Ly49P recognition of cytomegalovirus-infected cells expressing H2-Dk and CMV-encoded m04 correlates with the NK cell antiviral response

Natural killer (NK) cells are crucial in resistance to certain viral infections, but the mechanisms used to recognize infected cells remain largely unknown. Here, we show that the activating Ly49P receptor recognizes cells infected with mouse cytomegalovirus (MCMV) by a process that requires the presence of H2-Dk and the MCMV m04 protein. Using H2 chimeras between H2-Db and -Dk, we demonstrate that the H2-Dk peptide-binding platform is required for Ly49P recognition. We identified m04 as a viral component necessary for recognition using a panel of MCMV-deletion mutant viruses and complementation of m04-deletion mutant (Δm04) virus infection. MA/My mice, which express Ly49P and H2-Dk, are resistant to MCMV; however, infection with Δm04 MCMV abrogates resistance. Depletion of NK cells in MA/My mice abrogates their resistance to wild-type MCMV infection, but does not significantly affect viral titers in mice infected with Δm04 virus, implicating NK cells in host protection through m04-dependent recognition. These findings reveal a novel mechanism of major histocompatability complex class I–restricted recognition of virally infected cells by an activating NK cell receptor

Natural killer cell receptors and their MHC ligand interactions in innate resistance to mouse cytomegalovirus

Le premier but de mon projet de doctorat a ete la caracterisation moleculaire de l'interface entre les recepteurs activateurs des cellules Natural Killer (NK) et de leurs ligands exprimes dans les cellules infectees ainsi que l'implication de cette interaction sur la reponse a l'infection par le cytomegalovirus (MCMV).J'ai tire un avantage de la variation naturelle au sein des membres lies aux recepteurs Ly49C ainsi que de la disponibilite des structures cristallines des Ly49 afin de comprendre les determinants moleculaires des interactions Ly49H-m157 et egalement identifier les residus des acides amines qui permettent de discriminer entre les recepteurs qui se lient et ceux qui ne se lient pas a m157. Mes decouvertes suggerent que le "site 2" du contact entre le CMH de classe I et Ly49 n'est visiblement pas implique dans la liaison avec m157. Au contraire, les residus localises au niveau de l'interface homodimere-recepteur seraient probablement critiques a la reconnaissance fonctionnelle de la glycoproteine m157. Notre approche fonctionnelle et de modelisation tridimensionnelle suggerent que l'architecture du dimere Ly49H est cruciale pour l'accessibilite de m157 mais non pour les molecules de CMH de classe I et relient la variabilite dans l'homodimerisation des Ly49 a la reconnaissance directe des produits pathogeniques.Un autre mecanisme de la reponse de l'hote contre MCMV provient de l'etude de la souche de souris MA/My, laquelle, malgre l'absence du gene Ly49h ainsi que la proteine pour laquelle il code, y est hautement resistant. Des etudes anterieures ont demontre qu'une interaction epistatique entre un gene issu du groupe des genes Ly49 sur le chromosome 6 et le CMH (H2) sur le chromosome 17 est associee avec la resistance au virus. Utilisant une methode de co-culture de cellules reportrices NFAT-GFP exprimant les recepteurs activateurs Ly49 et de fibroblastes primaires infectes, j'ai montre que le recepteur activateur Ly49P des cellules NK reconnait specifiquement les cellules infectees par MCMV et que cette reconnaissance depend de la presence de l'haplotype H2k. Ce signal etait bloque par l'utilisation des anticorps anti-H2-D k mais non par anti-H2-Kk. Ces resultats indiquent l'existence d'un nouveau mecanisme des cellules NK implique dans la resistance au MCMV, lequel depend de l'interaction fonctionnelle entre le recepteur Ly49P et la molecule du MHC de classe I, H2-Dk, dans les cellules infectees par MCMV. Comme contribution directe de ce travail, nous avons demontre que la resistance chez MA/My est au moins partiellement dependante des interactions entre le recepteur Ly49P et la molecule H2-Dk modifiee par le virus dans les cellules infectees. Comme MCMV regule negativement l'expression des molecules du CMH de classe I, j'ai confirme la presence de H2-Dk dans les cellules infectees par l'utilisation d'un virus MCMV recombinant portant un gene rapporteur GFP. En permutant la plateforme peptidique de liaison, les domaines transmembranaires et intracellulaires entre les molecules H2-Db et H2-D k, j'ai demontre que la plateforme peptidique de liaison est critique pour la reconnaissance des cellules infectees. Par le criblage d'un panel de mutants MCMV portant des genes impliques dans l'evasion immune, j'ai demontre que l'infection de fibroblastes par le MCMV depourvu du gene m04 (Deltam04) abolit totalement l'activation de Ly49P. (Abstract shortened by UMI.

Ly49P recognition of cytomegalovirus-infected cells expressing H2-Dk and CMV-encoded m04 correlates with the NK cell antiviral response.

Natural killer (NK) cells are crucial in resistance to certain viral infections, but the mechanisms used to recognize infected cells remain largely unknown. Here, we show that the activating Ly49P receptor recognizes cells infected with mouse cytomegalovirus (MCMV) by a process that requires the presence of H2-D(k) and the MCMV m04 protein. Using H2 chimeras between H2-D(b) and -D(k), we demonstrate that the H2-D(k) peptide-binding platform is required for Ly49P recognition. We identified m04 as a viral component necessary for recognition using a panel of MCMV-deletion mutant viruses and complementation of m04-deletion mutant (Deltam04) virus infection. MA/My mice, which express Ly49P and H2-D(k), are resistant to MCMV; however, infection with Deltam04 MCMV abrogates resistance. Depletion of NK cells in MA/My mice abrogates their resistance to wild-type MCMV infection, but does not significantly affect viral titers in mice infected with Deltam04 virus, implicating NK cells in host protection through m04-dependent recognition. These findings reveal a novel mechanism of major histocompatibility complex class I-restricted recognition of virally infected cells by an activating NK cell receptor

Sequestosome-1/p62 Is the Key Intracellular Target of Innate Defense Regulator Peptide*

Innate defense regulator-1 (IDR-1) is a synthetic peptide with no antimicrobial activity that enhances microbial infection control while suppressing inflammation. Previously, the effects of IDR-1 were postulated to impact several regulatory pathways including mitogen-activated protein kinase (MAPK) p38 and CCAAT-enhancer-binding protein, but how this was mediated was unknown. Using a combined stable isotope labeling by amino acids in cell culture-proteomics methodology, we identified the cytoplasmic scaffold protein p62 as the molecular target of IDR-1. Direct IDR-1 binding to p62 was confirmed by several biochemical binding experiments, and the p62 ZZ-type zinc finger domain was identified as the IDR-1 binding site. Co-immunoprecipitation analysis of p62 molecular complexes demonstrated that IDR-1 enhanced the tumor necrosis factor α-induced p62 receptor-interacting protein 1 (RIP1) complex formation but did not affect tumor necrosis factor α-induced p62-protein kinase ζ complex formation. In addition, IDR-1 induced p38 MAPK activity in a p62-dependent manner and increased CCAAT-enhancer-binding protein β activity, whereas NF-κB activity was unaffected. Collectively, these results demonstrate that IDR-1 binding to p62 specifically affects protein-protein interactions and subsequent downstream events. Our results implicate p62 in the molecular mechanisms governing innate immunity and identify p62 as a potential therapeutic target in both infectious and inflammatory diseases