Current Work

Genetic determinants of reovirus spread from the murine respiratory tract

Reovirus type 3 strain Dearing is currently in stage I, II and III trials for treatment of human cancers. Understanding the molecular determinants of reovirus spread in the host is essential to developing a safe and efficacious oncolytic therapy. Genetic studies have associated reovirus spread with the S1 gene, which encodes two proteins: the cell attachment protein sigma-1 and a nonstructural protein, sigma-1s, in overlapping reading frames. Biochemical studies have correlated protease sensitivity of the cell attachment protein, sigma-1, with the failure of some reovirus strains to replicate and spread from the enteric tract. Recent studies have revealed that sigma-1s is required for hematogenous spread from the enteric tract. We are using reverse genetics to identify the determinants that regulate spread of two laboratory clones of the Dearing isolate: Dearing-Cashdollar and Dearing-Fields. We found that these Dearing isolates have a number of distinct biological properties, including their capacity to spread from the respiratory tract. The Fields and Cashdollar S1 genes differ at two positions, nucleotides 77 and 1234. Nucleotide 1234 impacts an amino acid in the sigma-1 head; nucleotide 77 impacts an amino acid in the sigma-1 tail, as well as one in sigma-1s. To determine if one or both of the S1 gene products contributes to viral spread from the respiratory tract, we engineered viruses on a Dearing-Fields background to express recombinant sigma-1 and sigma-1s proteins. Sigma-1s null versions of wild-type and recombinant viruses were also constructed. Following intranasal inoculation, we found that wild-type Dearing-Fields, and Dearing-Fields viruses with single Dearing-Cashdollar nucleotide changes in S1 replicated in the lungs to similar titers. Whereas Dearing-Fields did not efficiently spread to the lymph nodes or spleen, viruses with Dearing-Cashdollar changes at nucleotide 77 or 1234 both spread to these locations, but failed to achieve titers comparable to Dearing-Cashdollar. These results suggest that both sigma-1 and sigma-1s proteins play a role in reovirus spread from the respiratory tract. We are currently examining the spread phenotype of recombinant viruses containing both nucleotide changes in the S1 gene and viruses that do not express sigma-1s.