Glycolytic enzyme PGK1 restricts viral replication via a glycolysis-independent mechanism.

The development of effective and broad-spectrum antiviral therapies remains an urgent need. Identifying key host factors that participate in the viral life cycle may provide novel targets for the development of antiviral strategies. Herein, through tandem affinity purification-mass spectrometry and siRNA screening, we identified that a glycolytic enzyme, phosphoglycerate kinase 1 (PGK1), exerts a restrictive effect on the replication of influenza A virus (IAV). Furthermore, PGK1 was found to inhibit the replication of multiple other viruses, including Sendai virus, vesicular stomatitis virus, and herpes simplex virus 1. Although PGK1 is a glycolytic enzyme, its antiviral effect is independent of glycolysis and instead relies on decreasing the stability of multiple viral proteins. The kinase activity of PGK1 was essential for reducing the stability of viral proteins, but PGK1 did not directly catalyze their phosphorylation. Phosphoproteomic analysis revealed that the heat shock protein HSP90AA1 is a critical substrate phosphorylated by PGK1. By directly catalyzing the serine phosphorylation of HSP90AA1, PGK1 decreased the molecular chaperone activity and mediated the ubiquitin-proteasome degradation of HSP90AA1, leading to the instability of viral proteins and reduced viral replication. Interestingly, the IAV nucleoprotein (NP) competitively interacted with PGK1 to disrupt the PGK1-HSP90AA1 association, thereby antagonizing the antiviral effect of PGK1. Collectively, these findings suggest that PGK1 is a novel restrictive factor that inhibits viral replication by targeting the molecular chaperone HSP90AA1. Additionally, the intermolecular interaction between PGK1 and HSP90AA1 may provide a promising target for the development of broad-spectrum antiviral drugs.