Molecular Mechanism Of The Effect Of SUMOylation Of PB1 Protein On The Pathogenicity And Transmission Of Influenza A Virus

Influenza A virus(IAV)is an important zoonotic pathogen that causes frequent epidemics and occasional pandemics in humans.Based on the antigenicity of the surface glycoproteins hemagglutinin(HA)and neuraminidase(NA),IAVs are classified into 18 different HA subtypes and 11 different NA subtypes.H1N1 and H3N2 viruses are still actively circulating in humans globally The highly pathogenic H5 and H7 viruses often cause severe disease outbreaks in domestic poultry.Moreover,humans are also constantly facing the threats posted by avian influenza viruses(AIVs),with H5N1 and H7N9 human infections as the two prime examplesIAV takes advantage of host posttranslational modifications for their own benefit,such as the host SUMOylation system.However,the effect of SUMOylation on the adaptation,pathogenesis,and transmission of IAV remains largely unknown.Polymerase basic protein 1(PB1)is the catalytic subunit and the assembly core of the RNA-dependent RNA polymerase(RdRp),which is responsible for the transcription and replication of the IAV genome.In this study,we demonstrated that PB1 protein from different subtypes of IAV is a target of SUMOylation in both transfected and infected cells and identified that a conserved lysine residue at position 612(K612)of the PB1 of IAV was a bona fide SUMOylation site.SUMOylation of PB 1 at K612 had no effect on the stability or cellular localization of PB 1,but was critical for viral ribonucleoprotein(vRNP)complex activity and virus replication in vitro.We found that the vRNP complex activity and viral replication in vitro were dramatically attenuated when the SUMOylation-defective PB1/K612R mutation was introduced.When tested in vivo,we found that the virulence of SUMOylation-defective PB1/K612R mutant IAVs was highly attenuated in mice.Moreover,the airborne transmission of a 2009 pandemic H1N1 PB1/K612R mutant virus was impaired in ferrets,resulting in the reversion to wild-type PB 1 K612.Mechanistically,SUMOylation at K612 was essential for PB1 to act as the enzymatic core of viral polymerase by preserving its ability to bind viral RNA.Our study reveals an essential role of PB1 K612 SUMOylation in the pathogenesis and transmission of IAV.Our findings therefore thoroughly explore the contribution of PB1 SUMOylation on influenza infection and establish SUMOylation site PB1 K612 as a potential target for anti-influenza drug development.