Replication of mutant strains of oncolytic reovirus in different cell lines

906 Murine NIH-3T3 cells are poorly infected by mammalian reovirus but support productive infection upon cell transformation by H-Ras(G12V), leading to the idea that reovirus could be used as an “oncolytic” virus. It was initially believed that the inhibition of cellular interferon-inducible RNA-dependent protein kinase, PKR, was responsible for increased permissivity of Ras-transformed cells to reovirus. Other data later suggested an enhanced uncoating of the virions, due to increased levels of lysosomal proteases in transformed cells. Literature data also indicate that Ras activation is neither sufficient nor necessary for productive reovirus infection, depending on the cell types. --- Additional studies are thus needed to better comprehend the cellular and viral factors that ultimately determine the ability of reovirus to productively infect “normal” and/or “transformed” cells. --- A mutant reovirus (P4L-12) was previously isolated in our laboratory, based on increased sensitivity of this virus to interferon and on its better ability to discriminate between parental and Ras-transformed NIH-3T3 cells. Another mutant (SC1-PV) was obtained from persistently-infected murine fibroblasts; this virus requires only low levels of proteases to be uncoated, compared to the wild type virus. Our recent sequencing data revealed that both mutant viruses exhibit amino acid substitutions in their σ3 protein. This is consistent with the known importance of σ3 in the virus’ ability to be uncoated, as well as accumulating evidence suggesting the central role of σ3 in determining reovirus’ susceptibility to PKR. Mutant and wild-type viruses were used to infect a panel of murine and human cell lines; viral titers were then determined by TCID50 and plaque assays. Interestingly, both mutant viruses can infect cell lines that are relatively resistant to the wild type virus due to inefficient uncoating in these cells. Furthermore, the infectivity of P4L-12 was either unaffected or even slightly decreased by prior chymotrypsin treatment, while infectivity of wild type and SC1-PV was increased, as judged by virus titer and viral plaque size. However, P4L-12 was strongly restricted in cells that produce and respond to interferon, and thus this virus does not form plaques on these cells. We also observed a correlation between the ability of a given cell line to form colonies in soft agar and its ability to support reovirus replication, rather than a correlation with the Ras activation status of the cells. --- Altogether, our data thus support the idea that cellular factors affecting reovirus replication are complex and linked to pathways leading to cellular transformation. We further suggest that viral strains should be chosen or manipulated to optimize their oncolytic potential. The σ3 protein appears as the most likely viral factor to be involved in modulating the virus’ ability to replicate in different normal or transformed cell types.

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