Mapping the Mitochondrial Interactome Reveals Coordinated Control of Bioenergetic Reprogramming during HCMV Infection

Abstract

Underlying cellular form and function are complex networks of Protein-Protein Interactions (PPIs). These PPIs are constantly fluctuating, exhibiting dynamic responses to maintain normal cellular processes and respond to environmental stimuli. The introduction of disease-specific proteins often disrupts these already dynamic networks, broadly reprograming the cell and exacerbating disease progression. As obligate intracellular parasites, viruses are a prime example of such disease-driven remodeling of protein interaction networks, with viral proteins introducing new layers of PPIs that broadly alter cellular function to support viral replication. Human Cytomegalovirus (HCMV) presents one of the most profound examples of such virus-induced remodeling of the host cell, causing profound morphological changes throughout its replication cycle. Notably, HCMV infection induces severe mitochondrial fragmentation, simultaneously disrupting immune responses from this compartment while enhancing mitochondrial bioenergetics. This presents an interesting paradox to well established mitochondrial structure-function relationship paradigms across multiple model systems. Critical to our mechanistic understanding of mitochondrial dynamics during HCMV infection is elucidating the temporal PPI network disrupted by viral proteins localized to the mitochondria. Previous studies from our lab found that the viral protein pUL13 plays a key role in the HCMV-driven increase in mitochondrial bioenergetics. pUL13 alone is sufficient to upregulate oxidative phosphorylation and remodel the mitochondrial architecture. However, the mechanisms underlying the impact of pUL13 on mitochondrial bioenergetic and its contribution to HCMV-induced changes in mitochondrial structure have yet to be investigated. To understand how HCMV ‘s pUL13 alters mitochondrial protein interaction networks, we developed a novel platform integrating mitochondrial fractionation and Thermal Proximity Coaggregation Assay (TPCA) methods, herein referred to as mito-TPCA. We demonstrate that our optimized experimental framework improves the detection of mitochondrial-specific PPIs compared with whole-cell TPCA analysis. Following optimization, we leveraged this mito-TPCA workflow to compare the mitochondrial PPIs composing infections with wild-type HCMV strain AD169 or an AD169 virus lacking pUL13 (ΔUL13). Using these virus strains we uncovered the specific contribution of pUL13 on HCMV-driven changes in mitochondrial networks. Using this platform, we uncovered previously unreported pUL13 interactors, including the uncharacterized viral protein pUL15a, and reveal that pUL13 expression modulates cristae architecture and several ETC respiratory complexes during HCMV infection. We further validated these interactions between pUL13 and ETC complexes via orthogonal confocal microscopy and biochemical approaches. Furthermore, results from our mito-TPCA analysis revealed that the uncharacterized pUL15a is not only an important interactor of UL13 but also interacts with several subunits of Complex I, suggesting a role for this factor in the HCMV-driven rewiring of cellular metabolism. To better understand the contribution of pUL15a, we generated a stable pUL15a expressing cell line and mapped its impact on the host cell proteome via liquid-chromatography based mass spectrometry (LC-MS) analysis. Finally, to better understand how the clinical significance of HCMV-driven alterations in mitochondrial PPIs, we conduct a whole-cell TPCA analysis in cells infected with a clinical HCMV strain TB40/E and conducted a comparative analysis between our findings for both HCMV TB40/E and AD169 strains. Our findings have uncovered infection specific differences in mitochondrial complex architecture between the two strains.