Viral RNA N6-methyladenosine modification modulates both innate and adaptive immune responses of human respiratory syncytial virus

Author summaryRSV is the most important cause of upper and lower respiratory tract infection of infants, young children, immunocompromised individuals, and the elderly. Despite major efforts, no vaccine is available for RSV. A live attenuated vaccine is one of the most promising vaccine approaches for RSV. However, a major problem is that natural RSV infection does not induce long-lived protection. The reason likely lies in its NS1 and NS2 proteins' strong inhibition of type I interferon (IFN) induction, which impairs subsequent adaptive immunity. We found that RSV RNA contains N-6-methyladenosine (m(6)A) and that recombinant RSVs lacking m(6)A methylation induce significantly higher type I IFN in cell culture and in mice compared to the parental RSV. Despite the fact that these m(6)A-deficient RSVs are significantly attenuated, they induce higher RSV-specific antibody and T cell immune responses in mice and provide complete protection against RSV challenge in cotton rats. Thus, m(6)A-deficient RSVs are more immunogenic than the parental RSV, highlighting that suppression of viral RNA m(6)A methylation is a novel strategy to enhance innate immunity which, in turn, enhances adaptive immunity. Human respiratory syncytial virus (RSV) is the leading cause of respiratory tract infections in humans. A well-known challenge in the development of a live attenuated RSV vaccine is that interferon (IFN)-mediated antiviral responses are strongly suppressed by RSV nonstructural proteins which, in turn, dampens the subsequent adaptive immune responses. Here, we discovered a novel strategy to enhance innate and adaptive immunity to RSV infection. Specifically, we found that recombinant RSVs deficient in viral RNA N-6-methyladenosine (m(6)A) and RSV grown in m(6)A methyltransferase (METTL3)-knockdown cells induce higher expression of RIG-I, bind more efficiently to RIG-I, and enhance RIG-I ubiquitination and IRF3 phosphorylation compared to wild-type virion RNA, leading to enhanced type I IFN production. Importantly, these m(6)A-deficient RSV mutants also induce a stronger IFN response in vivo, are significantly attenuated, induce higher neutralizing antibody and T cell immune responses in mice and provide complete protection against RSV challenge in cotton rats. Collectively, our results demonstrate that inhibition of RSV RNA m(6)A methylation enhances innate immune responses which in turn promote adaptive immunity.