The reovirus μ2 C-terminal loop inversely regulates NTPase and transcription functions versus binding to factory-forming μNS and promotes replication in tumorigenic cells.

Wild type reovirus serotype 3 'Dearing PL strain' (T3wt) is being heavily evaluated as an oncolytic and immunotherapeutic treatment for cancers. Mutations that promote reovirus entry into tumor cells were previously reported to enhance oncolysis; herein we aimed to discover mutations that enhance the post-entry steps of reovirus infection in tumor cells. Using directed evolution, we identified that reovirus variant T3v10M1 exhibited enhanced replication relative to T3wt on a panel of cancer cells. T3v10M1 contains an alanine-to-valine substitution (A612V) in the core-associated μ2, which was previously found to have NTPase activities in virions and to facilitate virus factory formation by association with μNS. Paradoxically, the A612V mutation in μ2 from T3v10M1 was discovered to impair NTPase activities and RNA synthesis, leading to five-fold higher probability of abortive infection for T3v10M1 relative to T3wt. The A612V mutation resides in a previously uncharacterized C-terminal region that juxtaposes the template entry site of the polymerase μ2; our findings thus support an important role for this domain during virus transcription. Despite crippled onset of infection, T3v10M1 exhibited greater accumulation of viral proteins and progeny during replication, leading to increased overall virus burst size. Both Far-Western and co-immunoprecipitation approaches corroborated that the A612V mutation in μ2 increased association with the non-structural virus protein μNS and enhances burst size. Altogether the data supports that mutations in the C-terminal loop domain of μ2 inversely regulate NTPase and RNA synthesis versus interactions with μNS, but with a net gain of replication in tumorigenic cells.SIGNIFICANCEReovirus is a model system for understanding virus replication but also a clinically relevant virus for cancer therapy. We identified the first mutation that increases reovirus infection in tumorigenic cells by enhancing post-entry stages of reovirus replication. The mutation is in a previously uncharacterized c-terminal region of the M1-derived μ2 protein, which we demonstrated affects multiple functions of μ2; NTPase, RNA synthesis, inhibition of antiviral immune response and association with the virus replication factory-forming μNS protein. These findings promote a mechanistic understanding of viral protein functions. In the future, the benefits of μ2 mutations may be useful for enhancing reovirus potency in tumors.