Function Of Non-muscle Myosin Heavy Chain II-A Protein In Porcine Reproductive And Respiratory Syndrome Virus Infection Of Marc-145

Porcine reproductive and respiratory syndrome (PRRS) is a disease caused by PRRS virus (PRRSV) which belongs to the family Arteriviridae. PRRS mainly causes reproductive disorder, including premature, abortion, fetal death, mummy and porcine respiratory syndrome at variety of ages with high mortality. At present, PRRS has prevailed in the main world while pig-raising country and districts. Nowadays the study to pathogenic and immunologic mechanism of PRRSV is not clear. There is also no proper way for controling of PRRSV infection.PRRSV has a highly specific cell tropism, both in PAM and in the monkey kidney-derived cell lines; the virus enters through a mechanism of receptor-mediated endocytosis. Until now, two PRRSV receptors (CD163 and Vimentin) have been described on Marc-145 cells. CD163 is a cellular protein in the scavenger receptor cysteine-rich superfamily, functioned as interacting with PRRSV and may determine the replication levels of PRRSV. Vimentin is an intermediate-filament protein, which is widely expressed in various cell types. Anti-vimentin Abs block PRRSV infection of Marc-145 cells. When simian vimentin was delivered to BHK-21cells and CRFK cells, they became susceptible to PRRSV. Vimentin is also thought to be involved in the replication and transportation of PRRSV inside the cell by forming a complex with the other components of the intermediate filaments.An internal image Mab2-5G2 which was generated against idiotypic antibodies specific for GP5 antigen of PRRSV identified a soluable protein prepared from Marc-145 cells. The protein was identified to be non-muscle myosin heavy chain II-A (NMHC II-A). NMHC II-A is distributed ubiquitously throughout nature. It is one isoform of NM II heavy chain and it plays a role in a variety of cellular processes including cell migration, cytokinesis, cell adhesion and cell shape change both during development and in the adult organism. An association between the C-termininal of CXCR4 and CCR5 and the motor protein NMHC II-A has been found and this biochemical association may have a key role in lymphocyte migration. Soluble CD163 binds with T lymphocytes coeffected with NMHC II-A. These have proved the function of NMHC II-A in immuno-reaction. In this article, we mainly study the block function of C-terminus NMHC II-A in Marc-145 cells to PRRSV infection. At first, seven peptides which located on the C-terminal of NMHC II-A were synthesized. Virus neutralization experiment showed that three peptides among the seven ones can block PRRSV infection of Marc-145 cells. In order to study the biological function of C-terminus NMHC II-A, total RNA was extracted from Marc-145 cells and specific primes were synthesized. After RT-PCR, the C-terminus NMHC II-A named PRA was cloned and sequenced, the Gene bank number was HM490008. PRA was then cloned to pET-28a (+) vector, after expression, PRA protein was obtained. Expression of truncated proteins, western-blot and I-ELISA experiments showed that PRA protein can specifically bind with Mab2-5G2; also the specific binding site was identified. IFA experiment proved PRA protein can specifically bind the membrane of Marc-145 cells. What was more important was that VN, FFN and Real Time PCR experiments identified that PRA protein can block PRRSV infection of Marc-145 cells in some degree. PRA was also cloned to pEGFP-N1 vector; the ability of PRRSV infecting the transfected Marc-145 cells was decreased by 50% in comparison with non-transfected cells. Otherwise, (R)-(+)-Blebbistatin which was the inhibitor of NMHC II-A not only can not affect the cytokines of Marc-145 cells but also block PRRSV infection by VN experiment. These results showed that C-terminus NMHC II-A can block PRRSV infection of Marc-145 cells. They provide a noval approach for further understanding of PRRSV infection and control of PRRS.