Reduction of Virion-Associated σ1 Fibers on Oncolytic Reovirus Variants Promotes Adaptation toward Tumorigenic Cells

Abstract

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 oncolysis in a murine melanoma model relative to that of T3wt. Our current study explored how mutations in T3v1 and T3v2 promote infectivity. Reovirions contain trimers of 1, the reovirus cell attachment protein, at icosahedral capsid vertices. Quantitative Western blot analysis showed that purified T3v1 and T3v2 virions had ϳ2-and 4-fold-lower levels of 1 fiber than did T3wt virions. Importantly, using RNA interference to reduce 1 levels during T3wt production, we were able to generate wild-type reovirus with reduced levels of 1 per virion. As 1 levels were reduced, virion infectivity increased by 2-to 5-fold per cellbound particle, demonstrating a causal relationship between virion 1 levels and the infectivity of incoming virions. During infection of tumorigenic L929 cells, T3wt, T3v1, and T3v2 uncoated the outer capsid proteins 3 and 1C at similar rates. However, having started with fewer 1 molecules, a complete loss of 1 was achieved sooner for T3v1 and T3v2. Distinct from intracellular uncoating, chymotrypsin digestion, as a mimic of natural enteric infection, resulted in more rapid 3 and 1C removal, unique disassembly intermediates, and a rapid loss of infectivity for T3v1 and T3v2 compared to T3wt. Optimal infectivity toward natural versus therapeutic niches may therefore require distinct reovirus structures and 1 levels. IMPORTANCEWild-type reovirus is currently in clinical trials as a potential cancer therapy. Our molecular studies on variants of reovirus with enhanced oncolytic activity in vitro and in vivo now show that distinct reovirus structures promote adaptation toward cancer cells and away from conditions that mimic natural routes of infection. Specifically, we found that reovirus particles with fewer molecules of the cell attachment protein 1 became more infectious toward transformed cells. Reduced 1 levels conferred a benefit to incoming particles only, resulting in an earlier depletion of 1 and a higher probability of establishing productive infection. Conversely, reovirus variants with fewer 1 molecules showed reduced stability and infectivity and distinct disassembly when exposed to conditions that mimic natural intestinal proteolysis. These findings support a model where the mode of infection dictates the precise optimum of reovirus structure and provide a molecular rationale for considering alternative reovirus structures during oncolytic therapy. M ammalian orthoreovirus (reovirus) is a nonenveloped, icosahedral virus in the Reoviridae family (1). Reovirus is nonpathogenic in humans and has long served as a safe model system for understanding icosahedral virus structure and replication. Although reovirus encodes...

Novel High-throughput Approach for Purification of Infectious Virions

“…For example, many reovirus protein functions have been characterized by comparing phenotypes among natural reovirus variants, reovirus genome reassortants, and laboratory derived reovirus mutants10111213141516171819. In conjunction with reverse genetics approaches for these viruses202122232425, it is now possible to design copious virus mutants, purify them in-slurry, and make rapid comparisons of virus proteins, virion structure, and virus replication.…”

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Structural and Functional Features of the Reovirus σ1 Tail

“…Encapsidation and folding of 1 coiled-coil-to-body transition mutants. Reovirus variants and mutants with polymorphisms in the 1 sequence encapsidate different amounts of 1 fibers (31,32). Using quantitative immunoblotting, we found that the mutant viruses encapsidate approximately 50% (rsT3SA ϩ 1 R161A and rsT3SA ϩ 1 N182A) or 30% (rsT1L 1 R161A,N182A) of the amount of 1 encapsidated by the parental rsT3SA ϩ virus (Fig.…”

Genetic Polymorphisms and Molecular Mechanisms Mediating Oncolytic Potency of Reovirus Strains

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