Date of Award
Doctor of Philosophy
Veterinary Microbiology and Preventive Medicine
Cathy L. Miller
Mammalian orthoreoviruses (MRV) are non-fusogenic, nonenveloped, icosahedral, RNA viruses, containing a 10-segmented double-stranded RNA genome, belonging to the family Reoviridae. Infection with many mammalian orthoreovirus (MRV) strains results in shutoff of host, but not viral, protein synthesis via protein kinase R (PKR) activation and phosphorylation of translation initiation factor eIF2α. When cells are under stressful environments, such as heat shock, oxidative stress, nutritional starvation, and viral infection, several kinases (PKR, PERK, HRI, or GCN) are activated, which phosphorylate eIF2α( 51 ser), resulting in the formation of stress granules (SGs), discrete areas in the cytoplasm where cellular mRNAs are held in a translationally inactive state. We examined MRV-infected cells to characterize SG formation in response to MRV infection. We found SGs formed at early times following infection (2-6 h p.i.) in a manner dependent on phosphorylation of eIF2α. MRV induced SG formation in all four eIF2α kinase knockout cell lines, suggesting at least two kinases are involved in induction of SGs. Inhibitors of MRV disassembly prevented MRV-induced SG formation, indicating that viral uncoating is a required step for SG formation. Inactivation of MRV virions by ultraviolet (UV) light, or treatment of MRV-infected cells with the translational inhibitor, puromycin, did not prevent SG formation, suggesting that viral transcription and translation are not required for SG formation. Viral cores were found to colocalize with SGs, however, cores from UV-inactivated virions did not associate with SGs, suggesting viral core particles are recruited into SGs in a process that requires the synthesis of viral mRNA. These results demonstrate that MRV particles induce SGs in a step following viral disassembly but preceding viral mRNA transcription, and that core particles are themselves recruited to SGs, suggesting the cellular stress response may play an inhibitory role in viral translation.
As infection proceeds, MRV disrupts SGs despite sustained levels of phosphorylated eIF2α, and further, interferes with the induction of SGs by other stress inducers. MRV interference with SG formation occurs downstream of eIF2α phosphorylation suggesting the virus uncouples the cellular stress signaling machinery from SG formation. We additionally examined mRNA translation in the presence of SGs induced by eIF2α phosphorylation dependent and independent mechanisms. We found that irrespective of eIF2alpha phosphorylation status, the presence of SGs in cells correlated with inhibition of viral and cellular translation. In contrast, MRV disruption of SGs correlated with release of viral mRNAs from translational inhibition, even in the presence of phosphorylated eIF2α. Viral mRNAs were also translated in the presence of phosphorylated eIF2α in PKR-/- cells. These results suggest that MRV escape from host cell translational shutoff correlates with virus-induced SG disruption, and occurs in the presence of phosphorylated eIF2α in a PKR independent manner.
In order to escape from host cell translational shutoff induced by eIF2α phosphorylation, MRV must use a translational strategy different from that used by most cellular mRNA. We have recapitulated a 10-plasmid-based reverse genetics system in our lab and have established inducible viral protein-expressing cell lines to examine whether unique genomic sequences in the viral mRNA, or alternatively, virally encoded proteins, play a critical role in viral translation at late times in MRV infection. Taken together, these studies have added significantly to our knowledge on viral-host interactions and regulation of viral translation in members of the Reoviridae family, and further, have laid the groundwork for important studies examining the mechanisms of tumor oncolysis by MRV.
Qin, Qingsong, "Characterization of mammalian orthoreovirus (MRV) induced stress granules (SGs) and implications of eIF2α phosphorylation on viral translation" (2010). Graduate Theses and Dissertations. 11731.