Studying On The Molecular Mechanism That Porcine Reproductive And Respiratory Syndrome Virus(PRRSV) Non-structural Protein1α (nsp1α) Inhibited The Transcription Of IFN-β

Porcine reproductive and respiratory syndrome virus (PRRSV) could induce theimmunosuppression and persistent infection, which caused huge economic loss for the swineindustry. The study on the immunosuppression mechanism of PRRSV, would play animportant theoretical guiding role in inhibiting the activities of PRRSV so that to decreasethe infection.1. Recombinant IFN-β inhibited the replication of PRRSV in MARC-145cellsType Ⅰ interferons (IFN-α/β) are the important antiviral protein. Previous study hasshown that MARC-145cells pretreated with recombinant IFN-β didn’t develop cytopathiceffects (CPE) of PRRSV. However, up to date, whether MARC-145cells post-treated withIFN-β could develop CPE of PRRSV is not clear. The present work would explore whetherthe MARC-145cells could develop the CPE of PRRSV when the MARC-145cells werepre-treated with IFN-β and then infected with different dose PRRSV, and also explorewhether the MARC-145cells could develop the CPE of PRRSV when the MARC-145cellswere infected with different doses of PRRSV and then added the recombinant IFN-β atdifferent post infection time. The results showed that the MARC-145cells in Group1thatpre-treated with IFN-β and then infected with different doses of PRRSV, in Group2thatinfected with different doses of PRRSV after4h and then added recombinant IFN-β, and inGroup3that infected with different doses of PRRSV after8h and then added recombinantIFN-β, all couldn’t develop the CPE of PRRSV after120h post infection. But the high-doseinfected cells in Group4that infected with different doses of PRRSV and then addedrecombinant IFN-β at24h post infection could develop CPE。The indirect immunofluorescence assay showed that there were a few N proteinpositive cells in the high-dose infected cells in Group1, Group2and Group3， but thenumbers in each group didn’t increase in the course of infection, while there was no positiveN protein cells in the low-dose infected cells in Group1, Group2, Group3and Group4. Inaddition，the numbers of N protein positive cells in high-dose infected cells (MOI=10) inGroup1were less than that in Group2, while the numbers of N protein positive cells inhigh-dose infected cells (MOI=10) in Group2were less than that in Group3.The results above indicated that the recombinant IFN-β had the efficient antiviralactivity. Both pre-treatment of IFN-β and the earlier post-treatment of IFN-β could inhibit the PRRSV and the IFN-β could clear the low-dose infected PRRSV.2. Amino acid at position176and the zinc-finger domain took important roles fornsp1α as an inhibitor to the induction of IFN-βPRRSV nonstructural protein1α (nsp1α) possessed three domains, N-terminal zincfinger (ZF) domain, papain-like cysteine protease (PCP) domain and carboxyl-terminalextension (CTE)(167-180amino acid(aa)). Previous studies have shown that PRRSV nsp1αinhibited the poly(I:C)-induced transcription of IFN-β, and the carboxyl-terminal extension(CTE)(167-180amino acid(aa)) and the activity of PCP were essential for nsp1α to inhibitthe induction of IFN-β. However, it was not clear that the minimum area and which aminoacid of CTE was essential for nsp1α as the inhibitor to the induction of IFN-β, and whetherZF domain was essential for nspα as the inhibitor to the induction of IFN-β. A series ofC-terminal truncated mutants for nsp1α (Full length of180amino acid) in the presentexperiment showed that nsp1α1-176、 nsp1α1-177and nsp1α1-179was able to inhibitpoly(I:C)-or signal protein (VISA、TRIF or IKK-ε)-induced activation of IFN-β promoter,but the nsp1α1-174and nsp1α1-175couldn’t. The pIRF-3was a necessary component to theactivation of IFN-β promoter, and we found that nsp1α1-176、nsp1α1-177and nsp1α1-179was able to inhibit poly(I:C)-or signal protein (VISA、TRIF or IKK-ε)-induced activationof pIRF-3-dependent synthetic promoter but the nsp1α1-174and nsp1α1-175couldn’t,which indicated that the region representing167-176was the minimal region of the CTE fornsp1α to retain its suppressive activity to the induction of IFN-β and the amino acid F176may take an important role for nsp1α to inhibit the induction of IFN-β.Deleting or mutating the amino acid F176made nsp1α not inhibit poly(I:C)-or signalprotein (VISA、 TRIF or IKK-ε)-induced activations of IFN-β promoter and ofpIRF-3-dependent synthetic promoter. By deletion of the ZF domain of nsp1α, we foundthat it couldn’t inhibit poly(I:C)-or signal protein-(VISA、TRIF or IKK-ε)induced activationof IFN-β promoter and it also failed to block Poly(I:C) and signal protein-(VISA、TRIF orIKK-ε)induced activation of pIRF-3-dependent synthetic promoter. Crystal structure ofPRRSV nsp1a documented that the ZF domain of PRRSV nsp1α belonged to the4-Cys ZFsuperfamily, and the Cys8, Cys10, Cys25and Cys28participated in binding with the zincion，and mutating one of them could disrupt the ZF structure. By site-directed mutagenesisof the predicted zinc-coordinating residues of the zinc finger(ZF) domain of nsp1α, we foudthat mutagenesis of the predicted zinc-coordinating residues of the zinc finger(ZF) domaincouldn’t inhibit poly(I:C)-or signal protein-(VISA、TRIF or IKK-ε)-induced activation ofIFN-β promoter, and also couldn’t inhibit poly(I:C)-or signal protein-(VISA、 TRIF orIKK-ε) induced activity of pIRF-3-dependent synthetic promoter. And ZF domain itself could not inhibit the Poly(I:C) or the ectopic expression of VISA, TRIF, or IKK-ε-inducedactivations of the IFN-β promoter and of pIRF-3-dependent synthetic promoter.The results above indicated that ZF domain of nsp1α took an important role for nsp1αto inhibit the IFN-β induction.3. nsp1αinteracted with HnRNP A2/B1Our previous study has found that PRRSV nsp1α and nsp11inhibited IFN-β productionand other studies also indicated that PRRSV nsp1α and nsp11took important roles for thevirulence of PRRSV. But the mechanism was not clear. So in the view of protein interaction,the present study aimed to identify the proteins that interact with PRRSV nsp1α or withPRRSV nsp11by the methods of co-immunoprecipitation, SDS-PAGE， protein silverstaining and mass spectrometry sequencing, and also to explore the functions of this proteinto the signal transduction of the transcription of IFN-β and the effect of this protein to thePRRSV virulence. The results showed that the HSP60，WDR77and HnRNP A2/B1mayinteract with the nsp1α, while eIF4A and PDIA6may interact with the nsp11。 Next, theco-immunoprecipitation was conducted to identify that only HnRNP A2/B1interacted withthe nsp1α. Overexpression of HnRNP A2/B1enhenced the RRSV virulence, but didn’teffect on the signal transduction of the transcription of IFN-β, which indicated that HnRNPA2/B1enhanced the RRSV virulence probably not by the effect on the signal transductionof the transcription of IFN-β.In conclusion, Firstly, our present work indicated that the recombinant IFN-β had theefficient antiviral activity; Secondly, the region representing167-176was the minimalregion of the CTE for nsp1α to retain its suppressive activity to the induction of IFN-β, andthe amino acid F176and the ZF domain were critical for nsp1α as the IFN antagonist; Finaly,HnRNP A2/B1interacted with the nsp1α， while HnRNP A2/B1enhanced the RRSVvirulence probably not by affect on the signal transduction of the transcription of IFN-β.