Abstract
The influenza virus polymerase transcribes and replicates the viral genome. The proper timing and balance of polymerase activity is important for successful replication. Genome replication is controlled in part by phosphorylation of NP that regulates assembly of the replication machinery. However, it remains unclear whether phosphorylation directly regulated polymerase activity. Here we identified polymerase phosphosites that control its function. Mutating phosphosites in the catalytic subunit PB1 altered polymerase activity and virus replication. Biochemical analyses revealed phosphorylation events that disrupted global polymerase function by blocking the NTP entry channel or preventing RNA binding. We also identified a regulatory site that split polymerase function by specifically suppressing transcription. These experiments show that host kinases phospho-regulate viral RNA synthesis directly by modulating polymerase activity and indirectly by controlling assembly of replication machinery. Further, they suggest polymerase phosphorylation may bias replication versus transcription at discrete times or locations during the infectious cycle.
Highlights
All RNA viruses encode machinery both to express viral transcripts and to replicate genomes
We identified a phosphorylation site in the catalytic subunit PB1 that selectively disables transcription, but not replication
Negative sense RNA viruses must first transcribe using virally-encoded RNA-dependent RNA polymerases (RdRPs) that are packaged into virions
Summary
All RNA viruses encode machinery both to express viral transcripts and to replicate genomes. Negative sense RNA viruses must first transcribe using virally-encoded RNA-dependent RNA polymerases (RdRPs) that are packaged into virions. The viral RdRP subsequently replicates the genome, often with the help of protein products from the recently produced mRNA. Viruses employ diverse strategies to control the abundance of virally-derived RNAs. Many RNA viruses rely on RdRP co-factors whose activity is dynamically regulated by post-translational modifications. The Ebola virus polymerase is regulated by the viral transcription factor VP30. Dynamic phosphorylation of the M2-1 protein from respiratory syncytial virus regulates viral transcription. The dynamic and fully reversible nature of phosphorylation enables localized and temporal control of viral proteins and may help progression through the infectious cycle. Phosphorylation of polymerase co-factors is a common strategy to regulate transcription and replication. Influenza A virus and other members of Orthomyxoviridae do not encode polymerase co-factors and it remains unclear how their polymerases are regulated
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