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Mouse cytomegalovirus takes control of the host signaling STRIPAK hub

The mechanisms driving cell-to-cell spread of cytomegalovirus (CMV) are poorly understood, yet cytoskeletal proteins are thought to play a major role since CMV induces broad cytoskeletal changes, leading to characteristic rounding of infected cells. We identified the mouse CMV M25 protein as key regulator of virus-mediated cell rounding. Early in infection, M25 is confined to the nucleus, while later M25 localizes also to the viral assembly compartment in the cytoplasm. An M25 deletion mutant shows a small plaque phenotype and grows to lower titers in vitro and in vivo. We hypothesized that the M25 protein functionally connects the observed cytoskeletal changes with cell-to-cell spread of MCMV. To discover M25 host interacting partners we performed co-immunoprecipitation linked to SILAC based mass spectrometry, and subsequently verified interactions by co-IP of M25 and candidate proteins. Moreover, cytoskeletal remodeling was analyzed in detail over the MCMV infection cycle using confocal microscopy. We identified components of the host STRIPAK complex (Striatin interacting phosphatase and kinase) as interacting partners of M25. STRIPAK is a multi-protein signaling complex involved in regulation of focal adhesions and cell-cell contacts, cytoskeletal signaling, Golgi assembly and vesicular trafficking. The protein complex is assembled by striatins – scaffold proteins that bind protein phosphatase 2A (PP2A), germinal center-like kinases III (GCKIII) and a number of adaptor proteins. Interestingly, while we confirmed M25 interaction with these proteins early in infection, our data suggest reduced binding of GCKIII kinases to the M25-associated STRIPAK complex at later stages of infection. It is known that upon dissociation from the complex, the GCKIII kinases are activated by auto- phosphorylation, while within the complex they are inhibited by PP2A. Further, actin rearrangement in MCMV-infected cells was accompanied with a reduced number of focal adhesions as well as de-phosphorylation of paxillin and focal adhesion kinase. Conversely, we did not observe such alterations in ∆M25- infected cells. Our results indicate that M25 induces cell rounding of MCMV-infected cells and enhances cell-to-cell spread through interaction with the STRIPAK complex and modulating the activity of GCKIII kinases.

murine cytomegalovirus, tegument protein, M25, host-virus interaction, phosphatase, STRIPAK

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