Study On The Interaction Between Influenza Virus Protein NS1 And Host Protein DOK6

Influenza A virus(IAV)is an important zoonotic pathogen.Its genome consists of eight singlestranded negative sense RNA segments,which are prone to gene mutations and reassortments,leading to the frequent emergence of new viruses,and thereby posing great challenges for its prevention and control.The replication of IAV requires the use of host biosynthesis system,transportation system,and energy system,in which a large number of host factors are involved.The host immune system initiates antiviral signaling pathways to limit viral replication by identifying the invasive influenza viruses.Meanwhile,the viral proteins of IAV interact with certain host proteins to inhibit the activation of the host antiviral signaling pathways.Therefore,an in-depth study of the complex interaction network and process between IAV proteins and host proteins is helpful to the development of anti-influenza drugs.Non-structural protein 1(NS1)is one of the essential viral proteins encoded by IAV,playing important roles in the virus replication cycle.NS1 is abundantly expressed during the virus infection cycle,inhibiting the activation of the host interferon pathway.It can also selectively reduce the expression of the host proteins so as to enhance the expression of the viral proteins.Our group previously used NS1 as a bait to screen for the potential interactional host proteins by means of immunoprecipitation and mass spectrometry,and identified docking protein 6(DOK6)from HEK293 T cells.In the present study,we further confirmed the interaction between NS1 and DOK6 by co-immunoprecipitation(Co-IP).Confocal microscopy assay showed that NS1 and DOK6 co-localized in the cytoplasm.We also demonstrated that NS1 interacted with the PTB domain of DOK6 by bimolecular fluorescence complementation(BiFC)assay.Plaque assay showed that overexpression of DOK6 protein inhibited the replication of IAV,while knockout of DOK6 promoted the viral replication.Dual-luciferase reporter assay indicated that DOK6 protein enhanced the expression of IFN-?,antagonizing the inhibitory effect of NS1 protein on host interferon response,in which its PH domain played the major role.Moreover,DOK6 activated several critical transcription factors of interferon pathway in a dose-dependent manner.We found that DOK6 interacted with TBK1 and enhanced the activity of TBK1 by increasing its phosphorylation level.The PTB domain of DOK6 was critical for its interaction with TBK1.Nevertheless,NS1 can bind the PTB domain of DOK6 to competitively inhibit the interaction between DOK6 and TBK1.Our study enriched the interaction network between NS1 protein of IAV and host proteins,thus facilitating better understanding of the regulatory mechanism of IAV replication in the host.