| Porcine reproductive and respiratory syndrome(PRRS)is a fellow of the most economic damage pig diseases worldwide.The causative PRRS virus(PRRSV),an enveloped,positive sense,single-stranded RNA virus,evolving rapidly,resists the innate immune response.Over the past decades,the development of effective PRRSV vaccines has been challenged by prominent antigenic variability and the ability to manage the host immune system through several immunomodulatory activities.Hence,research on the interaction between PRRSV and host cell may provide new strategies for PRRSV control and prevention without restriction on immune escape and antigenic variability.Recently,Rab-directed vesicular trafficking pathways have gained prominence as a crucial part of intracellular membrane trafficking,and Rab proteins participating in the viral life cycle has been proved by plenty of studies.In this thesis,we strive to expand our knowledge on the Rab-directed vesicular trafficking during PRRSV infection,and the role it plays in host cell defense or in supporting viral replication.We performed lentiviral delivery of shRNA technology to construct Rab knockdown MARC-145 cell lines(named shRab-MARC-145),as MARC-145 is one of the cell lines permissive to PRRSV replication.Next,as a PRRSV model,PRRSV-GFP was launched to screen the replication of PRRSV in Rab knockdown cell lines by fluorescence activated cell sorting(FACS).We found that there are 29 kinds of shRab-MARC-145 cell lines repressed PRRSV-GFP infection,13 of them enhanced PRRSV-GFP infection and 20 of the remained cell lines did not affect PRRSV infection.Further bioinformatics analyses revealed that 29 kinds of Rab proteins were mainly distributed in the plasma membrane,endosomes,endoplasmic reticulum(ER),perinuclear region of cytoplasm,trans-Golgi network and exosomes among biological processes such as protein transportation,vesicle fusion,endocytosis and exocytosis.These results confirm that PRRSV infection indeed needs Rab-mediated endocytic and secretory membrane trafficking.Interestingly,Rab proteins correlated with lipid droplets(LDs)were also gathered(P<0.049193),which have not been notified to participate in PRRSV infection.To gain a better understanding of the roles of LDs in PRRSV,Rab 18 was nominated for thorough study,which is an LD-associated protein mediating LD biogenesis and lipolysis,as well as LD trafficking.Firstly,we confirmed Rab18 was involved in PRRSV infection,as the knockdown or non-function mutation of Rab18 pronouncedly represses PRRSV replication.Moreover,PRRSV replication increases mRNA and protein expression levels of Rab18 detected by quantitative real-time PCR(qPCR)and western blot respectively.Next,we endeavored to elucidate Rab18 participates in the exact stage of the PRRSV life cycle.We found Rab18 participates in PRRSV particles assembly.The results of the electron microscopy(EM)study further voted in favor of the prediction,as immature nucleocapsid-like particles accumulated in Rab18 knockdown cells.The discovery of Rab18 involved in PRRSV replication led us to excavate the functional mechanism of Rab18 in PRRSV infection.Analyses of lipid metabolism during virus infection showed increased LD biogenesis and lipolysis during PRRSV infection.However,PRRSV-induced LD lipolysis could be resisted by the knockdown of Rab18.Remarkably,we observed that increased adipose differentiation-related protein(ADRP)degradation during PRRSV infection,which is an initiation to allow access of adipose tissue lipase(ATGL)and autophagic proteins to the surface of LDs to promote LD catabolism,could also be retarded by Rab18 knockdown or knockout.Subsequently,we focused on the role of Rab18 in ADRP degradation.As reported,ADRP is directly identified by heat shock cognate protein of 70 kDa(Hsc70)with a pentapeptide motif in LDs and targeted to the lysosome for degradation.In the present study,we observed both ADRP-Hsc70 and ADRP-lysosomal associated membrane protein 2A(LAMP2A)colocalization were blocked in Rab18 depletion cells.Therefore,we proposed that Rab18 acts as a binding partner for the association of Hsc70 with LDs:once anchoring on LDs,Hsc70 initiates to binding ADRP for chaperone-mediated autophagy(CMA).Rab18 may be the joining point that enables Hsc70 action to degrade ADRP followed by raised LD lipolysis whose function is recruited for PRRSV particles assembly.Collectively,the results presented in this thesis provide a novel insight on the interaction between PRRSV and host intracellular membrane trafficking as well as insight on how LD lipolysis are modulated by Rab18 during PRRSV infection and will be undoubtedly useful for establishing the theoretical basis of innovative strategies for PRRSV control and prevention. |
