ABSTRACT
[This corrects the article DOI: 10.1016/j.omto.2023.100743.].
ABSTRACT
Wild-type reovirus serotype 3 Dearing (T3wt), a non-pathogenic intestinal virus, has shown promise as a cancer therapy in clinical trials, but it would benefit from an increased potency. Given that T3wt is naturally adapted to the intestinal environment (rather than tumors), we genetically modified reovirus to improve its infectivity in cancer cells. Various reovirus mutants were created, and their oncolytic potency was evaluated in vitro using plaque size as a measure of virus fitness in cancer cells. Notably, Super Virus 5 (SV5), carrying five oncolytic mutations, displayed the largest plaques in breast cancer cells among the mutants tested, indicating the potential for enhancing oncolytic potency through the combination of mutations. Furthermore, in a HER2+ murine breast cancer model, mice treated with SV5 exhibited superior tumor reduction and increased survival compared with those treated with PBS or T3wt. Intriguingly, SV5 did not replicate faster than T3wt in cultured cells but demonstrated a farther spread relative to T3wt, attributed to its reduced attachment to cancer cells. These findings highlight the significance of increased virus spread as a crucial mechanism for improving oncolytic virus activity. Thus, genetic modifications of reovirus hold the potential for augmenting its efficacy in cancer therapy.
ABSTRACT
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.
