Investigation On The Mechanisms Of Host Cell Proteases Involved In Porcine Reproductive And Respiratory Syndrome Virus Membrane Fusion

Porcine reproductive and respiratory syndrome(PRRS),caused by PRRS virus(PRRSV),is characterized by reproductive failures and respiratory diseases,and has led to huge economic losses to global swine industry.Due to the lack of effective prevention and control measures,PRRS still circulates worldwide.PRRSV is an enveloped,positive-sense,single stranded RNA virus.Therefore,PRRSV must undergo merging of its envelope with host cell target membranes(namely membrane fusion)before delivering the genetic materials into host cytoplasm to establish infection.This complicated process is mediated by viral fusion protein(s)together with low p H,host cell proteases,and other factors.However,the detailed mechanisms involved in PRRSV membrane fusion are not yet elucidated.In-depth studies on PRRSV membrane fusion will provide novel ideas and strategies for prevention and control of PRRS.This study focused on host cell proteases and unraveled the mechanisms of host cell proteases involved in PRRSV membrane fusion.Firstly,after viral invasion through clathrin-mediated endocytosis(CME),PRRSV membrane fusion was observed to occurr in Ras-related protein 11(Rab11)-recycling endosomes during early infection(45 min)with labeled virions and subcellular markers by laser confocal microscopy(LCM).Furthermore,transfection with wild type(WT),constitutive active(CA)or dominant negative(DN)Rab11showed that Rab11 activity played a vital role in PRRSV membrane fusion.Secondly,cathepsin E under low p H was determined to be critical for PRRSV membrane fusion using specific inhibitors and small interference RNAs(si RNAs).Lastly,PRRSV glycoprotein(GP)5 was identified to be recognized,bound and cleaved by cathepsin E during membrane fusion by immunoprecipitation(IP),pull-down and LCM.All these findings reveal how a host cell protease(cathepsin E)mediates PRRSV membrane fusion after CME.On the other hand,elastase was discovered to be significantly produced in the lung tissues of highly pathogenic PRRSV(HP-PRRSV)-infected pigs compared to the mock-infected ones by reverse transcription-PCR(RT-PCR)and immunoblotting(IB).Next,elastase was demonstrated to promote HP-PRRSV infection in a dose-dependent manner by RT-q PCR(quantitative real-time PCR),TCID₅₀(50%tissue culture infective dose)and IB.Furthermore,binding,entry and infection assay showed that elastase was involved in PRRSV entry.When CME was blocked by low p H or clathrin inhibitor,HP-PRRSV wasn't located in early endosomes as shown by co-localization assay and its infection was obviously abolished.In contrast,elastase treatment restored HP-PRRSV infection and promoted it even to a higher level in the presence of inhibitors.Moreover,elastase was shown to mediate HP-PRRSV entry into host cells at the cell surface via direct membrane fusion with labeled virions and cell membrane marker by LCM.Lastly,HP-PRRSV GP5 was also recognized,bound and cleaved by elastase during this process.All the above results provide the details of a host cell protease(elastase)mediating PRRSV membrane fusion on cell surface.In summary,this study uncovers the details involved in PRRSV membrane fusion for the first time.The results contribute to unraveling PRRSV pathogenesis,and development of vaccines against viral membrane fusion proteins as well as antibodies or small-molecule antiviral drugs against virus membrane fusion process,which will lay a foundation for prevention and control of PRRS.