Nef protein of human immunodeficiency virus type 1 binds its own myristoylated N-terminus

HIV-1 Nef is a small protein (approx. 25 kDa) that is posttranslationally modified by myristoylation. To explain its complex activities, a 'Nef-cycle' is discussed, which postulates different molecular conformations of Nef. Using recombinant full-length non-myristoylated Nef and synthetic peptides, we demonstrate by fluorescence titration experiments that a peptide representing the myristoylated N-terminus of Nef is specifically bound by Nef. A non-myristoylated N-terminal fragment of Nef or a myristoylated control peptide does not bind to Nef. These results are the first direct experimental evidence of the existence of a myristate-binding pocket in Nef, a prerequisite of the postulated 'closed' Nef conformation.     

Human immunodeficiency virus type 1 Nef: adapting to intracellular trafficking pathways.

The Nef protein of primate lentiviruses is a unique protein that has evolved in several ways to manipulate the biology of an infected cell to support viral replication, immune evasion, pathogenesis, and viral spread. Nef is a small (25- to 34-kDa), myristoylated protein that binds to a collection of cellular factors and acts as an adaptor to generate novel protein interactions to accomplish specific functions. Of the many biological activities attributed to Nef, the reduction of surface levels of the viral receptor (CD4) and antigen-presenting molecules (major histocompatibility complex class I) has been intensely examined; recent evidence demonstrates that Nef utilizes multiple, distinct pathways to affect these proteins. To accomplish this, Nef promotes the formation of multiprotein complexes, recruiting host adaptor proteins to commandeer intracellular vesicular trafficking routes. The altered trafficking of several other host molecules has also been reported, and an emerging theory suggests that Nef generates pleiotrophic effects in the secretory and endocytic pathways that reprogram intracellular protein trafficking and may ultimately provide an efficient platform for viral assembly. This review critically discusses some of the major findings regarding the impact of human immunodeficiency virus type 1 Nef on host protein transport and addresses some emerging directions in this area of human immunodeficiency virus biology.     

Human immunodeficiency virus type 1 Vif protein binds to the Pr55Gag precursor.

The Vif protein of human immunodeficiency virus type 1 is required for productive replication in peripheral blood lymphocytes. Previous reports suggest that vif-deleted viruses are limited in replication because of a defect in the late steps of the virus life cycle. One of the remaining questions is to determine whether the functional role of Vif involves a specific interaction with virus core proteins. In this study, we demonstrate a direct interaction between Vif and the Pr55Gag precursor in vitro as well as in infected cells. No interaction is observed between Vif and the mature capsid protein. The Pr55Gag-Vif interaction is detected (i) in the glutathione S-transferase system, with in vitro-translated proteins demonstrating a critical role of the NC p7 domain of the Gag precursor; (ii) with proteins expressed in infected cells; and (iii) by coimmunoprecipitation experiments. Deletion of the C-terminal 22 amino acids of Vif abolishes its interaction with the Pr55Gag precursor. Furthermore, point mutations in the C-terminal domain of Vif which have been previously shown to abolish virus infectivity and binding to cell membranes dramatically decrease the Gag-Vif interaction. These results suggest that the interaction between Vif and the pr55Gag precursor is a critical determinant of Vif function.     

Human immunodeficiency virus type 1 Vpr-dependent cell cycle arrest through a mitogen-activated protein kinase signal transduction pathway

The human immunodeficiency virus type 1 (HIV-1) Vpr protein has important functions in advancing HIV pathogenesis via several effects on the host cell. Vpr mediates nuclear import of the preintegration complex, induces host cell apoptosis, and inhibits cell cycle progression at G(2), which increases HIV gene expression. Some of Vpr's activities have been well described, but some functions, such as cell cycle arrest, are not yet completely characterized, although components of the ATR DNA damage repair pathway and the Cdc25C and Cdc2 cell cycle control mechanisms clearly play important roles. We investigated the mechanisms underlying Vpr-mediated cell cycle arrest by examining global cellular gene expression profiles in cell lines that inducibly express wild-type and mutant Vpr proteins. We found that Vpr expression is associated with the down-regulation of genes in the MEK2-ERK pathway and with decreased phosphorylation of the MEK2 effector protein ERK. Exogenous provision of excess MEK2 reverses the cell cycle arrest associated with Vpr, confirming the involvement of the MEK2-ERK pathway in Vpr-mediated cell cycle arrest. Vpr therefore appears to arrest the cell cycle at G(2)/M through two different mechanisms, the ATR mechanism and a newly described MEK2 mechanism. This redundancy suggests that Vpr-mediated cell cycle arrest is important for HIV replication and pathogenesis. Our findings additionally reinforce the idea that HIV can optimize the host cell environment for viral replication.     

Human immunodeficiency virus type 1 Vpr binds to the N lobe of the Wee1 kinase domain and enhances kinase activity for CDC2

Human immunodeficiency virus type 1 Vpr is a virion-associated accessory protein that has multiple activities within an infected cell. One of the most dramatic effects of Vpr is the induction of cell cycle arrest at the G(2)/M boundary, followed by apoptosis. This effect has implications for CD4(+) cell loss in AIDS. In normal cell cycle regulation, Wee1, a key regulator for G(2)-M progression, phosphorylates Tyr15 on Cdc2 and thereby blocks the progression of cells into M phase. We demonstrate that Vpr physically interacts with Wee1 at the N lobe of the kinase domain analogous to that present in other kinases. This interaction with Vpr enhances Wee1 kinase activity for Cdc2. Overexpression of Wee1 kinase-deficient mutants competes for Vpr-mediated cell cycle arrest, and deletion of the region of Wee1 that binds Vpr abrogates that competition. However, the Vpr mutants I74P and I81P, which fail to induce G(2) arrest, can bind to and increase the kinase activity of Wee1 to the same extent as wild-type Vpr. Therefore, we conclude that the binding of Vpr to Wee1 is not sufficient for Vpr to activate the G(2) checkpoint, and it may reflect an independent function of Vpr.     

Human immunodeficiency virus type 1 Vpr protein binds to the uracil DNA glycosylase DNA repair enzyme.

The role of the accessory gene product Vpr during human immunodeficiency virus type 1 infection remains unclear. We have used the yeast two-hybrid system to identify cellular proteins that interact with Vpr and could be involved in its function. A cDNA clone which encodes the human uracil DNA glycosylase (UNG), a DNA repair enzyme involved in removal of uracil in DNA, has been isolated. Interaction between Vpr and UNG has been demonstrated by in vitro protein-protein binding assays using translated, radiolabeled Vpr and UNG recombinant proteins expressed as a glutathione S-transferase fusion protein. Conversely, purified UNG has been demonstrated to interact with Vpr recombinant protein expressed as a glutathione S-transferase fusion protein. Coimmunoprecipitation experiments confirmed that Vpr and UNG are associated within cells expressing Vpr. By using a panel of C- and N-terminally deleted Vpr mutants, we have determined that the core protein of Vpr, spanning amino acids 15 to 77, is involved in the interaction with UNG. We also demonstrate by in vitro experiments that the enzymatic activity of UNG is retained upon interaction with Vpr.     

Human immunodeficiency virus type 1 Nef does not alter T-cell sensitivity to antigen-specific stimulation.

We have developed an in vitro model to study the influence that human immunodeficiency virus type 1 (HIV-1) may have on the ability of T cells to respond to antigenic challenge. We have examined consequences of HIV-1 gene expression on T-cell activation in antigen-dependent T cells that have stably integrated copies of replication-defective proviral HIV-1. Virus production by HIV-infected, antigen-dependent T cells was induced in response to antigenic stimulation and then decreased as infected cells returned to a state of quiescence. Contrary to the predictions of models proposing that Nef alters signal transduction pathways in T lymphocytes and thereby alters cellular activation, Nef expression in antigen-dependent T-cell clones did not influence their proliferative responses to low or intermediate concentrations of antigen and did not affect other measures of T-cell activation, such as induction of interleukin 2 receptor alpha-chain expression and cytokine production. In addition, we found no evidence for alteration of T-cell responsiveness to antigen by the gag, pol, vif, tat, or rev gene of HIV-1.     

Human immunodeficiency virus type 1 Nef associates with a member of the p21-activated kinase family.

Although human immunodeficiency virus (HIV) Nef is essential for the induction of AIDS, its biochemical function has remained an enigma. In this study, HIV Nef protein is shown to associate with a serine-threonine kinase that recognizes histone H4 as a substrate, is serologically related to rat p21-activated kinase (PAK), and is specifically activated by Rac and Cdc42. These characteristics define the Nef-associated kinase as belonging to the PAK family. PAKs initiate kinase cascades in response to environmental stimuli, and their identification as a target of Nef implicates these signaling molecules in HIV pathogenesis and provides a novel target for clinical intervention.     

Human immunodeficiency virus type 1 Nef activates p21-activated kinase via recruitment into lipid rafts

The Nef protein of human immunodeficiency virus type 1 is an important factor in AIDS pathogenesis. In addition to downregulating CD4 and major histocompatibility complex class I molecules from the cell surface, as well as increasing virion infectivity, Nef triggers activation of the T-cell receptor (TCR) cascade to facilitate virus spread. Signaling pathways that are induced by Nef have been identified; however, it is unclear how and in which subcellular compartment Nef triggers signaling. Nef recruits a multiprotein complex to activate the cellular Pak kinase that mediates downstream effector functions. Since a subpopulation of Nef is present in detergent-insoluble microdomains (lipid rafts) from where physiological TCR signaling is initiated, we tested whether lipid rafts are instrumental for Nef-mediated Pak activation. In flotation analysis, Nef-associated Pak activity exclusively fractionated with lipid rafts. Activation of Pak in the presence of Nef coincided with lipid raft recruitment of the kinase, which was otherwise excluded from detergent-insoluble microdomains. Experimental solubilization of lipid rafts interfered with the association of Pak activity with Nef. To analyze the importance of the raft localization for Nef function more rigorously, we generated a palmitoylated Nef (PalmNef). PalmNef was highly enriched in lipid rafts and associated with significantly higher levels of Pak activity than Nef. Notably, activation of Pak by its physiological activators, Cdc42 and Rac, also occurred in lipid rafts and required raft integrity. Together, these data suggest that Nef induces signal transduction via the recruitment of a signaling machinery including Pak into lipid rafts, thereby mimicking a physiological cellular mechanism to initiate the TCR cascade.     

Disulfide bond formation in the human immunodeficiency virus type 1 Nef protein.

Substitution of alanine for cysteine residues of the human immunodeficiency virus type 1 LAI (BRU) and ELI Nef proteins was used to determine pairing of the cysteine residues present in each protein. The results show that under nonreducing conditions, alternative pairing of the cysteines occurs. The preferred pairing of cysteine residues of the LAI and ELI proteins differs. In the experimental system used, viruses carrying the ELI nef allele are found to express Nef proteins which accelerate virus replication. Mutation in critical cysteine residues of the protein reduce the rate of virus replication. In the same system, viruses harboring the LAI nef allele fail to replicate. These observations raise the possibility that differences in the observed biological activity of nef alleles may be attributed, at least in part, to differences in the secondary structure of the proteins.     

Human immunodeficiency virus type 1 Rev-responsive element RNA binds to host cell-specific proteins.

RNase protection-gel retention studies show human host cell-specific ribonucleoprotein complexes with human immunodeficiency virus type 1 Rev-responsive element (RRE) RNA. Nuclear proteins from rodent or murine cells appear to lack the ability to form these complexes. Human-mouse somatic cell hybrids retaining a single human chromosome, either 6 or 12, form the RRE-nuclear-protein complexes. One of the complexes requires the entire RRE RNA, while the other needs RRE RNA stem-loops 1 and 2 only. Two major proteins with molecular masses of 120 and 62 kDa specifically bind to RRE RNA. Rodent cells (CHO) either lack or contain small amounts of these RRE-binding proteins.     

Nef binds p6* in GagPol during replication of human immunodeficiency virus type 1

The atypical Nef protein (NefF12) from human immunodeficiency virus type 1 strain F12 (HIV-1(F12)) interferes with virion production and infectivity via a mysterious mechanism. The correlation of these effects with the unusual perinuclear subcellular localization of NefF12 suggested that the wild-type Nef protein could bind to assembly intermediates in late stages of viral replication. To test this hypothesis, Nef from HIV-1(NL4-3) was fused to an endoplasmic reticulum (ER) retention signal (NefKKXX). This mutant NefKKXX protein recapitulated fully the effects of NefF12 on on Gag processing and virion production, either alone or as a CD8 fusion protein. Importantly, the mutant NefKKXX protein also localized to the intermediate compartment, between the ER and the trans-Golgi network. Furthermore, Nef bound the GagPol polyprotein in vitro and in vivo. This binding mapped to the C-terminal flexible loop in Nef and the transframe p6* protein in GagPol. The significance of this interaction was demonstrated by a genetic assay in which the release of a mutant HIV-1 provirus lacking the PTAP motif in the late domain that no longer binds Tsg101 was rescued by a Nef.Tsg101 chimera. Importantly, this rescue as well as incorporation of Nef into HIV-1 virions correlated with the ability of Nef to interact with GagPol. Our data demonstrate that the retention of Nef in the intermediate compartment interferes with viral replication and suggest a new role for Nef in the production of HIV-1.     

Human immunodeficiency virus type 1 Nef protein inhibits activation pathways in peripheral blood mononuclear cells and T-cell lines.

Human immunodeficiency virus type 1 (HIV-1) Nef protein causes the loss of cell surface CD4 and interleukin-2 (IL-2) receptor (Tac) from peripheral blood mononuclear cells (PBMC) and CD4+ T-cell lines. As both CD4 and the IL-2 receptor play crucial roles in antigen-driven helper T-cell signalling and T-cell proliferation, respectively, the role of Nef in the viral life cycle may be to perturb signalling pathways emanating from these receptors. However, the intracellular targets for Nef that result in receptor down-regulation are unknown. Using a recombinant glutathione S-transferase-full-length 27 kDa Nef (Nef27) fusion protein, produced in Escherichia coli by translation from the first start codon of HIV-1 nef clone pNL4-3, as an affinity reagent to probe cytoplasmic extracts of MT-2 cells and PBMC, we have shown interaction with at least seven host cell protein species ranging from 24 to 75 kDa. Immunoblotting identified four of these proteins as p56lck, CD4, p53, and p44mapk/erk1, all of which are intimately involved in intracellular signalling. To assess the relevance of these interactions and further define the biochemical activity of Nef in signal transduction pathways, highly purified Nef27 protein was introduced directly into PBMC by electroporation. Nef27-treated PBMC showed reduced proliferative responsiveness to exogenous recombinant IL-2. Normally, stimulation of T-cells by IL-2 or phorbol 12-myristate 13-acetate provokes both augmentation of p56lck activity and corresponding posttranslational modification of p56lck. These changes were also inhibited by treatment of PBMC with Nef, suggesting that Nef interferes with activation of p56lck and as a consequence of signalling via the IL-2 receptor. Further evidence for Nef interfering with cell proliferation was the decreased production of the proto-oncogene c-myb, which is required for cell cycle progression, in Nef-treated MT-2 cells. In contrast to the binding characteristics and biological effects of Nef27, the alternate 25-kDa isoform of Nef (Nef25) produced by translation from the second start codon of HIV nef pNL4-3 (57 nucleotide residues downstream) was shown to interact with only three cellular proteins of approximately 26, 28, and 56 kDa from PBMC and MT-2 cells, one of which was identified as p56lck. Also, proliferation and posttranslational modification of p56lck in response to IL-2 stimulation were not profoundly affected by treatment of PBMC with Nef25 compared with Nef27.(ABSTRACT TRUNCATED AT 400 WORDS)     

Human immunodeficiency virus type 1 Nef induces accumulation of CD4 in early endosomes.

We have studied the fate of CD4 in CEM T cells expressing a human immunodeficiency virus type 1 HIV-1 Nef protein. Nef triggered a rapid endocytosis and a degradation of CD4, while most of the p56lck was upheld at the cell membrane. In the presence of Nef, CD4 accumulated in acidic intracellular vesicles that were not stained by antibodies against rab6, a marker of the Golgi apparatus complex. Detection of transferrin in CD4-containing vesicles showed that CD4 was trapped in early endosomes, without significant accumulation of CD4 in late endocytic compartments. Internalization pathways taken by CD4 in Nef+ cells may therefore be different from those observed after treatment with phorbol esters.     

Leucine-specific, functional interactions between human immunodeficiency virus type 1 Nef and adaptor protein complexes

The human immunodeficiency virus type 1 virulence protein Nef interacts with the endosomal sorting machinery via a leucine-based motif. Similar sequences within the cytoplasmic domains of cellular transmembrane proteins bind to the adaptor protein (AP) complexes of coated vesicles to modulate protein traffic, but the molecular basis of the interactions between these motifs and the heterotetrameric complexes is controversial. To identify the target of the Nef leucine motif, the native sequence was replaced with either leucine- or tyrosine-based AP-binding sequences from cellular proteins, and the interactions with AP subunits were correlated with function. Tyrosine motifs predictably modulated the interactions between Nef and the mu subunits of AP-1, AP-2, and AP-3; heterologous leucine motifs caused little change in these interactions. Conversely, leucine motifs mediated a ternary interaction between Nef and hemicomplexes containing the sigma1 plus gamma subunits of AP-1 or the sigma3 plus delta subunits of AP-3, whereas tyrosine motifs did not. Similarly, only leucine motifs supported the Nef-mediated association of AP-1 and AP-3 with endosomal membranes in cells treated with brefeldin A. Functionally, Nef proteins containing leucine motifs down-regulated CD4 from the cell surface and enhanced viral replication, whereas those containing tyrosine motifs were inactive. Apparently, the interaction of Nef with the mu subunits of AP complexes is insufficient for function. A leucine-specific mode of interaction that likely involves AP hemicomplexes is further required for Nef activity. The mu and hemicomplex interactions may cooperate to yield high avidity binding of AP complexes to Nef. This binding likely underlies the unusual ability of Nef to induce the stabilization of these complexes on endosomal membranes, an activity that correlates with enhancement of viral replication.     

Human immunodeficiency virus type 1 Nef incorporation into virions does not increase infectivity

The viral protein Nef contributes to the optimal infectivity of human and simian immunodeficiency viruses. The requirement for Nef during viral biogenesis particles suggests that Nef might play a role in this process. Alternatively, because Nef is incorporated into viruses, it might play a role when progeny virions reach target cells. We challenged these hypotheses by manipulating the amounts of Nef incorporated in viruses while keeping its expression level constant in producer cells. This was achieved by forcing the incorporation of Nef into viral particles by fusing a Vpr sequence to the C-terminal end of Nef. A cleavage site for the viral protease was introduced between Nef and Vpr to allow the release of Nef fragments from the fusion protein during virus maturation. We show that the resulting Nef-CS-Vpr fusion partially retains the ability of Nef to downregulate cell surface CD4 and that high amounts of Nef-CS-Vpr are incorporated into viral particles compared with what is seen for wild-type Nef. The fusion protein is processed during virion maturation and releases Nef fragments similar to those found in viruses produced in the presence of wild-type Nef. Unlike viruses produced in the presence of wild-type Nef, viruses produced in the presence of Nef-CS-Vpr do not have an increase in infectivity and are as poorly infectious as viruses produced in the absence of Nef. These findings demonstrate that the presence of Nef in viral particles is not sufficient to increase human immunodeficiency virus type 1 infectivity and suggest that Nef plays a role during the biogenesis of viral particles.     

Regulation of human immunodeficiency virus type 1 infectivity by the ERK mitogen-activated protein kinase signaling pathway

ERK1 and ERK2 mitogen-activated protein kinases (MAPK) play a critical role in regulation of cell proliferation and differentiation in response to mitogens and other extracellular stimuli. Mitogens and cytokines that activate MAPK in T cells have been shown to activate human immunodeficiency virus type 1 (HIV-1) replication. Little is known about the signal transduction pathways that activate HIV-1 replication in T cells upon activation by extracellular stimulation. Here, we report that activation of MAPK through the Ras/Raf/MEK signaling pathway enhances the infectivity of HIV-1 virions. Virus infectivity was enhanced by treatment of cells with MAPK stimulators, such as serum and phorbol myristate acetate, as well as by coexpression of constitutively activated Ras, Raf, or MEK (MAPK kinase) in the absence of extracellular stimulation. Treatment of cells with PD 098059, a specific inhibitor of MAPK activation, or with a MAPK antisense oligonucleotide reduced the infectivity of HIV-1 virions without significantly affecting virus production or the levels of virion-associated Gag and Env proteins. MAPK has been shown to regulate HIV-1 infectivity by phosphorylating Vif (X. Yang and D. Gabuzda, J. Biol. Chem. 273:29879-29887, 1998). However, MAPK activation enhanced virus infectivity in some cells lines that do not require Vif function. The HIV-1 Rev, Tat, p17(Gag), and Nef proteins were directly phosphorylated by MAPK in vitro, suggesting that other HIV-1 proteins are potential substrates for MAPK phosphorylation. These results suggest that activation of the ERK MAPK pathway plays a role in HIV-1 replication by enhancing the infectivity of HIV-1 virions through Vif-dependent as well as Vif-independent mechanisms. MAPK activation in producer cells may contribute to the activation of HIV-1 replication when T cells are activated by mitogens and other extracellular stimuli.