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"KAPOSI`S SARCOMA-ASSOCIATED HERPESVIRUS (KSHV) LYTIC SWITCH RTA SUBVERTS TUMORIGENIC CELL SIGNALING REGULATORS PIN1 AND RBP-JK TO ENHANCE REACTIVATION FROM LATENCY"

by
Jonathan Colin Guito
Microbiology and Molecular Genetics Program
B.S., B.A., 2008, Rowan University, Glassboro, NJ



Thesis Advisor: David M. Lukac, Ph.D.
Associate Professor
Department of Microbiology and Molecular Genetics

Thursday, May 15, 2014
10:00 A.M., ICPH-Auditorium


Abstract

Kaposiís sarcoma-associated herpesvirus (KSHV) causes Kaposiís sarcoma (KS), an AIDS-defining illness, and primary effusion lymphoma (PEL). KSHV-infected cells contain predominantly latent virus, with a small subpopulation undergoing lytic reactivation. Rta protein is the lytic switch that reactivates virus, forming transactivation-competent complexes with Notch pathway effector RBP-JK and promoter DNA. Rta transactivation is essential for viral replication, but is functionally inefficient. We hypothesize that this balances viral lytic cycle oncogene expression with productive replication, the latter of which culminates in infectious virus release and host cell lysis.
Interaction with cellular and viral cofactors, such as RBP-JK, is also essential for Rta-mediated reactivation, and it is anticipated that others remain to be characterized. We previously demonstrated that Rta protein forms tetramers required for RBP-JK association. The broad Rta region that binds to RBP-JK localized to amino acids (aa) 414-530; however, the specific critical, non-canonical motif recognized by RBP-JK is unknown. Rta tetramer formation, meanwhile, was determined by prolines in an N-terminal leucine repeat (LR). Strikingly, gammaherpesvirinae comparison reveals prolines constitute 17% of conserved residues throughout Rta, which is also highly phosphorylated in vivo.
We thus hypothesize that proline-directed phosphorylation regulates Rta activity by controlling binding to peptidyl-prolyl cis/trans isomerases (PPIases). Cellular PPIase Pin1 binds specifically to phosphoserine- or phosphothreonine-proline motifs in target proteins. Pin1 dysregulation is implicated in myriad human cancers and can be subverted by viruses.
Our data show that KSHV Rta protein contains S/T-P motifs, binds directly to Pin1, is transcriptionally upregulated by Pin1, is relocalized in Pin1-coexpressing nuclei and has enhanced transactivation with Pin1 at two viral promoters in uninfected cells. Pin1ís effect, however, suggests a rheostat-like influence on Rta function. In infected cells, we found that endogenous Pin1 is active during reactivation and enhances Rta-dependent delayed-early gene expression and viral DNA replication. Surprisingly, Pin1 strongly inhibits late gene synthesis and infectious virion production. We thus propose that Pin1 is a unique, dose-dependent molecular timer that promotes viral lytic cycle gene expression in the absence of host cell lysis. In a parallel structure-function study, we identified five peptides within the Rta aa 414-530 region that, when mutated, have strongly diminished RBP-JK binding affinities. Intriguingly, most of these peptides are enriched in glutamines and prolines, and in silico analysis suggested the minimal residues QPPQ and PLP as promising candidates as the bona fide, novel RBP-JK recognition motif for Rta.


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