Sendai virus C proteins are categorically nonessential gene products but silencing their expression severely impairs viral replication and pathogenesis

Background: The P/C mRNA of Sendai virus (SeV), a prototypic member of the family Paramyxoviridae in the Mononegavirales superfamily comprising a large number of nonsegmented negative strand RNA viruses, encodes a nested set of accessory proteins, C', C, Y1 and Y2, referred to collectively as C proteins, initiating, respectively, at ACG/81 and AUGs/114, 183, 201 in the +1 frame relative to the ORF of phospho (P) protein, the smaller subunit of RNA polymerase. Among them, C is the major species expressed in infected cells at a molar ratio which is several-fold higher than the other three. However, their function has remained an enigma. It has not even been established whether or not the C proteins are essential for viral replication. Many other viruses in Mononegavirales encode C-like proteins, but their roles also remain to be defined. Results: By taking advantage of a recently developed reverse genetics system to recover infectious SeV from cDNA, we created mutants in which C protein frames were variously silenced. C/C'(-) viruses which did riot express C and C', but did express Y1 and Y2, were severely attenuated in replication in tissue culture cells of various species and tissues, as well as in embryonated chicken eggs. More notably, they were almost totally incapable of growing productively in-and hence nonpathogenic for mice-the natural host. Both gene expression and genome replication appeared to be impaired in C/C'(-) viruses. Additionally silencing the Y1 and Y2 expression was also possible, and a critically impaired but viable clone, the 4C(-) virus, was isolated which expressed none of the four C proteins. Conclusion: SeV C proteins are categorically nonessential gene products, but greatly contribute to full replication capability in vitro and are indispensable for in viva multiplication and pathogenesis. This study represents the first comprehensive functional assessment of the accessary C protein for Mononegavirales.