C Protein is Essential for Canine Distemper Virus Virulence and Pathogenicity in Ferrets

Paramyxoviruses, including members of the genus Morbillivirus, express accessory proteins with ancillary functions during viral replication. One of these, the C protein, is expressed from an alternative open reading frame (ORF) located in the P gene. The measles virus (MeV) C protein has been implicated in modulation of interferon signaling, but has more recently been shown to play a vital role in regulation of viral transcription and replication, preventing the excessive production of double-stranded RNA. Failure to do so, as seen with C-deficient MeV, leads to early activation of innate immune responses resulting in restriction of viral replication and attenuation in the host. One puzzling aspect of morbillivirus C protein biology has been the finding that a C-deficient canine distemper virus (CDV) generated by a similar mutagenesis strategy displayed no attenuation in ferrets, an animal model commonly used to evaluate CDV pathogenesis. To resolve how virus lacking this protein could maintain virulence, we revisited the CDV C protein and found that truncated C proteins are expressed from the CDV gene using alternative downstream start codons, even when the first start codon has been disrupted. We introduced an additional point mutation abrogating expression of these truncated C proteins. A new CDV with this mutation was attenuated in vitro and led to increased activation of protein kinase R. It was also strongly attenuated in ferrets, inducing only mild disease in infected animals, thus replicating the phenotype of C-deficient MeV. Our results demonstrate the crucial role of morbillivirus C proteins in pathogenesis. IMPORTANCE The measles (MeV) and canine distemper (CDV) viruses express accessory proteins that regulate the host’s immune response and enhance replication. The MeV C protein is critical in preventing the generation of excess immunostimulatory double-stranded RNA. C protein-deficient MeV is strongly attenuated compared to wild-type virus, whereas CDV with a similarly disrupted C open reading frame is fully pathogenic. Here, we show that CDV can compensate for the disrupting mutations by expression of truncated, but apparently functional, C proteins from several alternative start codons. We generated a new recombinant CDV that does not express these truncated C proteins. This virus was attenuated both in cell culture and in ferrets and finally resolves the paradox of the MeV and CDV C proteins, showing that both in fact have similar functions important for viral pathogenesis.