Role for the Phosphoprotein P Subunit of the Paramyxovirus Polymerase in Limiting Induction of Host Cell Antiviral Responses

Abstract

Six amino acid substitutions in the shared N-terminal region of the P subunit of the viral polymerase and the accessory V protein convert the noncytopathic paramyxovirus simian virus 5 (SV5), which is a poor inducer of host cell responses, into a P/V mutant (P/V-CPI-) that induces high levels of apoptosis, interferon-beta (IFN-beta), and proinflammatory cytokines. In this study, we addressed the question of whether these new mutant phenotypes are due to the presence of an altered P protein or of an altered V protein or of both proteins. By the use of the P/V-CPI- mutant as a backbone, new mutant viruses were engineered to express the wild-type (WT) V protein (+V-wt) or WT P protein (+P-wt) from an additional gene inserted between the HN and L genes. In human epithelial cell lines, the +V-wt virus showed reduced activation of apoptosis and lower secretion of IFN-beta and proinflammatory cytokines compared to the parental P/V-CPI- virus. The presence of a V protein lacking the C-terminal cysteine-rich domain (corresponding to the SV5 I protein) did not reduce these host cell responses to P/V-CPI- infection. Unexpectedly, the +P-wt virus, which expressed a WT P subunit of the viral polymerase, also induced much lower levels of host cell responses than the parental P/V-CPI- mutant. For both +V-wt and +P-wt viruses, reduced levels of IFN-beta synthesis correlated with reduced IRF-3 dimerization and nuclear localization of IRF-3 and NF-kappaB, suggesting that the WT P and V proteins acted at an early stage in antiviral pathways. Host cell responses induced by the various P/V mutants directly correlated with levels of viral mRNA accumulation but not with steady-state levels of genomic RNA. Our results support the hypothesis that WT P and V proteins limit induction of antiviral responses by controlling the production of key viral inducers. A model is presented for the mechanism by which both the P subunit of the viral polymerase and the V accessory protein contribute to the ability of a paramyxovirus to limit activation of antiviral responses.