Molecular Basis Of PLSCR1 Suppression Influenza A Virus And Biological Characterization Of H5N1 Isolated In Egypt

Influenza A viruses have caused several large outbreaks in human history,including 2009 H1N1 influenza pandemic.so,Influenza A viruses pose a serious threat to human health and life.Prevention and treatment of influenza is an important part of public health security.Strategies to deal with influenza pandemic including: strengthen the usual monitoring of the virus,predict the virus epidemic trendency accurately,update vaccine according to monitoring data,take urgent immunity and pharmaceutical treatment once outbreaks.dThe nucleoprotein(NP),a major component of the viral ribonucleoprotein(v RNP)complex,has a significant function in virus life cycle,including the nulear import of v RNPs,the transcription and replication,and mediated the export of v RNP together with M1 protein and NEP protein.NP can also be a excellent anti-influenza drug targets due to highly conserved in various subtypes of influenzaviruses.NP protein of influenza virus will inevitably interact with host factors during infections,such as importin α、CRM1、UAP56 and Tat-SF1 which have been reported ever.We performed yeast two-hybrid(Y2H)system to find host factors interacting with NP.PLSCR1(Phospholipid Scramblase 1)is one of the paterner interacting with NP during infection of influenza virus.PLSCR1 is II calcium-binding membrane protein that can be induced by interferon and growth factor,itexists in various kinds of tissues of human except of brain.There are many functions of PLSCR1 have been illuminated.It has been found that PLSCR1 can be substrate of many intracellular protein kinase(c-Abl、c-Src、 PKC),illustrating that PLSCR1 is related to signal transductions of cell proliferation,differentiation and apoptosis.PLSCR1 is an interferon responding gene(ISG)and related to antiviral activity.It has been reported that PLSCR1 can degrade X protein of hepatitis B virus(HBV)to inhibit the infection and replication of HBV.It also seriously interfere with the entering into cells of hepatitis c virus(HCV).PLSCR1 is screened for the fist time which interact with NP protein of influenza virus.In this paper,through the following experiment to study the effect of this intercatonon influenza virus replication.(1)The interaction between NP and PLSCR1 in vivo was futher confirmed by co-immunoprecipitation(co-ip)and confocal.The co-ip results showed that NP-PLSCR1 interaction was mediated by amino acids 250-318 of PLSCR1,and the interactions don’t require the participation of RNA.Imaging revealed that NP and PLSR1 co-located in cytoplasm of A549 cells byplasmids transfection.(2)293t cells were overexpressed or knockdown PLSCR1 by transfection p CAGGS-PLSCR1 or PLSCR1-si RNA to detectete the change of influenza polymerase activity,the results showed that overexpression PLSCR1 can makepolymerase enzyme activity decline only slightly,but knockdown PLSCR1 elevate polymerase enzyme activity about 15 folds.Those results reflect indirectly that PLSCR1 plays a inhibitory role for influenza virus replication during infection.(3)The overexpression cell line and control cell line wereestablished through retrovirus packaging system named A549-PQCXIN-PLSCR1 and A549-PQCXIN,respectively.Then the cell lines were inoculated with WSN virus at a multiplicity of infection(MOI)of 0.1.We found that the virus titer was reduced 100-fold on the overexpression cell line than that on the control cell line.The confocaldata showed consistent tendency,which displayed nuclear import prevention of NP at early stage of viral infection on overexpression cell line.Knockdown the endogenous PLSCR1 of A549 cell through si RNA interference,and then inoculate WSN at a MOI of 0.1.However,The virus titer only increased slightly.si RNAinterference complementary experimental of overexpression cell line showed similar result.(4)Mechanism research about PLSCR1 inhibition influenza virus replication was carried out from two aspects,as follows:(a).It has reported that introductionalanine(E)substitution(at S9 and Y10)to mimic thephosphorylated residueswhich prevent nuclear import of NP.This phenomenon was consistence with our confal data.So,we rescued mutant viruses(S9A,Y10 F,S9A/Y10F)to mimic the dephosphorylationthrough 12-plasmid reverse genetic system.Then infected overexpression and contron cell line with three muant viruses and WT virus.We found that the dephosphorylation mutant viruses did not alleviateinhibitory action on PLSCR1 overexpression cell line.Our resultssuggest that dephosphorylation of S9 and Y10 is independentof viral amplification on the overexpression.(b).it is known that NP is transported into the nucleus by the classicalimportin-α/β pathway,which recognizes the 3-13 nonclassical NLS.However,PLSCR1 also contains a nonclassical NLS(257GKISKHWTGI266)combined with importin-α.Wehypothesized a mechanism underlying the overexpression of PLSCR1 decreasing the binding of NP and importin-α.Totestthishypothesis,we co-transfected plasmids encoding WT NP or PLSCR1 and importin-αinto 293 T cells for co-IP assays.We found that PLSCR1 did not impairing the interaction of NP with importin-α,however,The result indicate that three proteins could form ternary complexessteadily.Furthermore,confal image clearly demonstratedthe cytoplasmic retention of PLSCR1 during the whole process of infectioncycle.entire infection caused by the Y296 E mutation and the accelerated nuclear export caused by the Y296 F mutation.So,We conclude that PLSCR1 inhibitnuclear import of NP by forming ternary complexes with NP/ importin-α,consequently reducing the virus titer during infection.Highly pathogenic avian influenza(HPAI)H5N1 virus was first isolated from goose in southern china in 1996[1].The viral progeny of A/goose/Guangdong/1996 not only caused considerable damage to poultry industry but also pose a threat to human health.Human cases of H5N1 were soon confirmed in Hong Kong in 1997[17].Since 2003,the H5N1 virus gradually spread to other regions,and in the past decade it has caused numerus outbreaks in domestic poultry in more than 60 countries in Asia,Europe and Africa,leading to 449 deaths among 846 human infections as of 20 January 2016(WHO,OIE).Influenza A viruses evolve via accumulation of mutations over time or rearrangement of viral RNA segments with different viruses [2].In 2005,a H5N1 outbreak in wild birds occurred in Qinghai Lake in western China,leading to ≈6,000 bird deaths [3,4].The Qinghai-lineage viruses(Clade 2.2)acquired a glutamic acid-to-lysine substitution at position 627(E627K)of PB2 protein,which was human-adapted molecular marker typically found in H1N1,H3N2,H5N1 and H7N9 human isolates [5-7].Fortunately,the current Qinghai-lineage viruses caused only sporadic human infections across the world(WHO).Reassortment is another most important reasons responsible for the emergence and spread of pandemic viruses,as proven by pandemic outbreaks in history and recent H7N9 human endemic[7-11].In Egypt,the HPAI H5N1 viruses transmit into Egypt in 2005 and spread quickly [12].Since then,the viruses originated from Qinghai-lineage developed to endemic in Egypt,with regular reported outbreaks,numerous human infections.Phylogenetically,H5N1 viruses circulating in Egpytian poultry was clade 2.2.1 and their descendants [13].By the end of 2014,the laboratory-confirmed cases reported to World Health Organization(WHO)was 210 with 77 deaths,however,the human cases dramatically increased in 2015,with 136 cases including 39 deaths.The number of human cases in 2015 was approximately 70% of that of past nine years.The current H5N1 do not get sustained human to human transimisibility,however,owing to its high evolution rates,it will adapted to mammal hosts when acquiring suitable mutations.Notably,the Egyptian H5N1 viruses have already acquired PB2 627 K,other mutations in Hemagglutinin(HA),polymerase proteins or reassort with other subtype influenza A viruses will facilitate the viruses to transmit efficiently in human beings.There were evidence from several studies indicated that by acquiring only three or four amino acid changes in HA protein,resulting in increasing binding affinity to Siaα2,6Gal,or by getting PA or NS segments from pandemic 2009(H1N1)virus,confers increased polymerase activity in human cells,the H5N1 will transmit efficiently between mammal models[14-16].The HPAIV in Egypt was serious.Thus,it is urgent to know the characterization the new clade 2.2.1.2 H5N1 viruses circulating in Egypt.In this study,we selected two viruses,one was isolated in 2014,and the other was isolated in 2015,evaluated the receptor property,pathogenicity and transmissibility of the two viruses.