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Molecular requirements for T cell recognition of N-myristoylated peptides derived from the simian immunodeficiency virus Nef protein.

ABSTRACT: We have recently isolated a rhesus macaque cytotoxic T cell line, 2N5.1, that specifically recognizes an N-myristoylated 5-mer peptide (C(14)-Gly-Gly-Ala-Ile-Ser [C14nef5]) derived from the simian immunodeficiency virus (SIV) Nef protein. Such C14nef5-specific T cells expand in the circulation of SIV-infected monkeys, underscoring the capacity of T cells to recognize viral lipopeptides; however, the molecular basis for the lipopeptide antigen presentation remains to be elucidated. Here, functional studies indicated that the putative antigen-presenting molecule for 2N5.1 was likely to have two separate antigen-binding sites, one for interaction with a C(14)-saturated acyl chain and the other for anchorage of the C-terminal serine residue. Mutants with alanine substitutions for the second glycine residue and the fourth isoleucine residue were not recognized by 2N5.1 but interfered with the presentation of C14nef5 to 2N5.1, indicating that these structural analogues retained the ability to interact with the antigen-presenting molecules. In contrast to the highly specific recognition of C14nef5 by 2N5.1, an additional cytotoxic T cell line, SN45, established independently from a C14nef5-stimulated T cell culture, showed superb reactivity to both C14nef5 and an N-myristoylated Nef 4-mer peptide, and therefore, the C-terminal serine residue was dispensable for the recognition of lipopeptides by the SN45 T cells. Furthermore, the mutants with alanine substitutions were indeed recognized by the SN45 T cells. Given that N-myristoylation of the Nef protein occurs in the conserved motifs and is critical for viral pathogenesis, these observations predict that the lipopeptide-specific T cell response is difficult for viruses to avoid by simply introducing amino acid mutations.

SUBMITTER: Morita D

PROVIDER: S-EPMC3536395 | biostudies-literature | 2013 Jan

REPOSITORIES: biostudies-literature

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Molecular requirements for T cell recognition of N-myristoylated peptides derived from the simian immunodeficiency virus Nef protein.

Morita Daisuke D Yamamoto Yukie Y Suzuki Juri J Mori Naoki N Igarashi Tatsuhiko T Sugita Masahiko M

Journal of virology 20121024 1

We have recently isolated a rhesus macaque cytotoxic T cell line, 2N5.1, that specifically recognizes an N-myristoylated 5-mer peptide (C(14)-Gly-Gly-Ala-Ile-Ser [C14nef5]) derived from the simian immunodeficiency virus (SIV) Nef protein. Such C14nef5-specific T cells expand in the circulation of SIV-infected monkeys, underscoring the capacity of T cells to recognize viral lipopeptides; however, the molecular basis for the lipopeptide antigen presentation remains to be elucidated. Here, function ...[more]

PMID: 23097434

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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.

Dataset Information

Molecular requirements for T cell recognition of N-myristoylated peptides derived from the simian immunodeficiency virus Nef protein.

Publications

Molecular requirements for T cell recognition of N-myristoylated peptides derived from the simian immunodeficiency virus Nef protein.

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