Study On The Function And Mechanism Of FAM46A In Regulating Influenza Virus Replication

The influenza virus is one of the most important pathogens which can cause severe infection of the human respiratory tract.In every year's influenza season,influenza viruses infect millions of persons,among which thousands die.The 1918 Spanish flu estimated to cause fifty million deaths during the pandemic from 1918 to 1920,which comprised 3%of the whole world population at that time.The 1918 Spanish flu pandemic was the worst public health incident in the 20th century.More highly transmissible and pathogenic new reassorted influenza viruses will emerge every decade or several decades due to gene reassortment,which result in pandemic and pose a great threat to human health and social stability.But until now,little is known about the mechanism of influenza virus replication and pathogenesis,which constrains the development of new antiviral drugs and vaccines.Influenza viruses are cellular parasites,and they strictly depend on host proteins during every step of the viral life cycle,including entry,replication,and release.It is important to investigate the interaction between influenza viruses and human beings,understand the pathogenesis,and develop new antiviral candidate proteins for the prevention and treatment of influenza virus infection.By a previously performed siRNA screen,we discovered that FAM46A was involved in influenza virus replication.We notice that FAM46A expression is induced after IFN-P treatment,which means it is an IFN-stimulated gene(ISG).Additionally,overexpression of FAM46A inhibited influenza virus replication,while CRISPR-Cas9 mediated knockout of FAM46A resulted in significantly enhanced viral replication,indicating FAM46A is a key host restriction factor.According to sequence and phylogeny analyses,FAM46A is a member of the nucleotidyltransferase fold superfamily.So,we constructed several different mutant forms of FAM46A.We discover that FAM46A mutants whose enzymatic activities were disrupted failed to inhibit influenza virus replication,suggesting the enzymatic activity of FAM46A plays an important role in inhibiting influenza virus replication.Because previous researches showed that the length of mRNA poly(A)tail plays an important role in mRNA stability,we infer that FAM46A could affect the length of the poly(A)tail of the influenza virus mRNA to make them unstable.By using TAIL-seq,we sequence the poly(A)tail of influenza virus mRNA.It turns out that the length of the influenza virus mRNA poly(A)tail and the composition of the 3' very end of poly(A)tail remained unaffected when overexpressing or depleting FAM46A.Moreover,the tethering assay and mRNA half-life assay indicate FAM46A does not affect the decay of influenza virus mRNA.Altogether,FAM46A could not modify the end of influenza virus mRNA and alter the decay of influenza virus mRNA.According to the kinetics of influenza virus replication in the wild type and FAM46A knockout cells,we find that FAM46A can inhibit influenza virus mRNA synthesis at a very early stage.Mini replicon assay suggests that FAM46A can inhibit the transcription activities of influenza RNA-dependent RNA polymerase.The Influenza mRNA synthesis is initiated when the end of host mRNA primers,which are acquired by cap-snatching,pair with the 3' end of influenza vRNA,so the nucleotides of the very 3' end of host mRNA primers that pair with the conserved vRNA 3' end play vital roles in transcription initiation.We postulate that FAM46A can interfere with this process by adding one or several nucleotides to the 3' end of host primers to make the pairings unfavorable.After sequencing the end of host mRNA primer used for influenza virus mRN A synthesis using 5'-RACE sequencing,no difference was observed at the end of the snatched mRNA primers in the presence or absence of FAM46A,indicating that FAM46A may not affect the initiation of influenza virus mRNA synthesis.Besides,FAM46A can interact with the three proteins that constitute the RdRp complex,i.e.,PA,PB1,and PB2,which suggests other mechanisms of action.Considering that FAM46A does not affect the initiation of influenza virus mRNA synthesis and the synthesis of influenza virus mRNA poly(A)tails,and that the enzymatic activity of FAM46A is responsible for its antiviral activity,we speculate that FAM46A may generate certain products to curb the influenza virus replication.We purified the FAM46A protein and performed in vitro assay to examine the enzymatic activity of FAM46A.FAM46A can de novo synthesize short oligomers using all four types of NTPs(ATP,UTP,CTP,and GTP),and the short oligomers are constituted of the same NTP.By using specific RNases and phosphodiesterases,we determine the linkage in the oligomers is 2'-5' linkage.Meanwhile,we find FAM46A synthesize additional oligomers with higher molecular weight when stimulated with short RNAs.The mechanism by which these oligomers inhibit influenza virus replication needs further studies.In summary,we identified FAM46A as a host restriction factor that can inhibit influenza virus replication.At a very early stage,FAM46A inhibits the mRNA synthesis of the influenza virus via its nucleotidyltransferase enzymatic activity.In vitro enzymatic assay shows FAM46A synthesize short oligomers with 2'-5' linkage.Our study provided insights into influenza virus-host interactions and shed light on the development of potential antiviral therapeutics.