Characterization of Murine cytomegalovirus dUTPase homolog, M72, and investigations into novel interacting host factors

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2018-08-17

Authors

Gopal, Sandhya

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Abstract

Human cytomegaloviruses (HCMV), a beta herpesvirus, presents a challenge in terms of morbidity and mortality associated with immunocompromised patients and congenital infections. One approach to tackle issues associated with HCMV infection is to understand multiple facets of host-pathogen interactions. However, since herpesviruses are species specific, it becomes imperative to utilize small animal model systems to investigate in the context of natural host. I utilized a genetically and biologically related Murine cytomegalovirus (MCMV) with mice as the small model system. For my dissertation research, I focused on the MCMV dUTPase homolog M72. The dUTPase homologs in the herpesvirus family are classified as core genes and significant roles are ascribed to them. However, little was known specifically about the role of dUTPase homologs among beta herpesviruses. Human cytomegalovirus (HCMV) UL72 and murine cytomegalovirus (MCMV) M72 were designated as dUTPases based on limited sequence and positional homology. I found that M72 is not enzymatically active as a dUTPase and is expressed as a leaky-late gene product with multiple protein isoforms. Additionally, M72 augments virus replication in vitro and in the acute phase in vivo. To begin to understand M72 function, I took a proteomics approach and identified interacting host protein partners. I identified and confirmed interaction of M72 with the eukaryotic chaperonin tailless complex protein-1 (TCP-1) ring complex (TRiC) or chaperonin containing tailless complex polypeptide 1 (CCT). Accumulating biochemical evidence indicates M72 forms homo-oligomers and is a substrate of TRiC/CCT. To explore the role of M72 beyond protein folding, I also identified components of Carbon catabolite repression 4 (CCR4)-negative on TATA-less (NOT) or CCR4-NOT complex, including the 182-kDa Tankyrase 1 binding protein (TAB182) as M72 candidate interacting proteins. My current work suggests that CCR4-NOT complex subunit 1 (CNOT1) is necessary for MCMV replication. Additionally, M72 mediates its own function at least partially via CNOT1 during virus replication. Taken together, this research provides the first evidence of a beta herpesvirus dUTPase homolog’s contribution to viral replication. My dissertation research has helped uncover host proteins novel for herpesviruses as interacting partners. In addition, one of these host factors, CNOT1, contributes to M72 mediated function

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