The Mechanism Of Heat Shock Protein Member 8 In Regulation Of Porcine Reproductive And Respiratory Syndrome Virus Infection

Porcine reproductive and respiratory syndrome(PRRS)is a serious infectious disease,and leads to enormous economic losses in the word swine industry.Its causative agent is PRRS virus(PRRSV).PRRSV is an enveloped,single-stranded,positive-sense RNA virus,which belongs to the arteriviridae family.As an acellular pathogen,PRRSV infection highly depends on host cells.Virus infection is a multistep process including binding,internalization,translation and replication,assembly,and release.Such a complex and unique process is one of important reasons why it is difficult to prevent and control PRRS.Understanding the mechanism of virus infection is an important theoretical basis for the development of vaccines and antiviral drug.Further exploration of these host factors involved in PRRSV infection will help clarify the mechanism of PRRSV infection and lay a foundation for PRRS effective prevention and control.This study,heat shock protein member 8(HSPA8)was firstly demonstrated to be associated with PRRSV infection.Further study focused on the important functions of HSPA8 in PRRSV infection which mainly includes three parts stated below:Part I The role of HSPA8 in the binding and internalization of PRRSVIn this part,HSPA8 was firstly identified to interact with GP4 protein of PRRSV by immunoprecipitation(IP)coupled with mass spectrometry(MS).Then,HSPA8 was conformed to bind PRRSV virions by GST pulldown and ELISA.Subsequently,HSPA8 knockdown or its ATPase activity inhibition could significantly suppress PRRSV infection in host cells.HSPA8 was observed to be on surface of host cells and co-located with PRRSV by confocal microscope at early stage of infection.Both HSPA8 soluble protein and polyclonal antibody can inhibit PRRSV infection.Our results demonstrated that HSPA8 was involved in the binding of PRRSV.HSPA8 was further found to be associated with clathrin-mediated endocytosis(CME)pathway.HSPA8 along with CD163 promoted PRRSV infection.Collectively,these results demonstrated that HSPA8 was important for PRRSV binding and internalization,and would be a potential target to prevent and control viral infection.Part II The function of HSPA8 in PRRSV replicationIn this part,utilizing confocal microscopy,HSPA8 was found to be specially-recruited to the preclear region and co-locate with viral replication and transcription complex(RTC).Subsequently,the inhibition of enzyme activity of HSPA8 could restrict the formation of RTC,our results showed that HSPA8 was associated with PRRSV replication.Furtherly,the binding of HSPA8 with the 3’untranslated region(3’UTR)of PRRSV genome was confirmed by RNA pulldown and RNA immunoprecipitation(RIP).As 3’UTR is a key element for PRRSV replication,HSPA8 regulated the gene expression mediated by 3’UTR.Part III HDAC4 regulated PRRSV replication via HSPA8In this part,utilizing anti-acetylated lysine antibody,we found that the level of acetylation of HSPA8 in PRRSV-infected cells was increased.Subsequently,histone deacetylase 4(HDAC4)was identified as a deacetylase of HSPA8.The further study showed that overexpression of HDAC4 impaired the formation of RTC induced by PRRSV,and negatively regulated the 3’UTR mediated gene expression,to inhibit viral replication.In turn,PRRSV infection down-regulated HDAC4 protein.These results demonstrated that PRRSV down-regulated HDAC4 protein,increased HSPA8 acetylation,to favor its replication.Collectively,during PRRSV infection,as an important chaperone protein,HSPA8 was involved in the regulation of host cell against viral infection.At the early stage of virus infection,HSPA8 was associated with viral binding and internalization through interacting with viral envelop protein.At the post-entry stage of virus infection,HSPA8 regulated PRRSV replication via recognizing viral RNA.In addition,our study also clarified that HDAC4 inhibited PRRSV replication via deacetylating HSPA8.This study expands insight into the molecular mechanisms of PRRSV infection,and provided a novel theoretical basis for the control of PRRS.