Complete Title ol Thesis:
"Cell Cycle Inhibitors in Control of Chronic Gammaherpesvirus 68 Infection"
Prepared under the direction of: Linda F. van Dyk, Ph.D.
γHVs are significant medical and veterinary pathogens that cause a wide variety of malignancies associated with chronic inflammation and reactivation from latency, and a better understanding of reactivation from latency is clearly needed. Previous studies have shown that the v-cyclin is required for efficient reactivation from latency but dispensable for lytic replication and the establishment of latency. The defect in reactivation comparing wt γHV68 and v-cyclin KO virus is consistently found regardless of mouse strain or deficiency status and is always at least ≥100 fold. In vitro, over-expression studies have been used to determine potential substrates and binding partners of the v-cyclin. These studies have provided critical insights into the capabilities of v-cyclins; however this work has been done outside the context of viral infection. The activities and true functions of the v-cyclins are likely to be further regulated in vivo during the course of viral infection.
Chapter III of this dissertation was started to understand at what point in reactivation from latency the v-cyclin was acting, thus help give insights into its mechanism of action. I wanted to address this question by using a lentiviral system to provide the v-cyclin in trans ex vivo. Work described in this chapter demonstrates that I was able to clone in my gene of interest and obtain protein expression that was functional; however I was unable to transduce B lymphocytes to a high enough efficiency to determine at what point the viral cyclin is required in reactivation from latency.
Given the global requirement for the viral cyclin in reaclivation from latency and the link between increased reactivation, the viral cyclin and chronic disease, it is important to identify host molecules that are working in this pathway. Based on the following observations: i) the viral cyclin is required for efficient reactivation from latency; ii) the viral cyclin can bypass CDK innibitors in vitro; and iii) p18 is required for cell cycle control in B lymphocytes, the main reservoir of latent γHV68 infection. I hypothesized that the viral cyclin bypasses or inaclivates p18 and/or 27 in reactivation from latency. The goal of this work is to better define the mechanism of the v-cyclin in reactivation from latency in vivo (Fig. 1 -9).
In the studies described in ihis dissertation. I have utilized wt γHV68 and a v-cyclin deficient virus to study the establishment of latency and reactivation in mice deficient for two CDKIs, p18 and p27. The work described in chapters IV and V of this dissertation identified two host proteins that are required in the reactivation process.
The work described in chapter IV identified p18 as a host molecule that is specifically bypassed by the γHV68 viral cyclin in reactivation from latency. Furthermore, an antagonist to p18 function partially overcomes the v-cyclin reactivation defect seen in C57BL/6 mice. These studies are the first to identify a host protein that restores the reactivation defect seen in mice infected with the v-cyclin deficient virus. These data identify a critical pathway in reactivation from latency, including a host negative regulator, p18, and a viral positive regulator, the v-cyclin.
Additionally, my studies described in chapter V, have found that p27 is a positive regulator of reactivation from latency, thus it is required for efficient reactivation from latency regardless of the viral cyclin status. The reduced reactivation from latency is not due to a decreased number of latently infected cells.
I have identified two important host molecules that have different and unique functions in reactivation from latency in infection with γHV68. Understanding the function of the v-cyclin and its mechanism of action during chronic infection will provide important insights regarding γHV pathogenesis thus providing critical information for the design of new therapeutics.