Date

7-13-2005

UMMS Affiliation

Graduate School of Biomedical Sciences

Document Type

Dissertation, Doctoral

Subjects

Vaccinia virus; GTPase-Activating Proteins; Orthopoxvirus; Academic Dissertations; Dissertations, UMMS

Disciplines

Life Sciences | Medicine and Health Sciences

Abstract

The KIL gene of vaccinia virus encodes for a host range protein; in the absence of which, the virus is unable to grow in certain cell lines (RK-13 and some human cell lines). KIL function can be complemented in RK-13 cells by the cowpox host range gene product CP77 despite a lack of homology between the two proteins except for ankyrin repeats. We investigated the role of ankyrin repeats ofthe K1L gene in the host-range restriction of growth in RK-13 cells. The growth of a recombinant vaccinia virus, with the K1L gene mutated in the most conserved ankyrin repeat, was severely impaired as evidenced by lack of plaque formation and reduction in viral titers. Infection of RK-I3 cells with the mutant recombinant vaccinia virus resulted in total shutdown of both cellular and viral protein synthesis early in infection, indicating that the host restriction mediated by the ankyrin repeat is due to a translational block. A comparison of the cellular localization of the K1L wild type and mutated forms showed no difference, as both localized exclusively in the cytoplasm of RK-I3 cells. We also investigated the interaction of the vaccinia virus K1L protein with cellular proteins in RK-13 cells and co-immunoprecipitated a 90 kDa protein identified as the rabbit homologue of human ACAP2, a GTPase-activating protein with ankyrin repeats. Our result suggests the importance of ankyrin repeat for host-range function of K1L in RK-13 cells and identifies ACAP2 as a cellular protein which may be interacting with K1L.

 
 

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