MYH9 Interacts With PRV Glycoproteins And Its Roles In The Virus Infection Of Permissive Cells

Porcine pseudorabies is caused by pseudorabies virus(PRV)and leads to decreased reproduction in sows,respiratory disease in adult pigs,disorders of the nervous system,and high mortality in piglets.It is one of the most devastating infectious diseases in pigs that brings great economic losses to the swine industry worldwide.The entry of PRV into permissive cells requires a cascade of events mediated by cooperative interactions of with cellular receptors or factors.There are at least five viral glycoproteins(g C,g B,g D,gH and gL,)involved in the process of PRV infection,and cellular heparin sulfate(HS),nectin-1,nectin-2 and CD155have been identified asPRV entry mediators.MYH9,non-muscle myosin heavy chain IIA,is involved in many biological processes,including cell migration,adhesion,division,polarity,and morphogenesis.It was also identified as a functional receptor for HSV-1,EBV,SFTSV and PRRSV.HSV-1 and PRV belong to the Alphaherpesvirinae subfamily,and have high similarities in genome sequence,structure,physical and chemical properties.However,the detailed mechanism of MYH9involved in PRV infection process is inadequately understood.In this study,we identified that MYH9 interacts with gH and gL of PRV and is an important factor for PRV infection.The main contents and results are as follows.1.In order to examine the expression and distribution of MYH9 in cells upon PRV infection,we exposed PK-15 cells to PRV at 4?for 2 h,followed by a temperature shift to37?,and used IFA to analyze the distribution of MYH9.To assess the cell surface expressed MYH9,we extract cell membrane proteins after PRV infection.The results show that MYH9in the cytoplasm significantly redistributed to the membrane after PRV infection,while the total amount of MYH9 is not obvious change.2.In order to identify the interaction between MYH9 and glycoprotein of PRV,we used eukaryotic expression plasmids containing viral glycoproteins(gB,gC,gD,gH or gL)with FLAG tag and MYH9 C-terminal with HA tag.After co-transfection into HEK 293T cells,co-immune precipitation assays were used to detect MYH9 interacting proteins.The result showed that PRV g C,gH and gL can interact with MYH9.We also constructed prokaryotic expression plasmids p ET-28a-g C,p ET-28a-gH,p ET-28a-gL,expressed and purified g C,gH and gL proteins.CO-IP assay was used to detect interaction between these proteins and endogenous MYH9 in PK-15 cells.The results showed that MYH9 interacted with PRV gH and gL proteins.3.In order to determine if MYH9 is required for PRV infection,we used MYH9 ATPase inhibitor(blebbistatin),si RNA^MYH9 and rabbit anti-sPRA.As expected,blebbistatin,si RNA^MYH9 and rabbit anti-sPRA can significantly inhibit PRV infection in a dose-dependent manner.In addition,sPRA was used to block PRV infection and the result showed that sPRA can block PRV infection in a dose-dependent manner.The above results preliminarily confirm that MYH9 has a certain role in the process of PRV infection,and provide new research ideas for the prevention and treatment of PRV.