Investigation On The Viral Apoptotic Mimicry Utilized By Porcine Reproductive And Respiratory Syndrome Virus To Invade Host Cells

Porcine reproductive and respiratory syndrome(PRRS),caused by PRRS virus(PRRSV),is a highly contagious disease.PRRS has caused huge economic losses to the global swine industry.Especially,PRRS is an economically critical factor in China.PRRS and PRRSV are the focuses of veterinary and virology research.However,efficient prevention and control of PRRS is hindered by the viral complicated infection.Invasion by PRRSV is previously shown to be via low p H-dependent clathrin-mediated endocytosis(CME)and CD163 functions as an essential receptor during the process.In-depth study on PRRSV invasion will contribute to understanding of PRRSV pathogenesis and providing a basis for the development of new antiviral drugs and vaccines.As a novel strategy,viral apoptotic mimicry plays a significant role in viral invasion.In the current work,a series of studies were carried out to eludidate the untilization of viral apoptotic mimicry by PRRSV.This work mainly includes four parts as follows:1.Phosphatidylserine(Ptd Ser)is found on the surface of PRRSV envelope.A variety of viruses incorporate Ptd Ser,a marker of apoptosis,on the surfaces of their envelopes and disguise as apoptotic debris.In this study,different strains from PRRSV-1 and PRRSV-2 were purified by sucrose density gradient centrifugation.Subsequently,the presence of Ptd Ser on the envelope of PRRSV was confirmed by flow cytometry and Dot-blot,using specific Ptd Ser-binding annexin V and anti-Ptd Ser antibody,respectively.The results determine that PRRSV externalizes Ptd Ser on the envelope as viral apoptotic mimicry.2.T-cell immunoglobulin and mucin domain(TIM)family is identified to recognize PRRSV as apoptotic mimicry.Ptd Ser exposed on viruses is recognized by Ptd Ser receptors.Here,TIM-1 and TIM-4 were identified in MARC-145 cells and PAMs,respectively,through reverse transcription-polymerase chain reaction(RT-PCR)and immunoblotting(IB).Subsequently,knockdown of TIM-1/4 decreased PRRSV RNA replication,N protein expression,progeny viral titers,and viral binding and entry,indicating that TIM played an important role in PRRSV invasion.By Dot-blot,pulldown and antibody blocking experiments,TIM-1/4 was proven to directly bind to PRRSV through Ptd Ser.3.PRRSV induces macropinocytosis via TIM-1 and utilizes macropinocytosis as an invasion pathway.Dextran is utilized as a marker of macropinocytosis.Knockdown of TIM-1 greatly influenced PRRSV-induced macropinocytosis as indicated by decreased dextran uptake.In addition,the membrane protrusions were observed using the F-actin marker of phalloidin,demonstrating that PRRSV induced macropinocytosis.Upon recognition by Ptd Ser receptors,the viruses are internalized by host cells to promote their infections.Ethylisopropyl amiloride(EIPA)specifically inhibits PRRSV macropinocytosis.Furthermore,cytochalasin D and latrunculin A which inhibit actin polymerization suppressed PRRSV invasion via macropinocytosis.Moreover,PRRSV co-localized with sorting nexin 5(SNX5),a marker ofspecific endosomes for macropinocytosis(macropinosomes).These experiments demonstrated that macropinocytosis is an alternative pathway for PRRSV to invade host cells.4.The involvement of Rac1/Cdc42-Pak1 signaling pathway and CD163 in PRRSV macropinocytosis.Another characteristic of macropinocytosis is its dependence on Rho GTPases,including Rac1 and cell division control protein 42(Cdc42).The p21-activated kinase 1(Pak-1)is an effector down-stream target of both Rac1 and Cdc42.Knockdown and inhibition of Rac1,Cdc42 and Pak1 all inhibited of macropinocytosis and PRRSV infection.As a consequence,Rac1/Cdc42-Pak1 signaling pathway was found to involved in PRRSV-induced macropinocytosis.CD163 is an essential receptor for PRRSV infection.Its role in macropinocytosis was further investigated.Exogenous expression of CD163 in BHK-21 cells was found to support PRRSV infection,while that of TIM-4 wasn't.Co-expression of CD163 and TIM-4 enhanced PRRSV infection to a higher level compared to that of CD163 alone.Furthermore,co-localization of CD163 and SNX5 was observed.Consequently,CD163 is essential during PRRSV infection via macropinocytosis.Collectively,the study demonstrated that PRRSV externalized Ptd Ser on the envelope as viral apoptotic mimicry,and clarified the mechanism by which PRRSV utilizes viral apoptotic mimicry to invade host cells,including the Ptd Ser receptors recognizing PRRSV as apoptotic mimicry(TIM),the downstream signaling pathway(Rac1/Cdc42-Pak1)and the invasion routes(macropinocytosis),and the involvement of CD163 and TIM in PRRSV invasion via macropinocytosis.The knowledge gained from this study will provide in-depth information for PRRSV infection and pathogenesis,and have important scientific significance and application value.