Investigation On The Mechanisms By Which Non-structural Protein 2 Of Porcine Reproductive And Respiratory Syndrome Virus Induces Autophagy To Facilitate Viral Proliferation

Porcine reproductive and respiratory syndrome（PRRS）is a highly contagious disease caused by porcine reproductive and respiratory syndrome virus（PRRS virus,PRRSV）infection,which has caused huge economic losses to swine industry worldwide.Autophagy,including macroautophagy/autophagy,chaperone-mediated autophagy（CMA）,and microautophagy,is a lysosome-dependent degradation pathway involved in maintaining intracellular environmental homeostasis.A growing number of studies have demonstrated that autophagy plays a key role in viral proliferation.PRRSV nonstructural protein 2（Nsp2）is a multifunctional protein consisted of multiple domains.Previous studies have revealed that PRRSV Nsp2 induces autophagy to promote viral self-proliferation,but the detailed molecular mechanisms remain to be fully elucidated.Therefore,this thesis focuses on investigation on the mechanisms by which PRRSV Nsp2 induces autophagy to facilitate viral proliferation.1.Investigation on the mechanisms by which PRRSV Nsp2 triggers Golgi apparatus fragmentation-mediated autophagy to facilitate viral self-replicationThis study firstly monitored Golgi apparatus（GA）fragmentation in the PRRSV-infected cells via laser confocal microscopy（LCM）and transmission electron microscope（TEM）.PRRSV nonstructural protein 2（Nsp2）was further identified to induce GA fragmentation using LCM,TEM and immunoblotting（IB）.Subsequently,the study found Golgi reassembly and stacking protein 65（GRASP65）as a host cell protein targeted by PRRSV Nsp2 to induce GA fragmentation using immunoprecipitation（IP）and liquid chromatograph-tandem mass spectrometer（LC-MS/MS）techniques.Nsp2 papain-like cysteine proteinse 2（PLP2）domain was determined to degradated GRASP65 as detected by IP,IB,LCM and real-time fluorescence quantitative PCR（RT-q PCR）.Next,this study demonstrated that PRRSV Nsp2 degradation of GRASP65 induced GA fragmentation to promote autophagy by LCM and IB.PRRSV Nsp2-induced GA fragmentation disassociated GA-resident Ras-like protein in brain 2（RAB2）from Golgi matrix protein 130（GM130）to enhance its interaction with unc-51 like kinase 1（ULK1）by LCM,IP and IB,which increased the phosphorylation of ULK1 and promoted autophagy,elucidating the molecular mechanism by which PRRSV Nsp2-induced GA fragmentation activated autophagy via the RAB2-ULK1 pathway.Finally,this study showed that PRRSV induced GA fragmentation-mediated autophagy to promote viral self-replication as determined by indirect immunofluorescence assay（IFA）,RT-q PCR,flow cytometry（FCM）,IB,and50%tissue culture infected dose(TCID₅₀).Taken together,this study reveals that PRRSV promotes viral proliferation by inducing GA fragmentation-mediated autophagy.These findings expand our understanding of PRRSV pathogenesis and autophagy.More importantly,these results provide the detailed mechanism of GA fragmentation-mediated autophagy,deepening the understanding of autophagic processes.2.Investigation on the mechanisms by which PRRSV promotes TANK-binding kinase 1 degradation via chaperon-mediated autophagy to suppress type I interferon production and support viral proliferationThis study firstly identified that PRRSV Nsp2 degraded TANK-binding kinase 1（TBK1）through IB,IP and LCM.Nsp2 was subsequently found to degrade TBK1 via the lysosomal pathway using mutation,addition of autophagy inhibitor 3-methyladenine（3-MA）,lysosomal inhibitor chloroquine（CQ）,and proteasomal inhibitor（MG132）.Next,this study determined that PRRSV Nsp2 interacted with heat shock protein 8（HSPA8）using IP,LC-MS/MS,LCM,and IB.PRRSV Nsp2 was further demonstrated to degrade TBK1 via CMA through IP,LCM,and IB.Finally,this study detected that reduced TBK1 protein levels led to down-regulation of TBK1phosphorylation levels,which inhibited downstream signaling pathway activation,suppressed the production of type I interferon（IFN-I）,and promoted viral proliferation through RT-q PCR,FCM,and IB.Taken together,this study reveals that PRRSV Nsp2suppresses host innate immunity through CMA for the first time,which provides a novel insight to PRRSV eradication.In conclusion,this study reveals the molecular mechanisms by which PRRSV Nsp2induces GA fragmentation to promote autophagy and inhibits innate immunity through CMA to jointly promote self-proliferation,providing a new theoretical basis for the prevention and control of PRRS.