The antiviral sirtuin 3 bridges protein acetylation to mitochondrial integrity and metabolism during human cytomegalovirus infection

Author summary Given their functions in cellular metabolism and immune responses, mitochondria are targeted and disrupted by numerous prevalent viral pathogens, including human cytomegalovirus (HCMV). To characterize mechanisms underlying mitochondrial regulation during HCMV infection in human fibroblasts, this study integrates enzyme-substrate interaction studies, mass spectrometry quantification of protein abundance and acetylation, mutagenesis, microscopy, and virology assays. These analyses establish a link between the mitochondrial acetylation status and mitochondrial structure and metabolism during HCMV infection. We demonstrate that the mitochondrial deacetylase SIRT3 acts in host defense by modulating proteins that regulate mitochondrial structure and fatty acid oxidation. SIRT3 helps to maintain mitochondrial integrity through several mechanisms, including regulation of mitochondrial pH, membrane potential, and the balance between mitochondrial fission and fusion. As excess mitochondrial acetylation is detrimental to mitochondrial metabolism, the virus-induced alterations in SIRT3 functions and mitochondrial acetylation may be linked to known HCMV pathologies, such as the metabolic syndrome and cardiac hypertrophy. Regulation of mitochondrial structure and function is a central component of infection with viruses, including human cytomegalovirus (HCMV), as a virus means to modulate cellular metabolism and immune responses. Here, we link the activity of the mitochondrial deacetylase SIRT3 and global mitochondrial acetylation status to host antiviral responses via regulation of both mitochondrial structural integrity and metabolism during HCMV infection. We establish that SIRT3 deacetylase activity is necessary for suppressing virus production, and that SIRT3 maintains mitochondrial pH and membrane potential during infection. By defining the temporal dynamics of SIRT3-substrate interactions during infection, and overlaying acetylome and proteome information, we find altered SIRT3 associations with the mitochondrial fusion factor OPA1 and acetyl-CoA acyltransferase 2 (ACAA2), concomitant with changes in their acetylation levels. Using mutagenesis, microscopy, and virology assays, we determine OPA1 regulates mitochondrial morphology of infected cells and inhibits HCMV production. OPA1 acetylation status modulates these functions, and we establish K834 as a site regulated by SIRT3. Control of SIRT3 protein levels or enzymatic activity is sufficient for regulating mitochondrial filamentous structure. Lastly, we establish a virus restriction function for ACAA2, an enzyme involved in fatty acid beta-oxidation. Altogether, we highlight SIRT3 activity as a regulatory hub for mitochondrial acetylation and morphology during HCMV infection and point to global acetylation as a reflection of mitochondrial health.

Automated summary

This study highlights the role of SIRT3 as a key regulator of mitochondrial acetylation and morphology during HCMV infection, affecting both the structural integrity and metabolism of mitochondria. The findings suggest that virus-induced alterations in SIRT3 functions and mitochondrial acetylation are linked to known HCMV pathologies, such as metabolic syndrome and cardiac hypertrophy. Through various analyses, the study demonstrates the importance of SIRT3 in maintaining mitochondrial health and suppressing virus production during infection.