Mitochondrial OXPHOS restricts SARS-CoV-2 replication

SARS-CoV-2 rewires host metabolism to optimize virus production. While glycolysis is necessary for virus production, the importance of mitochondrial oxidative phosphorylation (OXPHOS) is unknown. The mitochondrial DNA (mtDNA) codes for 13 critical OXPHOS polypeptides plus the 22 tRNAs and 2 rRNAs for mitochondrial protein synthesis. We discovered an approximately 5 to 100-fold greater SARS-CoV-2 virus production in infected human ACE2-expressing A549 lung cells when OXPHOS was inhibited by mtDNA depletion (ρ⁰ cells), inhibition of mitochondrial translation with chloramphenicol (CAP), or chemical inhibition of OXPHOS complexes. OXPHOS inhibition led to a marked increase in the size and distribution of viral replication centers and accelerated the production and release of infectious particles, occurring approximately two hours earlier than in parental A549-ACE2 (WT) cells. Subsequently, we found that increased glycolytic capacity was required for enhanced viral replication while differences in innate immune pathway activation were not. Reintroduction of mtDNA from a well-defined maternal lineage into the ρ⁰ cells reinstated OXPHOS, impaired SARS-CoV-2 replication, and reversed associated viral and glycolytic correlates. Thus, metabolic balance regulates SARS-CoV-2 replication, with OXPHOS exerting an antiviral effect.