alleles modulate cell surface expression of various human receptors and enhance viral infectivity and replication

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.     

CD4 down-modulation by human immunodeficiency virus type 1 Nef correlates with the efficiency of viral replication and with CD4(+) T-cell depletion in human lymphoid tissue ex vivo

The human immunodeficiency virus type 1 (HIV-1) Nef protein is an important virulence factor. Nef has several functions, including down-modulation of CD4 and class I major histocompatibility complex cell surface expression, enhancement of virion infectivity, and stimulation of viral replication in peripheral blood mononuclear cells. Nef also increases HIV-1 replication in human lymphoid tissue (HLT) ex vivo. We analyzed recombinant and primary nef alleles with highly divergent activity in different in vitro assays to clarify which of these Nef activities are functionally linked. Our results demonstrate that Nef activity in CD4 down-regulation correlates significantly with the efficiency of HIV-1 replication and with the severity of CD4(+) T-cell depletion in HLT. In conclusion, HIV-1 Nef variants with increased activity in CD4 down-modulation would cause severe depletion of CD4(+) T cells in lymphoid tissues and accelerate AIDS progression.     

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.     

Contribution of Vpu, Env, and Nef to CD4 down-modulation and resistance of human immunodeficiency virus type 1-infected T cells to superinfection

Human immunodeficiency virus type 1 (HIV-1) utilizes Vpu, Env, and Nef to down-modulate its primary CD4 receptor from the cell surface, and this function seems to be critical for the pathogenesis of AIDS. The physiological relevance of CD4 down-modulation, however, is currently not well understood. In the present study, we analyzed the kinetics of CD4 down-modulation and the susceptibility of HIV-1-infected T cells to superinfection using proviral HIV-1 constructs containing individual and combined defects in vpu, env, and nef and expressing red or green fluorescent proteins. T cells infected with HIV-1 mutants containing functional nef genes expressed low surface levels of CD4 from the first moment that viral gene expression became detectable. In comparison, Vpu and Env had only minor to moderate effects on CD4 during later stages of infection. Consistent with these quantitative differences, Nef inhibited superinfection more efficiently than Vpu and Env. Notably, nef alleles from AIDS patients were more effective in preventing superinfection than those derived from a nonprogressor of HIV-1 infection. Our data suggest that protection against X4-tropic HIV-1 superinfection involves both CD4-independent and CD4-dependent mechanisms of HIV-1 Nef. X4 was effectively down-regulated by simian immunodeficiency virus and HIV-2 but not by HIV-1 Nef proteins. Thus, maximal protection seems to involve an as-yet-unknown mechanism that is independent of CD4 or coreceptor down-modulation. Finally, we demonstrate that superinfected primary T cells show enhanced levels of apoptosis. Accordingly, one reason that HIV-1 inhibits CD4 surface expression and superinfection is to prevent premature cell death in order to expand the period of effective virus production.     

Modulation of different human immunodeficiency virus type 1 Nef functions during progression to AIDS

The human immunodeficiency virus type 1 (HIV-1) Nef protein has several independent functions that might contribute to efficient viral replication in vivo. Since HIV-1 adapts rapidly to its host environment, we investigated if different Nef properties are associated with disease progression. Functional analysis revealed that nef alleles obtained during late stages of infection did not efficiently downmodulate class I major histocompatibility complex but were highly active in the stimulation of viral replication. In comparison, functional activity in downregulation of CD4 and enhancement of HIV-1 infectivity were maintained or enhanced after AIDS progression. Our results demonstrate that various Nef activities are modulated during the course of HIV-1 infection to maintain high viral loads at different stages of disease progression. These findings suggest that all in vitro Nef functions investigated contribute to AIDS pathogenesis and indicate that nef variants with increased pathogenicity emerge in a significant number of HIV-1-infected individuals.     

Down-modulation of CD8αβ is a fundamental activity of primate lentiviral Nef proteins

It is well established that the Nef proteins of human and simian immunodeficiency viruses (HIV and SIV) modulate major histocompatibility complex class I (MHC-I) cell surface expression to protect infected cells against lysis by cytotoxic T lymphocytes (CTLs). Recent data supported the observation that Nef also manipulates CTLs directly by down-modulating CD8αβ (J. A. Leonard, T. Filzen, C. C. Carter, M. Schaefer, and K. L. Collins, J. Virol. 85:6867-6881, 2011), but it remained unknown whether this Nef activity is conserved between different lineages of HIV and SIV. In this study, we examined a total of 42 nef alleles from 16 different primate lentiviruses representing most major lineages of primate lentiviruses, as well as nonpandemic HIV-1 strains and the direct precursors of HIV-1 (SIVcpz and SIVgor). We found that the vast majority of these nef alleles strongly down-modulate CD8β in human T cells. Primate lentiviral Nefs generally interacted specifically with the cytoplasmic tail of CD8β, and down-modulation of this receptor was dependent on the conserved dileucine-based motif and two adjacent acidic residues (DD/E) in the C-terminal flexible loop of SIV Nef proteins. Both of these motifs are known to be important for the interaction of HIV-1 Nef with AP-2, and they were also shown to be critical for down-modulation of CD4 and CD28, but not MHC-I, by SIV Nefs. Our results show that down-modulation of CD4, CD8β, and CD28 involves largely overlapping (but not identical) domains and is most likely dependent on conserved interactions of primate lentiviral Nefs with cellular adaptor proteins. Furthermore, our data demonstrate that Nef-mediated down-modulation of CD8αβ is a fundamental property of primate lentiviruses and suggest that direct manipulation of CD8+ T cells plays a relevant role in viral immune evasion.     

Lentiviral vectors interfering with virus-induced CD4 down-modulation potently block human immunodeficiency virus type 1 replication in primary lymphocytes

CD4 down-modulation is essential for the production of human immunodeficiency virus (HIV) infectious particles. Disease progression correlates with enhanced viral induced CD4 down-modulation, and a subset of long-term nonprogressors carry viruses defective in this function. Despite multiple pieces of evidence highlighting the importance of this function in viral pathogenesis in vivo, to date, HIV-induced CD4 down-modulation has not been used as a target for intervention. We describe here HIV-based vectors that deliver truncated CD4 molecules resistant to down-modulation by the viral products Nef and Vpu. Infection of cells previously transduced with these vectors proceeded normally, and viral particles were released in normal amounts. However, the infectivity of the released virions was reduced 1,000-fold. Lentiviral vectors expressing truncated CD4 molecules were efficient at blocking HIV-1 infectivity and replication in several cell lines and in CD4-positive primary lymphocytes. The findings presented here provide proof-of-principle that approaches targeting the virus-induced CD4 down-modulation may constitute the basis for novel anti-HIV therapies.     

Nef-mediated downregulation of CD4 enhances human immunodeficiency virus type 1 replication in primary T lymphocytes

The accessory protein Nef plays a crucial role in primate lentivirus pathogenesis. Nef enhances human immunodeficiency virus type 1 (HIV-1) infectivity in culture and stimulates viral replication in primary T cells. In this study, we investigated the relationship between HIV-1 replication efficiency in CD4(+) T cells purified from human blood and two various known activities of Nef, CD4 downregulation and single-cycle infectivity enhancement. Using a battery of reporter viruses containing point mutations in nef, we observed a strong genetic correlation between CD4 downregulation by Nef during acute HIV-1 infection of activated T cells and HIV-1 replication efficiency in T cells. In contrast, HIV-1 replication ability was not significantly correlated with the ability of Nef to enhance single-cycle virion infectivity, as determined by using viruses produced in cells lacking CD4. These results demonstrate that CD4 downregulation by Nef plays a crucial role in HIV-1 replication in activated T cells and underscore the potential for the development of therapies targeting this conserved activity of Nef.     

Nef-mediated enhancement of virion infectivity and stimulation of viral replication are fundamental properties of primate lentiviruses

Nef is a multifunctional accessory protein of primate lentiviruses. Recently, it has been shown that the ability of Nef to downmodulate CD4, CD28, and class I major histocompatibility complex is highly conserved between most or all primate lentiviruses, whereas Nef-mediated downregulation of T-cell receptor-CD3 was lost in the lineage that gave rise to human immunodeficiency virus type 1 (HIV-1). Whether or not other Nef activities are preserved between different groups of primate lentiviruses remained to be determined. Here, we show that nef genes from a large variety of HIVs and simian immunodeficiency viruses (SIVs) enhance virion infectivity and stimulate viral replication in human cells and/or in ex vivo infected human lymphoid tissue (HLT). Notably, nef alleles from unpassaged SIVcpz and SIVsmm enhanced viral infectivity, replication, and cytopathicity in cell culture and in ex vivo infected HLT as efficiently as those from HIV-1 and HIV-2, their human counterparts. Furthermore, nef genes from several highly divergent SIVs that have not been found in humans were also highly active in human cells and/or tissues. Thus, most primate lentiviral Nefs enhance virion infectivity and stimulate viral replication. Moreover, our data show that SIVcpz and SIVsmm Nefs do not require adaptive changes to perform these functions in human cells or tissues and support the idea that nef alleles from other primate lentiviruses would also be capable of promoting efficient virus spread in humans.     

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.     

CD4 down-modulation during infection of human T cells with human immunodeficiency virus type 1 involves independent activities of vpu, env, and nef.

The human immunodeficiency virus type 1 (HIV-1) genes vpu, env, and nef have all been implicated in modulating the levels of cell surface CD4 on infected cells. To quantitatively assess the relative contribution of each gene product to the regulation of CD4 during HIV infection of Jurkat T cells and peripheral blood mononuclear cells, we have developed an infectious HIV reporter system which expresses different combinations of these genes. To distinguish infected cells in the early or late stages of infection from uninfected cells, these viruses were designed to express human placental alkaline phosphatase with the kinetics of either early or late viral genes. Flow cytometry to detect placental alkaline phosphatase and CD4 in infected cells showed that vpu, env, and nef are independently capable of down-modulation of CD4. As predicted by their respective expression patterns, nef down-modulated CD4 rapidly during the early phase of virus infection whereas vpu and env functioned late in the infection. In both Jurkat cells and peripheral blood mononuclear cells, a combination of the three genes was more efficient than any one or two genes, demonstrating that all three genes are required to achieve maximal CD4 down-modulation. In primary cells, down-modulation of CD4 was less efficient than in Jurkat cells and there was a stronger dependence on nef function for reducing cell surface CD4. HIV therefore has three genes that are able to independently down-modulate CD4; together, they can eliminate the bulk of cell surface CD4.     

The human immunodeficiency virus type 1 nef gene can to a large extent replace simian immunodeficiency virus nef in vivo.

The nef gene of the pathogenic simian immunodeficiency virus (SIV) 239 clone was replaced with primary human immunodeficiency virus type 1 (HIV-1) nef alleles to investigate whether HIV-1 Nef can substitute for SIV Nef in vivo. Initially, two rhesus macaques were infected with the chimeric viruses (Nef-SHIVs). Most of the nef alleles obtained from both animals predicted intact open reading frames. Furthermore, forms containing upstream nucleotide substitutions that enhanced expression of the inserted gene became predominant. One animal maintained high viral loads and slowly progressed to immunodeficiency. nef long terminal repeat sequences amplified from this animal were used to generate a second generation of Nef-SHIVs. Two macaques, which were subsequently infected with a mixture of cloned chimeric viruses, showed high viral loads and progressed to fatal immunodeficiency. Five macaques received a single molecular clone, named SHIV-40K6. The SHIV-40K6 nef allele was active in CD4 and class I major histocompatibility complex downregulation and enhanced viral infectivity and replication. Notably, all of the macaques inoculated with SHIV-40K6 showed high levels of viral replication early in infection. During later stages, however, the course of infection was variable. Three animals maintained high viral loads and developed immunodeficiency. Of the remaining two macaques, which showed decreasing viral loads after the acute phase of infection, only one efficiently controlled viral replication and remained asymptomatic during 1.5 years of follow-up. The other animal showed an increasing viral load and developed signs of progressive infection during later stages. Our data demonstrate that HIV-1 nef can, to a large extent, functionally replace SIVmac nef in vivo.     

Altered T cell activation and development in transgenic mice expressing the HIV-1 nef gene.

The nef gene, which encodes related cytoplasmic proteins in both human (HIV) and simian (SIV) immunodeficiency viruses is dispensable for viral replication in vitro. In contrast, in vivo experiments have revealed that SIV nef is required for efficient viral replication and development of AIDS in SIV infected rhesus monkeys, thus indicating that nef plays an essential role in the natural infection. We show that expression of the Nef protein from the HIV-1 NL43 isolate in transgenic mice perturbs development of CD4+ T cells in the thymus and elicits depletion of peripheral CD4+ T cells. Thymic T cells expressing NL43 Nef show altered activation responses. In contrast, Nef protein of the HIV-1 HxB3 isolate does not have an overt effect on T cells when expressed in transgenic animals. The differential effects of the two HIV-1 nef alleles in transgenic mice correlate with down-regulation of CD4 antigen expression on thymic T cells. The differential interactions of the NL43 and HxB3 nef alleles with CD4 were reproduced in a transient assay in human CD4+ CEM T cells. Down-regulation of CD4 by nef in both human and transgenic murine T cells indicates that the relevant interactions are conserved in these two systems and suggests that the consequences of Nef expression on the host cell function can be analyzed in vivo in the murine system. Our observations from transgenic mice suggest that nef-elicited perturbations in T cell signalling play an important role in the viral life cycle in vivo, perhaps resulting in elimination of infected CD4+ T cells.     

Cell surface CD4 interferes with the infectivity of HIV-1 particles released from T cells.

The CD4 protein is required for the entry of human immunodeficiency virus (HIV) into target cells. Upon expression of the viral genome, three HIV-1 gene products participate in the removal of the primary viral receptor from the cell surface. To investigate the role of surface-CD4 in HIV replication, we have created a set of Jurkat cell lines which constitutively express surface levels of CD4 comparable to those found in peripheral blood lymphocytes and monocytes. Expression of low levels of CD4 on the surface of producer cells exerted an inhibitory effect on the infectivity of HIV-1 particles, whereas no differences in the amount of cell-free p24 antigen were observed. Higher levels of cell surface CD4 exerted a stronger inhibitory effect on infectivity, and also affected the release of free virus in experiments where the viral genomes were delivered by electrotransfection. The CD4-mediated inhibition of HIV-1 infectivity was not observed in experiments where the vesicular stomatitis virus G protein was used to pseudotype viruses, suggesting that an interaction between CD4 and gp120 is required for interference. In contrast, inhibition of particle release by high levels of cell-surface CD4 was not overcome by pseudotyping HIV-1 with foreign envelope proteins. Protein analysis of viral particles released from HIV-infected Jurkat-T cells revealed a CD4-dependent reduction in the incorporation of gp120. These results demonstrate that physiological levels of cell-surface CD4 interfere with HIV-1 replication in T cells by a mechanism that inhibits envelope incorporation into viral membranes, and therefore provide an explanation for the need to down-modulate the viral receptor in infected cells. Our findings have important implications for the spread of HIV in vivo and suggest that the CD4 down-modulation function may be an alternative target for therapeutic intervention.     

Inefficient enhancement of viral infectivity and CD4 downregulation by human immunodeficiency virus type 1 Nef from Japanese long-term nonprogressors

It has been reported that patients infected with nef-defective human immunodeficiency virus type 1 (HIV-1) do not progress to AIDS; however, mutations that abrogate Nef expression are not common in long-term nonprogressors (LTNPs). We postulated that Nef function might be impaired in LTNPs, irrespective of the presence or absence of detectable amino acid sequence anomalies. To challenge this hypothesis we compared in vitro function of nef alleles that were derived from three groups of Japanese patients: LTNPs, progressors, and asymptomatic carriers (ACs). The patient-derived nef alleles were subcloned into a nef-defective infectious HIV-1 molecular clone and an expression vector. We first examined Nef-dependent enhancement of infection in a single-round infectivity assay by the use of MAGNEF cells, in which Nef is required more strictly for the infection than in the parent MAGI cells. All nef alleles from LTNPs showed reduced enhancement in the infectivity of nef-defective HIV-1 mutants compared to the nef alleles of progressors or ACs. Second, we found that nef alleles from LTNPs were less efficient in CD4 downregulation than those of progressors or ACs. Third, all nef alleles from LTNPs, progressors, and ACs reduced the cell surface expression of major histocompatibility complex class I to a similar level. Last, there was no correlation between Hck-binding activity of Nef and clinical grouping. In conclusion, we detected inefficient enhancement of HIV-1 infectivity and CD4 downregulation by HIV-1 nef alleles of LTNPs. It awaits further study to conclude that these characteristics of nef alleles are the cause or the consequence of the long-term nonprogression after HIV-1 infection.     

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.     

Nef proteins from simian immunodeficiency virus-infected chimpanzees interact with p21-activated kinase 2 and modulate cell surface expression of various human receptors

The accessory Nef protein allows human immunodeficiency virus type 1 (HIV-1) to persist at high levels and to cause AIDS in infected humans. The function of HIV-1 group M subtype B nef alleles has been extensively studied, and a variety of in vitro activities believed to be important for viral pathogenesis have been established. However, the function of nef alleles derived from naturally simian immunodeficiency virus (SIV)-infected chimpanzees, the original host of HIV-1, or from the HIV-1 N and O groups resulting from independent zoonotic transmissions remains to be investigated. In the present study we demonstrate that SIVcpz and HIV-1 group N or O nef alleles down-modulate CD4, CD28, and class I or II MHC molecules and up-regulate surface expression of the invariant chain (Ii) associated with immature major histocompatibility complex (MHC) class II. Furthermore, the ability of Nef to interact with the p21-activated kinase 2 was generally conserved. The functional activity of HIV-1 group N and O nef genes did not differ significantly from group M nef alleles. However, SIVcpz nef genes as a group showed a 1.8- and 2.0-fold-higher activity in modulating CD28 (P = 0.0002) and Ii (P = 0.016) surface expression, respectively, but were 1.7-fold less active in down-regulating MHC class II molecules (P = 0.006) compared to HIV-1 M nef genes. Our finding that primary SIVcpz nef alleles derived from naturally infected chimpanzees modulate the surface expression of various human cellular receptors involved in T-cell activation and antigen presentation suggests that functional nef genes helped the chimpanzee virus to persist efficiently in infected humans immediately after zoonotic transmission.     

Vpu exerts a positive effect on HIV-1 infectivity by down-modulating CD4 receptor molecules at the surface of HIV-1-producing cells

Human immunodeficiencey virus, type 1 (HIV-1) encodes three proteins, Nef, Vpu, and gp160, that down-modulate surface expression of the CD4 receptor during viral infection. In the present study, we have investigated the role of CD4 down-modulation in the HIV-1 infection cycle, primarily from the perspective of Vpu function. We report here that, like Nef, Vpu-mediated CD4 degradation modulates positively HIV-1 infectivity. Our data reveal that accumulation of CD4 at the cell surface of Vpu-deficient HIV-1-producing cells leads to an efficient recruitment of CD4 into virions and to an impairment of viral infectivity. This CD4-mediated inhibition of viral infectivity was not observed when a CD4 mutant unable to bind Env gp120 was used or when VSV-G glycoprotein was utilized to pseudotype viruses, suggesting that an interaction between CD4 and gp120 is required for interference. Indeed, protein analysis of Vpu-defective viral particles reveals that CD4 recruitment is associated with an increased formation of gp120-CD4 complexes at the virion surface. Interestingly, we did not detect any difference at the level of total virion-associated Env glycoproteins between wild-type and Vpu-defective virus, indicating that accumulation of CD4 at the cell surface and recruitment of CD4 into Vpu-defective HIV-1 particles exert a negative effect on viral infectivity, most likely by promoting the formation of nonfunctional gp120-CD4 complexes at the virion surface. Finally, we show that both Vpu- and Nef-induced CD4 down-modulation activities are required for production of fully infectious particles in CD4+ T cell lines and primary cells, an observation that has clear implications for viral spread in vivo.     

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.     

Nef alleles from human immunodeficiency virus type 1-infected long-term-nonprogressor hemophiliacs with or without late disease progression are defective in enhancing virus replication and CD4 down-regulation

Infection with human immunodeficiency virus (HIV)-encoding defective nef variants may contribute to a relatively benign course of disease in a minority of long-term nonprogressors (LTNP). We have examined the functions of nef alleles from six individuals belonging to the same cohort of hemophiliacs infected with HIV-1 prior to 1985 and classified as LTNP in 1995. Three out of six individuals have progressed to HIV disease (late progressors [LP]), whereas the three remainders have maintained their LTNP status at least up to 2003. The nef alleles were obtained from both plasma virus and peripheral blood mononuclear cells of all six individuals in 1995 and 1998. The proportion of sequences containing mutations not yielding Nef expression significantly diminished in 1998 versus that in 1995. Several previously defined functional regions of intact nef alleles were highly conserved. However, the major variant obtained in 1998 from plasma RNA of five out of six individuals significantly reduced HIV infectivity/replication and impaired Nef-mediated CD4 but not major histocompatibility complex class I antigen down-modulation from the cell surface. Thus, functional alterations of the nef gene are present in both LP and LTNP, suggesting that Nef defectiveness in vitro is not necessarily associated with the long-term maintenance of LTNP status. Of interest is the fact that isolates from three out of three LP showed a dual CCR5/CXCR4 coreceptor use (R5X4), in contrast to those from LTNP, which were exclusively R5. Thus, in vivo evolution of gp120 Env to CXCR4 use appears to be associated with HIV disease progression in individuals infected with nef-defective viruses.