Title

The alpha subunit of eukaryotic initiation factor 2B is requisite for eIF2-mediated translational suppression of vesicular stomatitis virus

Date of Award

2007

Availability

Article

Degree Name

Doctor of Philosophy (Ph.D.)

Department

Microbiology and Immunology (Medicine)

First Committee Member

Glen N. Barber, Committee Chair

Abstract

Eukaryotic initiation factor 2B (eIF2B) is a heteropentameric guanine nucleotide exchange factor (GEF) that converts inactive eIF2 GDP-bound binary complexes into active eIF2 GTP-bound complexes that can bind initiator t-RNA molecules and ribosomes to begin translation. eIF2B is functionally divided into two subcomplexes: the catalytic core comprised of elF2Bepsilon and eIF2Bgamma, and the regulatory core comprised of eIF2Balpha, eIF2Bbeta and elF2Bdelta. While the catalytic subunits are responsible for exerting GEF activity, the regulatory subunits recognize eIF2 and respond to eIF2alpha phosphorylation. Cellular stress, such as virus infection, inhibits host protein synthesis by activating specific kinases that are capable of phosphorylating the alpha subunit of elF2, which can then sequester eIF2B to stall guanine nucleotide exchange by a currently unresolved mechanism. Importantly, we demonstrate that loss of eIF2Balpha or expression of a variant of the human elF2Balpha subunit harboring a single point mutation (T41A) is sufficient to neutralize the consequences of eIF2alpha phosphorylation, and render primary MEFs significantly more susceptible to vesicular stomatitis virus infection. To extend this analysis, we further exhibit the vital function of elF2Balpha in protein synthesis through phenotypic studies in yeast. Here, we report that this subunit can sufficiently substitute for its yeast counterpart, GCN3, and reproduce similar growth phenotypes under normal and amino acid deprived conditions. In addition, the human eIF2Balpha-T41A variant was unable derepress GCN4 translation in response to an inhibitor of amino acid biosynthesis in yeast, an activity that requires sensitivity to phosphorylation of the yeast eIF2alpha homolog, SUI2.Previously, we have demonstrated that vesicular stomatitis virus can infect and replicate to high levels in tumor cells. Moreover, these cells appear to contain defects in eIF2alpha-mediated translational control, plausibly due to disregulation of eIF2B activity, which overcomes the inhibitory effects of eIF2alpha phosphorylation. Our data suggest a role for eIF2B, specifically elF2Balpha, in suppression of translation following virus infection, and imply that this complex may contribute to oncogenic transformation. These results emphasize the importance of eIF2Balpha in mediating eIF2 kinase translation inhibitory activity and may provide insight into the complex nature of viral oncolysis and cellular transformation.

Keywords

Biology, Microbiology; Health Sciences, Immunology

Link to Full Text

http://access.library.miami.edu/login?url=http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:3295185