Effect Of HSP27 On H5N1 Influenza Virus Proliferation And The Mechanism

Since twenty-first century,the pandemic of H5N1 highly pathogenic avian influenza virus(HPAIV)has greatly threaten the human health and public safety over the global,leading to great panic and serious damage to the global economy.Thus,our country has taken great efforts and carried a lot of research to prevent and control this disease.In this paper,We confirmed the result of previous protein group,the further research on the function of heat shock protein 27(HSP27)in the viral infection process was also conducted.It was found that HSP27 interacts with H5N1 HPAIV nucleoprotein(NP)in vivo.The N-terminal region of NP(181-498 a.a.)was defined to be the domain of interaction with HSP27,indicating that HSP27 can affect the virus's proliferation and the cells' death in a certain extent through controlling polymerase activity.Our work expanded the existing interaction networks beween influenza virus and host factors,provided new ideas and directions of future prevention and control of the virus.The main results were as follows:1 Identified the HSP27 expression at different time points in A549 cells infected with H5N1 HPAIVCollected protein at 4h,8h,18h,24h,30h,42h after A549 cells infecting H5N1 HPAIV and Western blotting analysised that HSP27 protein expressions were changed at different time points.We confirmed that HSP27 was of up-regulation expression in early stage of infection and downregulation of 24h.2 Discoveried that HSP27 has a positive regulation on virus proliferation in A549 cells infected with H5N1 HPAIV and other two virusesWe got HSP27 sequences by PCR amplification.Then it was cloned into the eukaryotic expression vector and identificated by Western blotting.At the same time,we also designed siRNA for HSP27,comfirmed that it could effectively silence this gene.We infected cells which overexpressing or silencing of HSP27 and discoveried that HSP27 has a positive regulation on virus proliferation.Overexpression of HSP27 can promote the proliferation of virus whereas silence suppression of viral proliferation.We use another two virus strains to infect cells,and found that the virus proliferation ability was also strengthened with overexpression of HSP27.3 Found the influenza protein that interacts with HSP27We confirmed that HSP27 can interact with influenza virus NP through the Co-IP experiment.Then we tried to truncate these two proteins and found that the domain of interaction with HSP27 is located at C terminal of NP,which does not depend on the RNA binding domain.4 The positive regulation role in virus proliferation of HSP27 depends on phosphorylationWe used inhibitor to block the phosphorylation of HSP27,therefore the key phosphorylation sites of HSP27 could not phosphorylate.Then we infected cells with influenza virus and found that cells immidiatly died if HSP27 could't phosphorylate and the virus could not better proliferate in viral stimulation.then virus titer in cells was very low.We confirmed that the positive regulation on virus proliferation of HSP27 depends on its phosphorylation.5 The effects of various HSP27 forms on viral polymerase activity has been identifiedIt was confirmed 'that effects of different HSP27 forms on the viral polymerase activity were different by polymerase activity experiment:the normal HSP27 can negatively regulate the activity of the influenza virus polymerase.If HSP27 is truncated,then its inhibition ability is weakened.The non-phosphorylation of HSP27 also weakened its inhibition of polymerase activity.Among them,serine 82 plays a leading role.