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SERINC5 is a host restriction factor that impairs infectivity of HIV-1 and other primate lentiviruses and is counteracted by the viral accessory protein Nef. However, the importance of SERINC5 antagonism for viral replication and cytopathicity remained unclear. Here, we show that the Nef protein of the highly divergent SIVcol lineage infecting mantled guerezas (Colobus guereza) is a potent antagonist of SERINC5, although it lacks the CD4, CD3 and CD28 down-modulation activities exerted by other primate lentiviral Nefs. In addition, SIVcol Nefs decrease CXCR4 cell surface expression, suppress TCR-induced actin remodeling, and counteract Colobus but not human tetherin. Unlike HIV-1 Nef proteins, SIVcol Nef induces efficient proteasomal degradation of SERINC5 and counteracts orthologs from highly divergent vertebrate species, such as Xenopus frogs and zebrafish. A single Y86F mutation disrupts SERINC5 and tetherin antagonism but not CXCR4 down-modulation by SIVcol Nef, while mutation of a C-proximal di-leucine motif has the opposite effect. Unexpectedly, the Y86F change in SIVcol Nef had little if any effect on viral replication and CD4+ T cell depletion in preactivated human CD4+ T cells and in ex vivo infected lymphoid tissue. However, SIVcol Nef increased virion infectivity up to 10-fold and moderately increased viral replication in resting peripheral blood mononuclear cells (PBMCs) that were first infected with HIV-1 and activated three or six days later. In conclusion, SIVcol Nef lacks several activities that are conserved in other primate lentiviruses and utilizes a distinct proteasome-dependent mechanism to counteract SERINC5. Our finding that evolutionarily distinct SIVcol Nefs show potent anti-SERINC5 activity supports a relevant role of SERINC5 antagonism for viral fitness in vivo. Our results further suggest this Nef function is particularly important for virion infectivity under conditions of limited CD4+ T cell activation., Competing Interests: The authors have declared that no competing interests exist.

The Nef protein is an essential factor for lentiviral pathogenesis in humans and other simians. Despite a multitude of functions attributed to this protein, the exact role of Nef in disease progression remains unclear. One of its most intriguing functions is the ability of Nef to enhance the infectivity of viral particles. In this review we will discuss current insights in the mechanism of this well-known, yet poorly understood Nef effect. We will elaborate on effects of Nef, on both virion biogenesis and the early stage of the cellular infection, that might be involved in infectivity enhancement. In addition, we provide an overview of different HIV-1 Nef domains important for optimal infectivity and briefly discuss some possible sources of the frequent discrepancies in the field. Hereby we aim to contribute to a better understanding of this highly conserved and therapeutically attractive Nef function.

Herpesvirus saimiri (HVS)-transformed human T cells expressing terminal membrane proteins (TMPs) tyrosine kinase interacting protein (Tip) and saimiri transformation associated protein strain C (StpC) are highly permissive for R5 and X4 strains of HIV-1. StpC expression enhances replication of R5 and X4 strains of HIV-1 and induces latent reservoirs of replication competent HIV-1 in cell lines derived from T cells or monocytes. Paradoxically Tip expression restricts replication and cytopathic effects of R5 and X4 strains of HIV-1 in T cells and monocytes post-retrotransposition. Understanding the canonical pathways whereby Tip and StpC alter HIV-1 replication may uncover novel therapeutic approaches to HIV-1 infection. Here we show Tip inhibits Tat-mediated transcriptional activation of the long terminal repeat (LTR). Tip mediated inhibition of Tat transactivation is reversed by Nef. Tip also mediates restriction of late-stage replication of HIV-1 by disrupting Nef interaction with lymphocyte-specific protein-tyrosine kinase (Lck) in lipid rafts. Specifically, in the presence of Tip, Lck does not localize to lipid rafts reducing Nef interaction with Lck within the lipid rafts. Finally, the permissive phenotype conferred by StpC is the result of synergy with Tat during transcriptional activation of the HIV-1 LTR. This transcriptional synergy between StpC and Tat requires Lck and NF-kappaB consensus binding sequences. These findings demonstrate that the HVS TMPs influence transcriptional and post-transcriptional stages in HIV-1 replication. We propose that HVS-encoded TMPs associated with T cell transformation have evolved ability to modulate the replication of competing retroviruses. Gene based approaches utilizing Tip and StpC may provide therapeutic models for treating acute and latent HIV-1 infections, respectively.

The human immunodeficiency virus-1 (HIV-1) Nef protein, originally identified as a negative factor, has now emerged as one of the most important viral proteins necessary for viral pathogenesis and disease progression. Nef has been also implicated in viral infectivity and replication, however, the molecular mechanism of Nef-induced viral gene expression and replication is not clearly understood. Although involvement of heat shock proteins in viral pathogenesis has been reported earlier, a clear understanding of their role remains to be elucidated. Here we report for the first time that Nef not only interacts with heat shock protein 40 (Hsp40) but it also induces the expression of Hsp40 in HIV-1-infected cells. The interaction between Nef and Hsp40 is important for increased Hsp40 translocation into the nucleus of infected cells, which seems to facilitate viral gene expression by becoming part of the cyclin-dependent kinase 9-associated transcription complex regulating long terminal repeat-mediated gene expression. The finding is consistent with the failure of the nef-deleted virus to induce Hsp40, resulting in reduced virus production. Our data further shows that, whereas, Hsp40 overexpression induces viral gene expression, silencing of Hsp40 reduces the gene expression in a Nef-dependent manner. Thus our results clearly indicate that Hsp40 is crucial for Nef-mediated enhancement of viral gene expression and replication.

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.

The relevance of the accessory vpr, vpu, and nef genes for human immunodeficiency virus type 1 (HIV-1) replication in human lymphoid tissue (HLT), the major site of viral replication in vivo, is largely unknown. Here, we show that an individual deletion of nef, vpr, or vpu significantly decreases HIV-1 replication and prevents CD4+ T-cell depletion in ex vivo HLT. However, only combined defects in all three accessory genes entirely disrupt the replicative capacity of HIV-1. Our results demonstrate that nef, vpr, and vpu are all essential for efficient viral spread in HLT, suggesting an important role in AIDS pathogenesis.

The human immunodeficiency virus type 1 (HIV-1) Nef protein has been shown to accelerate viral growth kinetics in primary human T-lymphocytes and macrophages; however, the specific function(s) of Nef responsible for this phenotype in macrophages is unknown. To address this issue, mutants of a molecularly cloned macrophage-tropic isolate, HIV-1(SF162), were generated expressing single point mutations that abrogate the ability of Nef to interact with cellular kinases or mediate CD4 down-regulation. Infection of primary monocyte-derived macrophages (MDM) with these mutant viruses revealed that residues in the PXXP motif contribute to efficient replication. Interestingly, viruses expressing alleles of Nef defective in CD4 down-modulation activity retain wild-type levels of infectivity in single-round assays but exhibited delayed replication kinetics and grew to lower titres compared to the wild-type virus in MDM. These data suggest that efficient HIV-1 replication is dependent on the ability of Nef to interact with cellular kinases and remove CD4 from the surface of infected macrophages.

The live, attenuated vaccine simian immunodeficiency virus SIVmac239Deltanef efficiently protects rhesus macaques against infection with wild-type SIVmac but occasionally causes CD4(+) T-cell depletion and progression to simian AIDS (SAIDS). Virus recovered from a vaccinated macaque (Rh1490) that progressed to SAIDS had acquired an additional deletion in the nef gene, resulting in a frameshift that restored the original nef open reading frame (R. I. Connor, D. C. Montefiori, J. M. Binley, J. P. Moore, S. Bonhoeffer, A. Gettie, E. A. Fenamore, K. E. Sheridan, D. D. Ho, P. J. Dailey, and P. A. Marx, J. Virol. 72:7501-7509, 1998). Intravenous inoculation of the Rh1490 viral isolate into four naive rhesus macaques induced CD4(+) T-cell depletion and disease in three out of four animals within 2 years, indicating a restoration of virulence. A DNA fragment encompassing the truncated nef gene amplified from the Rh1490 isolate was inserted into the genetic backbone of SIVmac239. The resulting clone, SIVmac239-Delta2nef, expressed a Nef protein of approximately 23 kDa, while the original SIVmac239Deltanef clone expressed a shorter protein of 8 kDa. The revertant form of Nef did not cause downregulation of CD4, CD3, or major histocompatibility complex class I. The infectivity of SIVmac239-Delta2nef was similar to that of SIVmac239Deltanef in single-cycle assays using indicator cell lines. In contrast, SIVmac239-Delta2nef replicated more efficiently than SIVmac239Deltanef in peripheral blood mononuclear cell (PBMC) cultures infected under unstimulated conditions. The p27 Gag antigen levels in SIVmac239-Delta2nef-infected cultures were still lower than those obtained with wild-type SIVmac239, consistent with a partial recovery of Nef function. The transcriptional activity of long terminal repeat (LTR)-luciferase constructs containing the nef deletions did not differ markedly from that of wild-type LTR. Introduction of a premature stop codon within Nef-Delta2 abolished the replicative advantage in PBMCs, demonstrating that the Nef-Delta2 protein, rather than the structure of the U3 region of the LTR, was responsible for the increase in viral replication. Taken together, these results show that SIV with a deletion in the nef gene can revert to virulence and that expression of a form of nef with multiple deletions may contribute to this process by increasing viral replication.

In contrast to most animal viruses, infection with the human and simian immunodeficiency viruses results in prolonged, continuous viral replication in the infected host. Remarkably, viral persistence is not thwarted by the presence of apparently vigorous, virus-specific immune responses. Several factors are thought to contribute to persistent viral replication, most notably the destruction of virus-specific T helper cells, the emergence of antigenic escape variants, and the expression of an envelope complex that structurally minimizes antibody access to conserved epitopes. Not as well understood, though potentially important, is the ability of at least one viral encoded protein (Nef) to prevent presentation of viral antigens in the context of major histocompatibility complex. The future success of antiviral therapies and vaccination strategies may depend largely on understanding how and to what degree each of these factors (and presumably others) contributes to immune evasion.

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.

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.

The ability of human immunodeficiency virus type-1 (HIV-1) to establish a persistent infection is critically dependent on the cellular signals that regulate HIV-1 replication within target cells. The balance between numerous host factors that either enhance or suppress viral infection determines the clinical outcome. Perturbation of the steady-state level of viral replication can significantly influence the course and the speed at which the infection develops into clinical disease. Activation signals delivered to T cells by cytokines and antigen-presenting cells (APC), are key modulators of viral replication. Our laboratory seeks to decipher how HIV-1 exploits T cell signaling mechanisms and host factors that regulate viral replication. Elucidation of the molecular mechanisms by which cellular signals regulate the HIV-1 life cycle within target cells will significantly advance our understanding of host-virus interactions.

Although initially recognised as essential for the entry of certain viruses, endocytosis is now known to also play important roles in the replication of, and adaptation to, the host cell of a number of viruses. Here we consider several aspects of this association and recent results that have emerged to support this view.

SIVmac Nef contains two N-terminal tyrosines that were proposed to be part of an SH2-ligand domain and/or a tyrosine-based endocytosis signal and a putative SH3-ligand domain (P(104)xxP(107)). In the present study, we investigated the effects of combined mutations in these tyrosine and proline residues on simian immunodeficiency virus (SIV) Nef interactions with the cellular signal transduction and endocytic machinery. We found that mutation of Y(28)F, Y(39)F, P(104)A, and P(107)A (FFAA-Nef) had little effect on Nef functions such as the association with the cellular tyrosine kinase Src, downregulation of cell surface expression of CD4 and class I major histocompatibility complex, and enhancement of virion infectivity. However, mutations in the PxxP sequence reduced the ability of Nef to stimulate viral replication in primary lymphocytes. Three macaques infected with the SIVmac239 FFAA-Nef variant showed high viral loads during the acute phase of infection. Reversions in the mutated prolines were observed between 12 and 20 weeks postinfection. Importantly, reversion of A(107)-->P, which restored the ability of Nef to coprecipitate a 62-kDa phosphoprotein in in vitro kinase assays, did not precede the development of a high viral load. The Y(28)/Y(39)-->F(28)/F(39) substitutions did not revert. In conclusion, mutations in both the tyrosine residues and the putative SH3 ligand domain apparently do not disrupt major aspects of SIV Nef function in vivo.

The primate lentiviruses encode a protein, Nef, which is required for efficient viral replication in their host. Several biological activities of nef identified in vitro may contribute to this requirement in vivo, including receptor modulation and interference with cellular signaling pathways. We show that HIV- and SIV-encoded Nef can enhance virus production within a single viral replication cycle, not only by increasing viral infectivity, as previously reported, but also by acting through the efficiency of viral transcription and of viral release., (Copyright 1999 Academic Press.)

The stage in the human immunodeficiency virus type 1 (HIV-1) replication cycle that is affected by Nef has been investigated. Nef is dispensable during the transcription to virion production step. In contrast, Nef is critical in the early phase of the virus replication cycle, from virion adsorption to integration. Furthermore, virions produced in the absence of Nef have been demonstrated to be inefficient to complete the virus DNA synthesis. When virions are produced in particular cells without Nef, they exhibit a severe defect in the virus entry process into cells. Taken together, it is concluded that HIV-1 Nef acts via modulation of viral particles to enhance virus infectivity in a cell-dependent manner.

A proviral nef gene mutant of human immunodeficiency virus type 1 (HIV-1) was evaluated for its defective early replication step. Virus stocks were prepared from six CD4-positive and -negative cell lines transfected with wild-type (wt) or the nef mutant clone and inoculated into two target CD4-positive cell lines to monitor the efficiency of viral entry process. The nef mutant virions produced in one cell line exhibited a severe defect in the entry process, although those produced in the other five cell lines were only slightly less efficient than the wt virions at entering into cells. These results have demonstrated that the HIV-1 Nef is critical for efficient viral entry in a producer cell-dependent manner., (Copyright 1998 Academic Press.)

Human immunodeficiency virus type 1 (HIV-1) accessory genes including nef, vif, and vpr are important factors that determine the replication and pathogenesis of HIV-1. The state of activation is also important for the replication of HIV-1. We evaluated the properties of nef-, vif-, and vpr-minus macrophage-tropic HIV-1(JR) CSF in primary CD4+ Th1- or Th2-like cell cultures which had been activated through CD3 molecules in the presence of interleukin-2 (IL-2) and IL-12 (Th1-like culture) or IL-4 (Th2-like culture), respectively. In activated Th1- or Th2-like cultures, replication of nef-minus HIV-1(JR-CSF) was markedly lower than that of wild-type HIV-1. Subsequent analysis by site-directed mutagenesis showed that (i) the presence of an acidic amino acid-rich domain (amino acid residues 72 to 75) in the Nef protein was critical for the enhancement of viral DNA synthesis, resulting in increased virus growth rate, and (ii) prolines that form part of Src homology 3 binding domain were not essential for viral replication. We also confirmed the importance of sites by using an HIV-1-infected animal model, the hu-PBL-SCID mouse system, representing HIV-1 replication and pathogenesis in activated CD4+ T cells in vivo. These results indicate that Nef accelerates viral replication in activated CD4+ T cells.

Wild-type HIV-1 is more infectious than nef-deleted HIV-1 in both limiting dilution and single-cycle infectivity assays. Moreover, Nef expression from a separate plasmid in the virus-producing cells is capable of restoring the infectivity of genetically nef-deficient HIV-1. These observations indicate that the virion itself is altered by Nef expression to promote viral infectivity. Sucrose gradient-purified HIV-1 virions contain full-length Nef protein and its inclusion is dependent on N-terminal myristylation of Nef. As myristylation-defective mutants of Nef do not enhance infectivity, incorporation of Nef into virions may mediate the enhanced infectivity. Studies with recombinant Nef have further shown that HIV-1 protease can cleave Nef into two polypeptides, a 20-kDa C-terminal core domain and a small N-terminal domain. Our analysis of purified HIV-1 virions also showed a 20-kDa form of Nef. The generation of this 20-kDa form of Nef was inhibited by an HIV-1 protease inhibitor, and its C-terminal core domain identity was confirmed through epitope-tagging. Immunoblots of virions demonstrated that 60-80% of the incorporated Nef is cleaved by the HIV-1 protease. This finding raised the possibility that the Nef core domain, which may no longer be tethered to the membrane due to absence of an N-terminal myristyl anchor, might mediate the enhanced infectivity. Therefore, a panel of mutants surrounding the proteolytic cleavage site in Nef were analyzed for effects on cleavage and enhancement of viral infectivity. Although some Nef mutants both failed to cleave and did not enhance viral infectivity, other mutants proved discordant in these functions. Specifically, two mutants that contained point mutations in the N-terminal domain cleaved normally, hence generating wild-type Nef core domain, yet failed to enhance infectivity. Thus, although the majority of the Nef protein in HIV-1 virions is cleaved by the viral protease into a 20-kDa C-terminal core domain, generation of this core domain of Nef appears insufficient to enhance HIV-1 infectivity. These findings suggest that protease cleavage of the Nef protein in virions is irrelevant for the infectivity function of Nef.

We comparatively analyzed the replication kinetics of wild-type (wt) and nef mutant human immunodeficiency virus type 1 (HIV-1) in several CD4-positive cell lines, in order to clarify the molecular function of Nef protein. The delayed growth of nef mutant virus was observed at the initial stage of replication in all cell lines examined. This phenomenon was greatly amplified in the absence of vpu gene. In order to determine the infection stage in viral replication cycle which is specifically affected on virus replication rate in the presence of the Nef protein, we first examined the difference between wt and nef mutant viruses in the virus production rate from transfected cells, and found that the both viruses were produced with equal efficiency. This result showed that Nef protein could be dispensable for virion production. Therefore, early infection stages were focused by single-round infection assay, and the nef mutant virus was found to be much less infectious than wt virus. This indicated that the effect of Nef protein was exhibited in the early phase of a virus replication cycle, during viral adsorption to integration. By entry assay using wt and nef mutant virions, it was revealed that the Nef protein was required for efficient viral entry. These data suggest that the Nef protein might play a role in efficient incorporation of the Env protein into the virions, leading to enhanced viral infectivity.

The nef genes of HIV and SIV are dispensable in vitro, but are essential for viral spread and disease progression in vivo. Nef-induced down-regulation of CD4, the viral receptor, may be the key to this requirement.

The nef gene is conserved among all human and simian lentiviruses. However, the amino acid similarity between simian immunodeficiency virus (SIV) and human immunodeficiency virus type 1 NEF is only 38%. To assess the role of SIV NEF on virus replication and compare its activity with that of its human immunodeficiency virus type 1 counterpart, we examined the activity of an intact nef gene from proviral clone pSIV 102, an isolate from SIV-MAC-251-infected cells. Proviral clone pSIV BA was constructed by introducing a premature termination codon at codon 40 of the nef gene without altering the predicted amino acid sequence of the overlapping env gene. These two clones were transfected into CD4- COS cells, and virus replication was monitored by p27 enzyme-linked immunosorbent assay kits. In seven independent experiments, clone pSIV BA afforded two- to sixfold greater levels of viral antigen compared with those in clone pSIV 102 and two- to sixfold-increased levels of viral mRNAs as indicated with Northern (RNA) blot and S1 nuclease protection analyses. Nuclear run-on assays demonstrated a two- to threefold increased rate of RNA synthesis with nuclei isolated from cells transfected with pSIV BA compared with that from cells transfected with pSIV 102. In contrast, there was no apparent destabilization of SIV mRNAs by NEF, as measured in dactinomycin-treated cells. This study demonstrates that SIV NEF is a negative regulator of virus replication and acts by suppressing the level of mRNA synthesis and accumulation in COS cells.

Stable lymphoid cell lines expressing the human immunodeficiency virus type 1 (HIV-1) nef gene product, p27, were established. The presence of p27 in the lymphoid cells suppressed replication of some strains of both HIV-1 and HIV-2. This observation indicates that nef could be important in the establishment of HIV latency. In contrast, fast replicating and highly cytopathic HIV-1 isolates recovered from patients with advanced disease states were not affected by the negative effect of nef present in these lymphoid cell lines. This lack of response to nef appears to constitute another viral feature that correlates with disease progression. Thus, manipulating expression of the nef gene in vivo might influence pathogenesis in the host.