Roles of muNS in reovirus factory formation
By: by Michelle Marie Arnold
Series: Collections of the Harvard University Archives
Published: 2007
Language: eng
Number of pages: 299
Description: The sites of mammalian orthoreovirus (reovirus) replication and assembly are large nonenveloped phase-dense structures in cells called viral factories. The nonstructural protein μNS serves as the matrix of factories, and forms factory-like structures (FLS) when expressed alone in cells. μNS has been shown to recruit several viral proteins to factories during infection and to FLS when cotransfected with vectors expressing the other proteins, suggesting the viral factories play a role in viral replication and assembly. By deletion analysis, we have shown several regions of μNS to be required for formation of FLS, including the extreme C terminus and the first of two predicted coiled-coils. We also showed by mutational analysis that two conserved residues in the coiled-coil linker region are required for FLS formation, and predict that these two residues form an intramolecular zinc hook. We then determined the minimal contiguous region sufficient to form FLS to be the C-terminal third of μNS, which became the basis for a platform in which we assayed protein-protein interactions by fusing a protein or protein fragment of interest to GFP-μNS(471-721).

In order to study the requirement for μNS and the effect of specific mutations in the context of infection, we developed an siRNA knock-down and complementation assay. We showed that the expression of μNS was essential for viral infection, and concluded that the formation of FLS was required for rescue of infection. Finally, we hypothesized that in order to form the large three-dimensional structure of the factory there must be more than one region of μNS that interacts with another molecule of μNS. Using differentially tagged μNS constructs, we investigated the regions that mediated the recruitment of μNS to FLS, and showed that the predicted coiled-coils were sufficient to associate with μNS in FLS.
Notes: "September 2007."

Thesis (Ph.D., Division of Medical Sciences (Virology))--Harvard University, 2007.

Includes bibliographical references.