Infectivity Enhancement by Human Immunodeficiency Virus Type 1 Nef Is Independent of Its Association with a Cellular Serine/Threonine Kinase (original) (raw)
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Biology of the HIV Nef protein
The Indian journal of medical research, 2005
The accessory Nef protein is expressed by all primate lentiviruses--HIV-1,HIV-2 and simian immune deficiency virus (SIV). Its expression in the early stages of the viral life cycle ensures two basic attributes of HIV infection. These are T-cell activation and the establishment of a persistent state of infection. Nef has a positive effect on viral infection and replication by promoting the survival of infected cells. Its role in HIV persistence is based largely on the ability of Nef to downmodulate the surface levels of important molecules at the immune synapse. These include major histocompatibility complex-I (MHC I) and (MHC II) present on antigen-presenting cells (APCs) and target cells, and CD4 and CD28 present on helper T cells. In this review we present these biological properties of Nef from a mechanistic point of view, and relate them to the structural attributes and interactions of the Nef protein. A brief outline of the limited studies on Nef from Indian subtype C HIV-1 iso...
Nef from Human Immunodeficiency Virus Type 1F12 Inhibits Viral Production and Infectivity
Journal of Virology, 2001
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.
Functional Characterization of the Human Immunodeficiency Virus Type 1 Nef Acidic Domain
Journal of Virology, 2010
The human immunodeficiency virus type 1 (HIV-1) accessory protein Nef downregulates major histocompatibility complex class I (MHC-I) from the cell surface. It has been proposed that the direct interaction of the acidic cluster (AC) of Nef, 62 EEEE 65 , with the furin binding region (fbr) of PACS-1 is crucial for this Nef function. Contrary to this proposal, evidence is presented here that the four glutamates in Nef do not functionally engage the PACS-1 fbr. (i) The binding of Nef to the PACS-1 fbr in vitro is much weaker than the binding of the canonical furin AC to the PACS-1 fbr. (ii) The mutation of two of the four glutamates in Nef's AC to alanines does not alter Nef's ability to downregulate MHC-I, and triply mutated Nefs exhibit 50% activity.
1998
The nef gene of the human and simian immunodeficiency viruses (HIV and SIV) is dispensable for viral replication in T-cell lines; however, it is essential for high virus loads and progression to simian AIDS (SAIDS) in SIV-infected adult rhesus macaques. Nef proteins from HIV type 1 (HIV-1), HIV-2, and SIV contain a proline-Xaa-Xaa-proline (PxxP) motif. The region of Nef with this motif is similar to the Src homology region 3 (SH3) ligand domain found in many cell signaling proteins. In virus-infected lymphoid cells, Nef interacts with a cellular serine/threonine kinase, designated Nef-associated kinase (NAK). In this study, analysis of viral clones containing point mutations in the nef gene of the pathogenic clone SIVmac239 revealed that several strictly conserved residues in the PxxP region were essential for Nef-NAK interaction. The results of this analysis of Nef mutations in in vitro kinase assays indicated that the PxxP region in SIV Nef was strikingly similar to the consensus sequence for SH3 ligand domains possessing the minus orientation. To test the significance of the PxxP motif of Nef for viral pathogenesis, each proline was mutated to an alanine to produce the viral clone SIVmac239-P 104 A/P 107 A. This clone, expressing Nef that does not associate with NAK, was inoculated into seven juvenile rhesus macaques. In vitro kinase assays were performed on virus recovered from each animal; the ability of Nef to associate with NAK was restored in five of these animals as early as 8 weeks after infection. Analysis of nef genes from these viruses revealed patterns of genotypic reversion in the mutated PxxP motif. These revertant genotypes, which included a second-site suppressor mutation, restored the ability of Nef to interact with NAK. Additionally, the proportion of revertant viruses increased progressively during the course of infection in these animals, and two of these animals developed fatal SAIDS. Taken together, these results demonstrated that in vivo selection for the ability of SIV Nef to associate with NAK was correlated with the induction of SAIDS. Accordingly, these studies implicate a role for the conserved SH3 ligand domain for Nef function in virally induced immunodeficiency.
Structural and Functional Correlates between HIV-1 and SIV Nef Isolates
Virology, 1996
The nef genes of HIV-1 and SIV encode 27-34 kDa myristoylated proteins which have been shown to induce cell surface CD4 downregulation and bind to a cellular protein kinase. To identify regions of Nef important for function, structurefunction correlates of HIV SF2 nef (Nef) and SIV mm239open nef (SNef) were sought by constructing Nef/SNef hybrids. Metabolic labeling with 35 [S]methionine/cysteine demonstrated similar amounts of 35 [S] incorporation into all but one hybrid, SNeftll, in which the C-terminus of SNef was replaced by that of Nef. The weak protein expression of SNeftll was attributable to its short half-life of approximately 45 min. Nef, SNef, and SHSNef, a hybrid containing the internal sequences of Nef and the N-and C-terminal sequences from SNef, downregulated CD4 in human CEM cells. Only Nef and SHSNef downregulated CD4 in mouse AKR1-G1 cells. Nef, SNef, and SHSNef also effectively bound phosphoproteins of MW Å 62,000 and 78,000 in CEM cells. Two additional hybrids, in which the Nef sequences of SHSNef were replaced with additional SNef sequences, were essentially ineffective in both assays. Thus, in two different assays of Nef function, swapping the SIV and HIV internal nef sequences were shown to be greatly deleterious to Nef function while SHSNef remained functional. ᭧ 1996 Academic Press, Inc.
Functional characterization of HIV-1 Nef mutants in the context of viral infection
Virology, 2006
Nef is an important pathogenesis factor of HIV-1 with a multitude of effector functions. We have designed a broad panel of isogenic viruses encoding defined mutants of HIV-1 SF2 Nef and analyzed their biological activity in the context of productive HIV-1 infection. Analysis of subcellular localization, virion incorporation, downregulation of cell surface CD4 and MHC-I, enhancement of virion infectivity and facilitation of HIV replication in primary human T lymphocytes mostly confirmed the mapping of Nef determinants previously reported upon isolated expression of Nef. However, reduced activity in downregulation of CD4, infectivity enhancement and virion incorporation of a Nef variant (Δ12-39) lacking an amphipatic helix required for binding of a cellular kinase complex and the association of Nef with MHC-I/AP-1 suggested a novel role of this N-terminal motif. The SH3 binding motif of Nef was partially required for infectivity enhancement and replication but not for receptor downmodulation. In contrast to previous results obtained using other Nef alleles, non-myristoylated SF2-Nef was only partly defective when expressed during HIV infection and was present in HIV-1 particles. Importantly, incorporation of Nef into HIV-1 virions was not required for any of the tested Nef activities. Altogether, this study provides a broad characterization and mapping of multiple Nef activities in HIV-infected cells. The results emphasize that multiple activities govern Nef's effects on HIV replication and argue against a role of virion incorporation for Nef's activity as pathogenicity factor.
Journal of Virology, 2008
Nef is an accessory protein of human immunodeficiency virus type 1 (HIV-1) that enhances the infectivity of progeny virions when expressed in virus-producing cells. The requirement for Nef for optimal infectivity is, at least in part, determined by the envelope (Env) glycoprotein, because it can be eliminated by pseudotyping HIV-1 particles with pH-dependent Env proteins. To investigate the role of Env in the function of Nef, we have examined the effect of Nef on the infectivity of Env-deficient HIV-1 particles pseudotyped with viral receptors for cells expressing cognate Env proteins. We found that Nef significantly enhances the infectivity of CD4chemokine receptor pseudotypes for cells expressing HIV-1 Env. Nef also increased the infectivity of HIV-1 particles pseudotyped with Tva, the receptor for subgroup A Rous sarcoma virus (RSV-A), even though Nef had no effect if the pH-dependent Env protein of RSV-A was used for pseudotyping. However, Nef does not always enhance viral infectivity if the normal orientation of the Env-receptor interaction is reversed, because the entry of Env-deficient HIV-1 into cells expressing the vesicular stomatitis virus G protein was unaffected by Nef. Together, our results demonstrate that the presence of a viral Env protein during virus production is not required for the ability of Nef to increase viral infectivity. Furthermore, since the infectivity of Tva pseudotypes was blocked by inhibitors of endosomal acidification, we conclude that low-pH-dependent entry does not always bypass the requirement for Nef.