The Mechanism Of PRRSV GP5 Inhibits The Antivirus Effects Of Chaperone-mediated Autophagy By Targeting LAMP2A

Porcine reproductive and respiratory syndrome virus（PRRSV）could cause reproductive system disorders in sows and respiratory system disorders in piglets,bring great distress to the pig industry.PRRSV is a typical immunosuppressive virus which could escape the host antiviral effect through a variety of strategies.Autophagy plays an important role in host defense against viral infection and immune recognition due to its degradation function.Nevertheless,many viruses have evolved a variety of strategies to manipulate certain steps in the autophagy process to escape from degradation and clearance.At present,studies on autophagy induced by PRRSV infection have focused on the mechanism by which PRRSV or its nonstructural proteins（NSPs）underlie autophagosomes and autolysosomes formation.However,studies on the escape mechanism of PRRSV in the autophagy process,especially through chaperone-mediated autophagy（CMA）,are limited.Therefore,this study explored the escape mechanism of PRRSV in CMA,in order to provide new insight into the escape mechanism of immunosuppressive viruses and provide some theoretical basis for the prevention and control of PRRS.Firstly,the effects of PRRSV and its structural proteins on the autophagy were analyzed by western blotting and immunofluorescence assays.The results showed that PRRSV could up-regulate the expression of microtubule associated protein 1 light chain 3 II（LC3-II）and sequestosome 1（SQSTM1）,inhibit the fusion of autophagosome and lysosome,and then induce incomplete autophagy.Among its structural proteins,GP5 significantly up-regulated the expression of LC3-II and SQSTM1 and inhibited the fusion of autophagosome and lysosome.In addition,GP5 had a greater binding affinity for lysosomal marker lysosome-associated membrane protein type 2A（LAMP2A）than for autophagosome marker LC3.These results demonstrate that PRRSV inhibits autophagic flux and then induces incomplete autophagy mainly by GP5,and the GP5-mediated inhibition of autophagy might be mainly related to the lysosome.Secondly,the autophagy-related host proteins that interact with GP5 in primary porcine alveolar macrophages（PAMs）were screened by immunoprecipitation coupled with liquid chromatography-tandem mass spectrometry（LC-MS/MS）assay,and then western blotting,immunofluorescence,and co-immunoprecipitation（Co-IP）assays were used to verify the protein interactions between them.The results showed that GP5 could interact with two markers of CMA,heat shock cognate protein 70（HSC70）and LAMP2A.Whereas,the CMA substrate specific KFERQ-like motif of GP5 is not the region where GP5 interacts with HSC70.Moreover,GP5could compete with HSC70 for binding LAMP2A to impede substrate translocation.These results suggest that GP5 is not a substrate of CMA and it might regulate the activity of CMA by interacting with LAMP2A.Subsequently,the regulatory mechanism of GP5 on CMA activity was explored by western blotting,immunofluorescence,and Co-IP assays.The results showed that GP5 could inhibit m TORC2/PHLPP1/GFAP pathway to destroy the stability of LAMP2A-GFAP complex,promote the dissociation of p GFAP-EF1αcomplex to disrupt the formation of LAMP2A-GFAP complex,and inhibit K63 ubiquitylation of LAMP2A to promote LAMP2A degradation,thereby inhibiting CMA.These results reveal that GP5 could inhibit CMA by targeting LAMP2A.Finally,the effect of GP5 on the antiviral effect of CMA was further explored by western blotting,immunofluorescence,RT-q PCR and TCID₅₀assays.The results showed that the activated CMA inhibited PRRSV proliferation,while inhibited CMA promoted PRRSV proliferation.Moreover,GP5 could promote PRRSV proliferation by inhibiting CMA.Furthermore,activated CMA suppressed the expression of PRRSV NSP11,thus inhibiting NSP11-mediated negative regulation of type I interferon（IFN-I）signaling pathway.These results indicate that GP5 could inhibit the IFN-I signaling pathway by reducing the activity of CMA,thereby inhibiting the antiviral effect of CMA.In conclusion,this study resolves the antiviral mechanism by which PRRSV GP5 inhibiting CMA by targeting LAMP2A.Mechanistically,GP5 inhibits m TORC2/PHLPP1/GFAP pathway,promotes the dissociation of p GFAP-EF1αcomplex,and facilitates the degradation of LAMP2A,thus destroys the formation of GFAP-LAMP2A complex and subsequently inhibits CMA,and then strengthen the inhibitory effect of NSP11-mediated IFN-I signaling pathway to inhibit the antiviral effect of CMA,therefore facilitates PRRSV replication.This study provides new ideas for exploring the potential host targets of antiviral drugs and the immune escape mechanism of immunosuppressive viruses in CMA.