SARS-CoV-2 3CLpro inhibits the replication of influenza viruses through the cleavage of NP and PA.
The co-circulation of multiple viruses can lead to distinct pathological outcomes, yet how severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection influences other viral infections remains poorly understood, despite its documented high frequency during the pandemic. In this study, we investigated how the proteolytic activity of SARS-CoV-2 3C-like protease (3CLpro) influences the replication of influenza A virus. In silico analysis identified candidate 3CLpro cleavage sites across numerous viral proteins, and biochemical assays confirmed that 3CLpro catalyzes the degradation of influenza virus nucleoprotein (NP) and polymerase acidic protein (PA) in a manner requiring its protease activity. This degradation of NP and PA, which are essential for viral genome packaging and transcription, disrupted the influenza replicative cycle and suppressed viral replication, both upon ectopic 3CLpro expression and during SARS-CoV-2 infection. Our data uncover a direct, enzyme-based mechanism by which SARS-CoV-2 can suppress influenza virus replication during coinfection. We provide a molecular explanation for the sharp, global decline in influenza activity observed during the COVID-19 pandemic and illustrate how enzymatic weapons of one virus can be repurposed to restrain a competing pathogen.