The Study On The Mechanism Of Porcine Reproductive And Respiratory Syndrome Virus Nsp12 Inhibiting The Expression Of Type I Interferon

Porcine reproductive and respiratory syndrome virus(PRRSV)is a kind of RNA virus prone to mutation and recombination.It can cause persistent infection and immunosuppression after PRRSV infection,which is extremely harmful to the pig industry.Natural immune response is an important way of host anti-virus infection.PRRSV can use its encoded protein to antagonize host natural immune response and escape host immune surveillance.Nsp12 is a non-structural protein related to PRRSV replication.Whether Nsp12 participates in the antagonism of innate immune response of the host has not yet been elucidated.This study focused on the mechanism of Nsp12 of HP-PRRSV HuN4 strain in inhibiting type I IFN signaling pathway.Preliminary studies in the laboratory found that Nsp12 of HuN4 strain could inhibit IFN-I expression.In order to explore the molecular pathway of Nsp12 inhibiting IFN-I expression,the effects of Nsp12 of HuN4 strain on IFN-? transcription initiation activity were analyzed by double luciferase reporting system and fluorescence quantitative PCR.The results showed that Nsp12 of HuN4 strain could inhibit IFN-? transcription and promoter activity,and this inhibition was also observed.The effect was dose-dependent.There were four amino acid differences between HP-PRRSV and classical strain Nsp12,but these differences did not affect the inhibition of IFN-? promoter activity by Nsp12.After a series of truncation,the main functional domains of Nsp12 inhibiting IFN-I expression were analyzed.The results showed that the main domain of Nsp12 inhibiting IFN-I expression was located in its N-terminal domain.To determine whether Nsp12 inhibits IFN-? signaling pathway,we examined the effects of Nsp12 on RIG-I signaling pathway and TLR3 signaling pathway.The results showed that Nsp12 significantly inhibited IFN-? induced by RIG-I and MAVS,but did not inhibit IFN-? induced by TRIF,suggesting that Nsp12 inhibited IFN-? expression through RIG-I signaling pathway,rather than TLR3 signaling pathway.Because the expression of IFN-? can be regulated by transcription factors IRF3 and NF-?B,we detected the promoter activity of Nsp12 on IRF3 and NF-?B binding domain.The results showed that Nsp12 could inhibit the promoter activity of transcription factor NF-?B binding domain.Nsp12 also inhibited p65 nuclear migration induced by TNF-? stimulation.These results confirmed that Nsp12 of HP-PRRSV HuN4 strain could inhibit IFN-? expression through the activation pathway of NF-?B.In order to further explore the function of Nsp12 protein,we screened the host cell protein galactose lectin 3(LGALS3)interacting with Nsp12 by yeast two-hybrid,and identified the interaction between Nsp12 and LGALS3 by immunoprecipitation and laser confocal assay.The results confirmed the interaction between LGALS3 and Nsp12,and confirmed that the region of interaction between LGALS3 and Nsp12 was located in LGALS3(125-166aa).Overexpression of LGALS3 inhibited the proliferation of HuN4 strain in Marc-145 cells,while the proliferation of HuN4 strain in Marc-145 LGALS3 deleted cell line could be promoted.Interestingly,we found that over-expression of LGALS3 in Marc-145 cells not only inhibited IFN-? promoter activity,but also inhibited RIG-I-induced IFN-? production,and also inhibited the promoter activity of transcription factor NF-?B binding domain.In Marc-145 LGALS3 deleted cell lines,the activity of IFN-? promoter induced by poly(I:C)stimulation was significantly stronger than that of normal Marc-145 cells.Although Nsp12 could inhibit the activity of IFN-? promoter in both cell lines,its inhibition in Marc-145 LGALS3 deleted cell lines was significantly weaker than that in normal Marc-145 cells.These studies suggest that LGALS3 may play a synergistic role in the inhibition of IFN-? expression by Nsp12,but the inhibition of IFN-? expression by Nsp12 is not entirely dependent on LGALS3.In conclusion,this study confirms that PRRSV Nsp12 can inhibit IFN-I expression through RIG-I/NF-? B pathway,and its interaction protein LGALS3 can also assist Nsp12 to inhibit IFN-I expression through RIG-I/NF-?B pathway,which provides a new theoretical basis for elucidating the mechanism of PRRSV immunosuppression.