NSP5 phosphorylation regulates the fate of viral mRNA in rotavirus infected cells

Elucidation of the function of the non-structural rotavirus proteins during infection is difficult in the absence of a reverse genetic system. To study the role of NSP5, nonstructural phosphoprotein NSP5, we constructed a reassortant strain (SACC11) in the SA 11 background that harbours a heterologous segment 11 encoding a variant protein (h-NSP5). Cells infected by SACC11 produced viral polypeptides at earlier times than SA 11 infected cells while showing less accumulation of genomic dsRNA. These changes suggested that NSP5 might direct viral messenger RNA to protein synthesis or genome replication. Distinct patterns of proteins were shown to form complexes with NSP5 in co-immunoprecipitation studies with SA 11 and SACC 11 infected cells. Recombinant h-NSP5 from either bacteria or eucaryotic cells migrated faster in PAGE suggesting that it was hypophosphorylated. Indeed, the kinase inhibitor H-7 enhanced translation of viral proteins in SA11 but not SACC11 infected cells suggesting that NSP5 function in the regulation of the fate of viral positive strand RNA is mediated by phosphorylation.