Avian reovirus nonstructural protein microNS forms viroplasm-like inclusions and recruits protein sigmaNS to these...

The M3 genome segment of avian reovirus 1733, which encodes the nonstructural protein microNS, 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 microNS revealed the presence of two regions near its carboxyl terminus with a high probability of forming alpha-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 microNS-specific antiserum. Only the larger microNS isoform, but not the smaller one, interacts with the nonstructural protein sigmaNS 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 microNS and the nonstructural protein sigmaNS 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 microNS is able to form inclusions in the absence of other viral proteins, sigmaNS distributes diffusely throughout the cytoplasm in the absence of microNS. These data suggest that microNS is the minimal viral factor required for inclusion formation during avian reovirus infection. On the other hand, our findings that sigmaNS associates with microNS in infected cells, and that sigmaNS colocalizes with microNS in viroplasm-like inclusions when the two proteins are coexpressed in transfected cells, suggest that microNS mediates the association of sigmaNS to inclusions in avian reovirus-infected cells.