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Evolutionary plasticity of SH3 domain binding by Nef proteins of the HIV-1/SIVcpz lentiviral lineage.

ABSTRACT: The accessory protein Nef of human and simian immunodeficiency viruses (HIV and SIV) is an important pathogenicity factor known to interact with cellular protein kinases and other signaling proteins. A canonical SH3 domain binding motif in Nef is required for most of these interactions. For example, HIV-1 Nef activates the tyrosine kinase Hck by tightly binding to its SH3 domain. An archetypal contact between a negatively charged SH3 residue and a highly conserved arginine in Nef (Arg77) plays a key role here. Combining structural analyses with functional assays, we here show that Nef proteins have also developed a distinct structural strategy-termed the "R-clamp"-that favors the formation of this salt bridge via buttressing Arg77. Comparison of evolutionarily diverse Nef proteins revealed that several distinct R-clamps have evolved that are functionally equivalent but differ in the side chain compositions of Nef residues 83 and 120. Whereas a similar R-clamp design is shared by Nef proteins of HIV-1 groups M, O, and P, as well as SIVgor, the Nef proteins of SIV from the Eastern chimpanzee subspecies (SIVcpzP.t.s.) exclusively utilize another type of R-clamp. By contrast, SIV of Central chimpanzees (SIVcpzP.t.t.) and HIV-1 group N strains show more heterogenous R-clamp design principles, including a non-functional evolutionary intermediate of the aforementioned two classes. These data add to our understanding of the structural basis of SH3 binding and kinase deregulation by Nef, and provide an interesting example of primate lentiviral protein evolution.

SUBMITTER: Zhao Z

PROVIDER: S-EPMC8629392 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundA nef gene is present in all primate lentiviral genomes and is important for high viral loads and progression to AIDS in human or experimental macaque hosts of HIV or SIV, respectively. In these hosts, infection of the thymus results in a decreased output of naive T cells that may contribute to the development of immunodeficiency. We have previously shown that HIV-1 subtype B Nef proteins can block human T-cell development. However, the underlying mechanism(s) and the conservation of this Nef function between different groups of HIV and SIV remained to be determined.ResultsWe investigated whether reduction of thymic output is a conserved function of highly divergent lentiviral Nef proteins including those from both types of human immunodeficiency viruses (HIV-1 and HIV-2), their direct simian counterparts (SIVcpz, SIVgor and SIVsmm, respectively), and some additional SIV strains. We found that expression of most of these nef alleles in thymocyte progenitors impaired T-cell development and reduced thymic output. For HIV-1 Nef, binding to active p21 protein (Cdc42/Rac)-activated kinase (PAK2) was a major determinant of this function. In contrast, selective disruption of PAK2 binding did not eliminate the effect on T-cell development of SIVmac239 Nef, as was shown by expressing mutants in a newly discovered PAK2 activating structural motif (PASM) constituted by residues I117, H121, T218 and Y221, as well as previously described mutants. Rather, down-modulation of cell surface CD3 was sufficient for reduced thymic output by SIVmac Nef, while other functions of SIV Nefs contributed.ConclusionsOur results indicate that primate lentiviral Nef proteins impair development of thymocyte precursors into T cells in multiple ways. The interaction of HIV-1 Nef with active PAK2 by HIV-1 seem to be most detrimental, and downregulation of CD3 by HIV-2 and most SIV Nef proteins sufficient for reduced thymic output. Since the reduction of thymic output by Nef is a conserved property of divergent lentiviruses, it is likely to be relevant for peripheral T-cell depletion in poorly adapted primate lentiviral infections.

Project description: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-P104A/P107A. 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.

Project description:UNLABELLED:Human immunodeficiency virus type 1 (HIV-1) Nef enhances virus replication and contributes to immune evasion in vivo, but the underlying molecular mechanisms remain incompletely defined. Nef interferes with host cell actin dynamics to restrict T lymphocyte responses to chemokine stimulation and T cell receptor engagement. This relies on the assembly of a labile multiprotein complex including the host kinase PAK2 that Nef usurps to phosphorylate and inactivate the actin-severing factor cofilin. Components of the exocyst complex (EXOC), an octameric protein complex involved in vesicular transport and actin remodeling, were recently reported to interact with Nef via the same molecular surface that mediates PAK2 association. Exploring the functional relevance of EXOC in Nef-PAK2 complex assembly/function, we found Nef-EXOC interactions to be specifically mediated by the PAK2 interface of Nef, to occur in infected human T lymphocytes, and to be conserved among lentiviral Nef proteins. In turn, EXOC was dispensable for direct downstream effector functions of Nef-associated PAK2. Surprisingly, PAK2 was essential for Nef-EXOC association, which required a functional Rac1/Cdc42 binding site but not the catalytic activity of PAK2. EXOC was dispensable for Nef functions in vesicular transport but critical for inhibition of actin remodeling and proximal signaling upon T cell receptor engagement. Thus, Nef exploits PAK2 in a stepwise mechanism in which its kinase activity cooperates with an adaptor function for EXOC to inhibit host cell actin dynamics. IMPORTANCE:Human immunodeficiency virus type 1 (HIV-1) Nef contributes to AIDS pathogenesis, but the underlying molecular mechanisms remain incompletely understood. An important aspect of Nef function is to facilitate virus replication by disrupting T lymphocyte actin dynamics in response to stimulation via its association with the host cell kinase PAK2. We report here that the molecular surface of Nef for PAK2 association also mediates interaction of Nef with EXOC and establish that PAK2 provides an essential adaptor function for the subsequent formation of Nef-EXOC complexes. PAK2 and EXOC specifically cooperate in the inhibition of actin dynamics and proximal signaling induced by T cell receptor engagement by Nef. These results establish EXOC as a functionally relevant Nef interaction partner, emphasize the suitability of the PAK2 interaction surface for future therapeutic interference with Nef function, and show that such strategies need to target activity-independent PAK2 functions.

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Evolutionary plasticity of SH3 domain binding by Nef proteins of the HIV-1/SIVcpz lentiviral lineage.

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