Production Of Porcine TNFα By ADAM17-mediated Cleavage Negatively Regulates Porcine Reproductive And Respiratory Syndrome Virus Infection

In China in 2006, a highly pathogenic strain of PRRSV caused an unparalleled large-scale and atypical PRRS outbreak, characterized with high fever, morbidity and mortality, damaging the pig farming industry and causing great economic losses each year. As an important inflammatory mediator, TNFα plays a key role in inflammatory reaction caused by PRRSV infection. However, there is no any report about the secretion mechanism of porcine TNFα. With the further research on TNFα biological activities, more and more people pay attention to the antiviral effect of TNFα.In order to analyze the effect of TNFα on PRRSV infection, we examined the levels of soluble TNFα from PAMs at 4, 8, 12, 24 and 48 h post-infection by ELISA. The data suggest that HP-PRRSV infection stimulates PAMs to produce TNFα. To evaluate the physiological consequences of TNFα on PRRSV infection, we used Marc-145 cells as a basic research cell line. We transfected Marc-145 cells with pcDNA3.1/TNFα or pcDNA3.1 vector control for 24 h, followed by PRRSV infection for 24 h. The results of qRT-PCR, TCID50 and IFA indicate that TNFα suppresses PRRSV infection in vitro.ADAM17 is a key metalloprotease, which is involved in cleaving ectodomains of various thransmembrane proteins. First, we observed that the presence of BB94 inhibitor markedly attenuated TNFα production upon PAMs treated with PMA, a cell activator that induces robust TNFa production. The data suggest that ADAM17 might be involved in the production of porcine TNFα in PAMs. We next examined the TNFα production in HEK293 by transient transfection with porcine TNFα gene. Western blot verifies the expression of porcine TNFα in the cells. We use the inhibitor BB94 and overexpression of ADAM17 to determine the role of ADAM17 in soluble porcine TNFα. To further determine the involvement of ADAM17, HEK293 cells were transduced with a bicistronic vector that expressed GFP and ADAM17 shRNA or negative shRNA. Western blot analysis confirmed that ADAM17 expression was decreased in HEK293 cells expressing ADAM17 shRNA1# & 2#. Subsequently, these cells were transfected with TNFα and the production of soluble TNFα was examined by ELISA. The data showed that soluble TNFα production was significantly inhibited in ADAM17-silenced HEK293 cells compared with parental HEK293 cells and negative shRNA-transduced cells. Collectively, these findings demonstrate that ADAM17 mediates the production of soluble porcine TNFα. Furthermore, we examined the critical cleavage region in porcine TNFα by using site-directed mutagenesis. The findings indicate that the 78 RSS motif is essential for efficient cleavage of porcine TNFα.In summary, the research first reported that TNFα mediated by ADAM17 inhibits PRRSV infection, which suggests that ADAM17 might serve as a therapeutic option against PRRSV infection and inflammatory storm. So, the study has many great potential applications in anti-virus infection and developing targeted inflammation therapies.