Interaction And Regulatory Mechanism Of Influenza Virus Ribonucleoprotein Complex(vRNP) With Host

Influenza A viruses effectively hijack the intracellular "resources" to complete transcription and replication,which involve extensive interactions between the viral and host proteins.Herein,we screened the host factors,which belong to DEx D/H-box protein family members,RNA-binding proteins or mitochondrial anchoring proteins,to investigate their effects on polymerase activity.We observed DDX39 B and DDX39 A,DEAD-box RNA-Helicases,exert a dual effect on regulating polymerase activity and replication of influenza A viruses.We further revealed that DDX39 B and DDX39 A interact with viral NP and NS1 proteins.Interestingly,the viral NP proteins could reverse the inhibitory effect of excess DDX39 B or DDX39 A on polymerase activity.Mechanistically,the TREX complex subunits,THOC1,THOC4 and CIP29,were recruited to DDX39B-DDX39A-NP complex in an ATP-dependent manner,probably via the interaction with DDX39 B or DDX39 A,followed by excess TREXNP complexes interfere with the normal oligomerization state of NP depending on the ratio between the viral and host proteins.On the other hand,the TREX complex,an evolutionarily conserved protein complex,is responsible for the integration of several m RNA processing steps to export viral m RNA.Knockdown of TREX complex subunits significantly down-regulated viral titers and protein levels,accompanied by retention of viral m RNA in the nucleus.To fully explore the role of polymerase complex-related host factors,we combined high-throughput transcriptome data to analyze the changes in m RNA levels of these factors during viral infection.Transcriptome data showed that viral infection caused down-regulation of MYH9,HNRNPU,SRSF3 and RPS24 m RNA levels.We confirmed the changes in m RNA and protein levels of MYH9,HNRNPU and SRSF3 by q PCR and WB.Then their effects on virus replication were tested through overexpression and knockdown experiments.We emphatically explained the mechanism of SRSF3 during influenza virus replication.Results showed that SRSF3 promoted influenza virus replication and regulated viral protein expression at the post-transcriptional level.Further analysis found that SRSF3 regulated viral replication depends on the 88 th amino acid.RIP and FISH experiments further proved that SRSF3 bound to viral m RNA and participated in the nuclear and cytoplasmic transport of viral m RNA.Collectively,these findings suggested that virus infection regulated the expression of many host factors and SRSF3 positively regulated virus replication.DDX5,a member of DEx D/H-box helicases,is known to participate in all aspects of RNA metabolism.However,its regulatory effect in antiviral innate immunity during replication of influenza virus remains unclear.Herein,we found that human DDX5 promotes replication of influenza virus in A549 cells.Moreover,our results further revealed that DDX5 relies on its N-terminus to interact with the NP protein of influenza virus,which is independent of RNA.Of course,we also observed colocalization of DDX5 with NP in the context of transfection or infection.However,influenza virus infection had no significant effect on the protein expression and nucleocytoplasmic distribution of DDX5.Importantly,we found that DDX5 suppresses antiviral innate immunity induced by influenza virus infection.Mechanistically,DDX5 down-regulated the m RNA levels of IFNβ,IL6,and DHX58 via METTL3-METTL14/YTHDF2 axis.We revealed that DDX5 bound antiviral transcripts and participated in m6 A modification of m RNAs,which in turn regulate immune responses through YTHDF2-dependent m RNA decay.Taken together,our data demonstrate that DDX5/METTL3-METTL14/YTHDF2 axis regulates replication of influenza A virus.