Reovirus Chaperone Interaction And Apoptosis Regulation
Many viruses require chaperones during viral replication. I investigated the involvement of HSP70 family chaperones in reovirus replication and assembly. Assembly and replication occurs in the cytosol of reovirus-infected cells within structures called viral factories (VFs). The nonstructural protein [mu]NS forms the matrix of VFs. The eight structural proteins that assemble the inner core of the double-layered virion are recruited to VFs by specific interactions with [mu]NS. However, it is unknown how the outer capsid proteins [MICRO SIGN]1 and [sigma]3 are recruited to VFs. Using biochemical and cell biological approaches I found that recruitment of [mu]1 to VFs requires assembly with its partner protein [sigma]3. In addition, I found that the [mu]1:[sigma]3 complex and [MICRO SIGN]1 alone, interact with the cellular chaperone Hsc70. Moreover, the subcellular distribution of Hsc70 correlated with that of [mu]1 in reovirus infected cells and Hsc70 also co-localized with ectopically expressed [mu]NS. The Hsc70-[MICRO SIGN]NS interaction did not require the chaperone ATPase function, suggesting that [MICRO SIGN]NS is not a substrate for Hsc70. The formation of viral factory-like structures by [MICRO SIGN]NS was not affected after siRNAmediated knockdown of Hsc70. These findings suggest that Hsc70 facilitates assembly of [MICRO SIGN]1:[sigma]3 complexes and is likely involved in their subsequent recruitment to VFs. The specific interaction of Hsc70 with [mu]NS may be involved in assembly of viral particles, but as in other viruses could also be involved in other replicative processes. The last part of my thesis focused on reovirus-induced apoptosis. Apoptosis is induced by [MICRO SIGN]1 and occurs late in infection, but the regulatory mechanisms that prevent premature apoptosis induction by [MICRO SIGN]1 are poorly understood. I found that [MICRO SIGN]1 was cleaved by caspases upon [MICRO SIGN]1-induced apoptosis. This represents a negative feedback loop where [MICRO SIGN]1-induced apoptosis leads to degradation of [MICRO SIGN]1 by caspase cleavage. I also found that [MICRO SIGN]1 was ubiquitinated, suggesting proteasomal degradation as another means to regulate [MICRO SIGN]1-induced apoptosis. Interestingly, the extent of [MICRO SIGN]1 ubiquitination was influenced by caspase activity, indicating a link between the two phenotypes. These findings suggest caspase cleavage and ubiquitination of [MICRO SIGN]1 as mechanisms to regulate [MICRO SIGN]1-induced apoptosis, and they reveal a link between these so far unrelated events.
Parker, John Stuart Leslie
Vogt, Volker M; Leifer, Cynthia Anne; Dubovi, Edward Joseph
Ph.D. of Veterinary Medicine
Doctor of Philosophy
dissertation or thesis