Comparision Of Influenza HA Proteolytic Cleavage Efficiency By Different Type Ⅱ Transmembrane Serine Proteases And The Establishment Of MDCK Cell Lines Expressing TMPRSS2and MSPL

Hemagglutinin (HA) mediates binding to cell receptors and fusion of the viral envelope with theendosomal membrane are prerequisite processes for influenza replication. HA is synthesized as aprecursor protein HA0and needs to be cleaved by host cell protease(s) into the subunits HA1and HA2to gain its fusion capacity. Thus, proteolytic cleavage of HA plays a key role in virus entry and spreadin host cells. The specificity of HA cleavage site partially determines virus pathogenicity. HA of thehighly pathogenic avian influenza viruses could be cleaved at the multibasic motif by ubiquitouscellular proteases and cause lethal systemic infection. All other influenza viruses, including human andmammalian influenza strains, contain monobasic cleavage sites; these viruses can only be activated bycertain host serine proteases, subsequently infect a limited number of tissues. Human type IItransmembrane serine proteases(TTSPs) are host serine proteases involved in influenza proteolyticactivation.To investigate HA cleavage efficiency of different TTSPs, we conduct proteolytic activationexperiment using HA protein of A/Sichuan/1/2009(H1N1)(SC), a2009H1N1pandemic isolation fromSichuan in China. We constructed five plasmids pCA-TTSPs-Flag expressing transmembrane proteaseserine2(TMPRSS2), transmembrane protease serine3(TMPRSS3), transmembrane protease serine4(TMPRSS4), human airway trypsin-like protease (HAT) and transmembrane protease serine13(TMPRSS13, also named MSPL) respectively. Western blot indicated the expression of TTSP intransfected293T cells. Co-transfection of293T cells with pCA-TTSPs-Flag and plasmid pCA-SCHAexpressing the HA protein of the2009H1N1pandemic strain (SC) were performed. Western blotshowed the HA of SC were cleaved by TMPRSS2, TMPRSS4, HAT and MSPL. Confocal immuno-staining revealed that co-location of HA and TTSPs at cell membrane. Correctly cleaved HA couldinduce cell fusion under low pH condition (pH5.0), the pH dependent cell fusion assay andimmunofluorescence results indicated that TMPRSS2and HAT have relatively higher cleavageefficiency than TMPRSS4and MSPL.As the cleavage efficiency and cleavage site specificity varies in different TTSPs, TMPRSS2, mainlyproteolytic cleave monobasic HA with high efficieny, and MSPL, highly activate multibasic HA werechose to establish MDCK stable cell lines. Two stable cell lines designated as MDCK-TMPRSS2-eGFPand MDCK-MSPL-eGFP were obtained by Amaxa neucleofection and Geneticin selection. Theexpression and stability of cell lines were verified by Western blot, RT-PCR and cytometry.Technology breakthrough in cell suspension culture made avian influenza vaccine scaledmanufacturing possible while the production require the addition of exogenous trypsin. We conductinfection experiment of three HA-modified low pathogenetic avian influenza vaccine strains, namely Re-5(H5N1/PR8), Re-6(H5N1/PR8) and Re-20(H9N2/PR8) in MDCK-TMPRSS2-eGFP and MDCK-MSPL-eGFP cells. The results indicated that two stable cell lines support replication of these avianinfluenza without exogenous trypsin. The TCID50assay of those vaccine strains revealed that theexpression of TMPRSS2and MSPL do not affect virus sensitivity of cell lines. Our results showed thatMDCK-TMPRSS2-eGFP and MDCK-MSPL-eGFP cells have great potential in industrialized cellculture-based influenza vaccine production. Beside this, two stable cell lines could be helpful toinfluenza basic research and facilitate the anti-influenza drug design targeting at host serine proteases.