Sendai virus C proteins regulate viral genome and antigenome synthesis to dictate the negative genome polarity.

The order Mononegavirales comprises a large number of nonsegmented negative-strand RNA viruses (NNSVs). How the genome polarity is determined is a central issue in RNA virus biology. Using a prototypic species, vesicular stomatitis virus (VSV), it has been established that the negative polarity of the viral genome is defined solely by different strengths of the cis-acting replication promoters located at the 3' ends of the genome and antigenome, resulting in the predominance of the genome over the antigenome. This VSV paradigm has long been applied for the Mononegavirales in general without concrete proof. We now found that another prototypic species, Sendai virus (SeV), undergoes a marked shift from the early antigenome-dominant to the late genome-dominant phase during the course of infection. This shift appeared to be governed primarily by the expression of the accessory C protein, because no such shift occurred in a recombinant SeV with the C gene deleted, and antigenomes were dominant throughout infection, generating antigenome-dominant and noninfectious progeny virions. Therefore, we proposed for the first time a trans-regulatory mechanism, the SeV paradigm, to dictate the genome polarity of an NNSV. A series of promoter-swapped SeV recombinants suggested the importance of the primary as well as secondary structures of the promoters in this trans-regulation.

Conserved charged amino acids within Sendai virus C protein play multiple roles in the evasion of innate immune responses.

One of the accessory proteins of Sendai virus (SeV), C, translated from an alternate reading frame of P/V mRNA has been shown to function at multiple stages of infection in cell cultures as well as in mice. C protein has been reported to counteract signal transduction by interferon (IFN), inhibit apoptosis induced by the infection, enhance the efficiency of budding of viral particles, and regulate the polarity of viral genome-length RNA synthesis to maximize production of infectious particles. In this study, we have generated a series of SeV recombinants containing substitutions of highly conserved, charged residues within the C protein, and characterized them together with previously-reported C'/C(-), 4C(-), and F170S recombinant viruses in infected cell cultures in terms of viral replication, cytopathogenicity, and antagonizing effects on host innate immunity. Unexpectedly, the amino acid substitutions had no or minimal effect on viral growth and viral RNA synthesis. However, all the substitutions of charged amino acids resulted in the loss of a counteracting effect against the establishment of an IFN-alpha-mediated anti-viral state. Infection by the virus (Cm2') containing mutations at K77 and D80 induced significant IFN-beta production, severe cytopathic effects, and detectable amounts of viral dsRNA production. In addition to the Cm2' virus, the virus containing mutations at E114 and E115 did not inhibit the poly(I:C)-triggered translocation of cellular IRF-3 to the nucleus. These results suggest that the C protein play important roles in viral escape from induction of IFN-beta and cell death triggered by infection by means of counteracting the pathway leading to activation of IRF-3 as well as of minimizing viral dsRNA production.