Characterizing the Mitochondrial Virus-Host Interactome During HCMV Infection

Abstract

In order to successfully infect its host, a virus must both evade the cellular immune response and hijack cellular machinery and metabolites that it subsequently uses for the efficient production of viral progeny. The dynamic virus-host interactions that achieve these goals often lead to phenotypic effects in infected cells. For example, during human cytomegalovirus (HCMV) infection, mitochondrial changes such as increased fragmentation and motility have been observed, as well as changes in the regulation of a variety of metabolic pathways. A recent study that characterized changes in cellular organelles as well as their proteomes during infection resulted in the identification of nine viral proteins which localize to the mitochondria during infection. The majority of these mitochondrial-localized viral proteins have not been characterized, except for pUL37, a viral protein most known for its role in cell death suppression during infection. In order to characterize virus-host interactions in the mitochondria in the context of natural infection, a platform employing tagged viral strains in conjunction with immunoaffinity purification (IP)-mass spectrometry (MS)-based techniques was validated by comparison of these tagged viruses with WT and Free GFP viruses. The use of this IP-MS system to explore the protein interactions of pUL37 both confirmed the findings of previous studies and expanded our understanding of this protein's multifaceted role in infection, as it appears to modulate apoptosis, mitochondrial fragmentation, the MICOS complex, as well as antiviral signaling. Follow-up study of functionally promising pUL37 interactors will be conducted using CRISPR/Cas9 knockout cell lines that are currently being validated. After the efficacy of this approach in the study of protein interactions of mitochondrial-localized viral proteins was thus confirmed, the system was used for the study of pUL13, a viral protein lacking any characterization aside from localization studies. Whole cell IP analysis was then employed to provide the first characterization of pUL13, identifying its diverse functions in the modulation of cell-cell adhesion at the plasma membrane, N-linked glycosylation in the ER/Golgi, and apoptotic signaling in the mitochondria. The long-term goal of this project is to create a global mitochondrial virus-host interactome during HCMV infection. Though only two of the nine mitochondrial-localized viral proteins were characterized in this thesis, evidence for significant overlap in the protein interactions and functions of these viral proteins in the mitochondria is already apparent. This interactome resource will prove to be invaluable in improving our understanding of viral modulation and remodeling of the mitochondria during HCMV infection.