Reduction of Virion-Associated 1 Fibers on Oncolytic Reovirus Variants Promotes Adaptation toward Tumorigenic Cells

Wild-type mammalian orthoreovirus serotype 3 Dearing (T3wt) is nonpathogenic in humans but preferentially infects and kills cancer cells in culture and demonstrates promising antitumor activity in vivo. Using forward genetics, we previously isolated two variants of reovirus, T3v1 and T3v2, with increased infectivity toward a panel of cancer cell lines and improved in vivo onco-lysis in a murine melanomamodel relative to that of T3wt. Our current study explored howmutations in T3v1 and T3v2 pro-mote infectivity. Reovirions contain trimers of1, the reovirus cell attachment protein, at icosahedral capsid vertices. Quantita-tiveWestern blot analysis showed that purified T3v1 and T3v2 virions had2- and 4-fold-lower levels of1 fiber than did T3wt virions. Importantly, using RNA interference to reduce1 levels during T3wt production, we were able to generate wild-type reovirus with reduced levels of1 per virion. As1 levels were reduced, virion infectivity increased by 2- to 5-fold per cell-bound particle, demonstrating a causal relationship between virion1 levels and the infectivity of incoming virions. During infection of tumorigenic L929 cells, T3wt, T3v1, and T3v2 uncoated the outer capsid proteins3 and1C at similar rates. How-ever, having started with fewer1 molecules, a complete loss of1 was achieved sooner for T3v1 and T3v2. Distinct from intra-cellular uncoating, chymotrypsin digestion, as a mimic of natural enteric infection, resulted in more rapid3 and1C removal, unique disassembly intermediates, and a rapid loss of infectivity for T3v1 and T3v2 compared to T3wt. Optimal infectivity to-ward natural versus therapeutic niches may therefore require distinct reovirus structures and1 levels