Investigating Sirtuin-3 in Host Defense Against HCMV Infection

Abstract

Successful viral infection relies upon virus modulation of host cell processes for both progeny replication and immune response evasion. In human cytomegalovirus (HCMV) infection, impacted processes include altered regulation of metabolic pathways and mitochondrial biogenesis and respiration, as well as morphological disruption. Recent work has found NAD+ -dependent deacetylase sirtuin-3 (SIRT3) to act in host defense against RNA and DNA viruses, including HCMV, though the mechanism through which this is accomplished remains unknown. Given the well-established role of SIRT3 in metabolic regulation, this thesis investigated the hypothesis that SIRT3 assists antiviral response via counter-regulation of host factors in pathways modulated by viral infection. Characterization of SIRT3 interactions at different stages of infection was conducted using an immunoaffinity purification (IP)-mass spectrometry (MS)-based methodology to analyze infected cells with overexpression of tagged SIRT3. Functional classification of identified interactors emphasized oxidative stress response, mitochondrial protein synthesis, ATP synthesis, the TCA cycle, glucose metabolism, amino acid metabolism, and fatty acid metabolism as key cellular processes of SIRT3 and viral association. Viral titer assays of infected nigericin-pretreated cells additionally highlighted regulation of mitochondrial membrane potential as a pathway of interest, supported by the presence of ATP synthase interactions. Comparison of interaction networks across time points of infection indicate the role of SIRT3 to be temporally dynamic, with most enhanced associations during early infection at 24 hpi. Furthermore, IP-MS uncovered compelling interactors found to be of interest due to strong changes in interaction abundance compared to mock at every stage of infection. The role of deacetylase activity in SIRT3 2 antiviral function was studied through comparison of interaction abundance findings and previously established acetylation data for known substrates. Results suggest the presence of strong deacetylase function during early infection that is attenuated as infection progresses. This substrate comparison was supported by visualized inhibition of mitochondrial fragmentation by catalytically active SIRT3 in contrast to a catalytically inactive mutant. Taken altogether, the findings of this thesis begin to characterize SIRT3 function in host defense as an early mechanism mediated through deacetylase activity in identified mitochondrial pathways. Continuation of this initial work will further elucidate the underlying mechanism behind the antiviral role of SIRT3 to hopefully assist in the design of novel HCMV therapies.