Avian reovirus nonstructural protein μNS forms viroplasm-like inclusions and recruits protein σNS to these structures

Abstract

The M3 genome segment of avian reovirus 1733, which encodes the nonstructural protein μNS, is 1996 nucleotides long and contains a long open reading frame that is predicted to encode a polypeptide of 635 amino acid residues. Examination of the deduced amino acid sequence of μNS revealed the presence of two regions near its carboxyl terminus with a high probability of forming α-helical coiled coils. Expression of the M3 gene in both infected and transfected cells revealed that this gene specifies two protein isoforms that are recognized by a μNS-specific antiserum. Only the larger μNS isoform, but not the smaller one, interacts with the nonstructural protein σNS in infected cells, suggesting that the two isoforms play different roles during avian reovirus infection. In the second part of this study, we show that μNS and the nonstructural protein σNS colocalize throughout the viral life cycle in large and small phase-dense globular cytoplasmic inclusions, which are believed to be the sites of viral replication and assembly. Individual expression of these proteins in transfected cells of avian and mammalian origin revealed that while μNS is able to form inclusions in the absence of other viral proteins, σNS distributes diffusely throughout the cytoplasm in the absence of μNS. These data suggest that μNS is the minimal viral factor required for inclusion formation during avian reovirus infection. On the other hand, our findings that σNS associates with μNS in infected cells, and that σNS colocalizes with μNS in viroplasm-like inclusions when the two proteins are coexpressed in transfected cells, suggest that μNS mediates the association of σNS to inclusions in avian reovirus-infected cells.