Investigation of Human Cytomegalovirus-Driven Modulation of Membrane Contact Sites and Organelle Remodeling Across Virus Strains and Infected Cell Types

Abstract

Subcellular organelles are dynamic regulators of cellular functions that constantly reorganize their composition and morphology in response to cellular cues. As obligate intracellular parasites, viruses commonly co-opt organelles to repurpose them for the subversion of host cell defenses and promotion of viral replication. This process is exemplified by infection with human cytomegalovirus (HCMV), which induces a pro-viral cellular state by remodeling of all major organelles. These virus-induced modulations of organelle morphology and cellular processes are reminiscent of functions controlled by membrane contact sites (MCSs). MCSs are regions of close organelle contact that are fundamental for inter-organelle communication and the coordinated control of critical cellular processes across cellular space and time. Our lab has previously shown that HCMV exhibits a nearly global increase in MCS protein abundance throughout infection. However, much of our understanding of HCMV pathogenesis, organelle remodeling, and MCS modulation has been performed using the AD169 HCMV strain. A highly passaged laboratory-adapted strain, AD169 has been shown to exhibit significant regions of genomic deletions and alterations resulting in phenotypic differences such as loss of broad cell tropism compared to HCMV infection in vivo. We therefore turn to the more clinically relevant TB40/E HCMV strain that retains its broad cell tropism with a more intact genome. Here we investigate the conservation of HCMV-mediated modulations of MCSs across different virus strains and infected cell types. We show that while infection with AD169 and TB40/E in MRC5 fibroblasts both exhibit a nearly global increase in changes to MCS protein abundance throughout infection, the extent to which they are upregulated differ, particularly later during infection. Furthermore, we discover that the ER-PM MCS protein ESYT1 is differentially regulated by infection with AD169 and TB40/E HCMV in fibroblasts. In contrast to our comparison of HCMV strains, MCS alterations in epithelial cells are more distinct than those in fibroblasts. To validate these findings and assess the relationship between MCS modulation and organelle remodeling, we turned to super-resolution microscopy. We show that while peroxisome morphological changes are also conserved between AD169 and TB40/E HCMV infection, peroxisome enlargement is more robust with infection by AD169 HCMV. Overall, our work begins to uncover the distinct modulation of MCSs and organelle structure and function by different HCMV strains and in different infected cell types.