Effects Of Host Protein Mx2 And Chemokine Cxcl12 On The Replication Of Procine Reproductive And Respiratory Syndrome Virus

Porcine reproductive and respiratory syndrome virus(PRRSV)is one of the most important pathogens that causes significant economic losses in the swine industry worldwide.Current commercial vaccines are not well satisfied in protective efficacy.Innate immunity is the first line against viral infection.Suppression of innate immunity may be an important contributing factor to PRRSV modulation of host immune responses.In addition,the genome of PRRSV is highly variable,and it usually causes persistent infection in herds.It would benefit a lot for the control of PRRSV by deepening the knowledge of the interaction between PRRSV and innate immune response,screening of effective antiviral proteins,and clarifying the mechanism involved in the inhibition of virus replication.This research contain three parts as following:1.Examination the effects of Mx2 on the replication of PRRSV and analysis of the key functional region in Mx2Mx2 has been identified as a novel interferon(IFN)-induced protein that inhibits virus replication.The role of Mx2 in PRRSV infection is not well understood.In this study,we cloned the full-length monkey Mx2(mMx2)complementary DNA(cDNA)from IFN-3 treated Marc-145 cells,and found that over-expression of mMx2 inhibited PRRSV replication.IFN-3 induced expression of mMx2 in Marc-145 cells and suppressed PRRSV replication in a dose-dependent manner.Knockdown of mMx2 impaired the antiviral activity mediated by IFN-?.Confocal imaging and immunoprecipitation assays indicated that mMx2 interacted with PRRSV N protein in virus-infected cells.By constructing and expressing of mMx2 truncations and mutant,we found that GTPase activity of mMx2 is necessary.This study laid important foundation for the development of novel antiviral drugs for PRRSV infection.2.The N-terminal 62 amino acid of porcine Mx2 was the key functional site for inhibiting PRRSV replicationThe identity between mMx2 and pMx2 was merely about 78%,and the effects of pMx2 on the replication of PRRSV were scarcely known.In this study,we cloned the full-length pMx2 complementary DNA(cDNA)from IFN-? treated PBMCs,and found that over-expression of pMx2 inhibited PRRSV replication.According to the structural characteristic of pMx2,we constructed pMx2 truncations with different sizes.It was found that N-terminal 60-64aa mediated the antiviral activity of pMx2.The site-directed mutagenesis results of N-terminal 60-64 aa showed that 62aa(Met)was the key site for the antiviral activity.Confocal imaging and immunoprecipitation assays indicated that pMx2 interacted with PRRSV N protein in PRRSV-infected cells.pMx2 truncations and mutants with different antiviral activities showed similar intracellular distribution,and they all interacted with PRRSV N protein.In addition,pMx2 could inhibit the replication of different PRRSV strains.The results indicated that pMx2 N-terminal 62aa was the key site for the antiviral activity.pMx2 could interact with PRRSV N protein,but the binding domain was not related with the 62aa(Met)which was assumed to be the key functional site for the inhibition of PRRSV replication.3.The role of chemokine CXCL12 in the replication of PRRSVChemokines are key regulators of innate and adaptive immunity,the secretion of which is an important mechanism against virus infection.However the details in the interaction between chemokines and PRRSV are still unclear.In this study,we found that two PRRSV strains with different virulence could both induce the expression of monkey CXCL12(mCXCL12)in Marc-145 cells.The expression of mCXCL12 was induced by PRRSV through MAPK(P38)and NF-?B signaling pathways,and non-structure protein Nsp12 of PRRSV mediated the expression of mCXCL12.In addition,we cloned the full-length mCXCL12 cDNA from Marc-145 cells,and over-expressed mCXCL12 by eukaryotic expression system.The result showed that PRRSV replication was inhibited by over-expressing of mCXCL12.RNA interference-mediated knockdown of mCXCL12 enhanced PRRSV replication in Marc-145 cells.Meanwhile,PRRSV replication was also inhibited by porcine CXCL12(pCXCL12).In conclusion,the replication of PRRSV was significantly inhibited by the interferon-induced Mx2 and chemokine CXCL12.It showed important theoretical significance and prospects for PRRSV control.