Cleavage susceptibility of reovirus attachment protein σ1 during proteolytic disassembly of virions is determined by a sequence polymorphism in the σ1 neck

A requisite step in reovirus infection of the murine intestine is proteolysis of outer-capsid proteins to yield infectious subvirion particles (ISVPs). When converted to ISVPs by intestinal proteases, virions of reovirus strain type 3 Dearing (T3D) lose 90% of their original infectivity due to cleavage of viral attachment protein sigma 1. In an analysis of eight field isolate strains of type 3 reovirus, we identified one additional strain, type 3 clone 31 (T3C31), that loses infectivity and undergoes sigma 1 cleavage upon conversion of virions to ISVPs. We examined the sigma 1 deduced amino acid sequences of T3D and the eight field isolate strains for a correlation between sequence variability and sigma 1 cleavage. The sigma 1 proteins of T3D and T3C31 contain a threonine at amino acid position 249, whereas an isoleucine occurs at this position in the sigma 1 proteins of the remaining strains. Thr(249) occupies the d position of a heptad repeat motif predicted to stabilize sigma 1 oligomers through alpha-helical coiled-coil interactions. This region of sequence comprises a portion of the fibrous tail domain of sigma 1 known as the neck Substitution of Thr(249) with isoleucine or leucine resulted in resistance to cleavage by trypsin, whereas replacement with asparagine did not affect cleavage susceptibility. These results demonstrate that amino acid position 249 is an independent determinant of T3D sigma 1 cleavage susceptibility and that an intact heptad repeat is required to confer cleavage resistance. We performed amino-terminal sequence analysis on the sigma 1 cleavage product released during trypsin treatment of T3D virions to generate ISVPs and found that trypsin cleaves sigma 1 after Arg(245). Thus, the sequence polymorphism at position 249 controls cleavage at a nearby site in the neck region. The relevance of these results to reovirus infection in vivo was assessed by treating virions,vith the contents of a murine intestinal wash under conditions that result in generation of ISVPs. The pattern of sigma 1 cleavage susceptibility generated by using purified protease was reproduced in assays using the intestinal wash. These results provide a mechanistic explanation for sigma 1 cleavage during exposure of virions to intestinal proteases and may account for certain strain-dependent patterns of reovirus pathogenesis.