The Characters Of Porcine 2'-5'oligoadenylate Synthetases Against Japanese Encephalitis Virus

Japanese encephalitis virus and WNV are very closely related and are included,along with DENV,TBEV,YFV,in the genus Flavivirus,which caused severe diseases in human.Japanese encephalitis(JE),caused by JEV,is one of the most important mosquito transmitted diseases of human and pigs,affecting acute inflammation of the central nervous system and swine reproductive disorder.Its outbreak significantly related to seasonality and regionality.Along with the spread of JEV pathogen,JE would be a global infectious disease to threaten the health of human and animals seriously.Therefore,to prevent JEV infection and cure JE effectively would be positive and beneficial to global public health.2'-5' Oligoadenylate Synthesis(OAS)is a kind of Interferon-induced antiviral protein,which plays a major role in the IFN-mediated antiviral pathway,and OAS protein also associate to several cellular processes,such as apoptosis,cell growth and differentiation,gene regulation,DNA replication,and RNA clipping.At present,there are more and more studies on characters of mammalian OAS protein,especially on the antiviral functions,but the mechanism of these OAS activities have not been clarified and would be studied further.OAS belongs to a highly conserved family that shares no significant overall sequence homology with other interferon-stimulated proteins.The OAS family consists of four genes encoding four proteins with different isoforms:OAS1,OAS2,OAS3,and OASL(OAS-like protein).Porcine OASs show highly sequence homology to those expressed in human and mouse.The first OAS to be crystallized successfully was porcine OAS1,which is 73%identical in sequence to human OAS1 and has similar enzymatic characteristics.The OAS proteins are characterized by their ability to catalyze the synthesis of 2',5'-linked oligoadenylates(2-5 A)using ATP.The synthesis of intracellular 2-5A results in the activation of RNase L and the activated RNase L degrades both viral and cellular RNAs,which results in the inhibition of global protein synthesis and the suppression of viral replication during infection.The flavivirus resistance(FlvT)gene was identified as OAS1b simultaneously by two groups that observed a restriction of West Nile virus(WNV)replication in wild mice by OAS1b.Lin et al demonstrated that human OAS1(p42/p46)and OAS3(p100)possessed antiviral activity against dengue virus(DENV).Kwon et al.verified that OAS1(p46)and OAS3(p100)defense against the hepatitis C virus(HCV)was dependent upon RNase L.A single nucleotide polymorphism(SNP)in the OAS1 gene was also associated with an increased risk of WNV disease in humans.Furthermore,extracellular OAS1 entered into cells and possessed strong antiviral activity in both in vitro and in vivo studies and was found to have its affect at an earlier step in the viral replication cycle.The 'A' allele at SNP rs10774671 of OAS1 has been shown to alter splicing of OAS1 and to be associated with reduced OAS activity,and was also considered as a host genetic risk factor for initial infection with WNV in humans.Bigham et al suggested that the OAS1 SNP rs34137742 was also associated with both risk for symptomatic WNV infection and WNV disease progression.The equine OAS1 also plays a role in resistance to severe WNV infection and naturally occurring single nucleotide mutations in equine OAS1 contribute to host susceptibility.To consider the closely relationship between JEV and WNV,we could presume that the SNP of OAS genes may relate to the susceptivity in JEV infection.1.Porcine OAS inhibit JEV replication in vitroThe expression kinetics and antiviral functions of porcine OAS family(OAS1,OAS2,and OASL)in PK-15 cells following infection by Japanese encephalitis virus(JEV)were evaluated in the present study.The endogenous expression of the three OAS genes was efficiently induced by JEV infection in PK-15 cells in a time-dependent manner.However,expression of pOAS1 responded more quickly than pOASL and pOAS2.pOAS1 expression resulted in 5.7-fold increase,whereas 3.1-and 1.7-fold increases were in pOAS2 and pOASL.Transient over-expression of pOASL and pOASl inhibited JEV replication more efficiently than OAS2 overexpression.Interestingly,knockdown of pOAS2 expression by siRNA treatment led to the 7.99-fold increase in JEV multiplication,which was significantly higher than?4.5-fold increases in OAS2 and OASL groups.Co-silencing of RNase L and each pOAS revealed that the anti-JEV function of pOAS1 and pOAS2 were RNase L dependent,while the antiviral activity of pOASL was not.In conclusion,all pOAS isoforms play a significant role in the response to JEV infection,and are differentially induced by stimuli.The alternative pathways of antiviral activity stimulated by OASL require further study.2.Impact of porcine OAS1 genetic variation on viral infectionIn man,polymorphisims at OAS1 have been shown to correlate with differential susceptibility to several infections of' great public health significance,including hepatitis C virus,SARS coronavirus,and West Nile virus.In PK-15 cells,we have demonstrated the anti-JEV activity of porcine OAS1 in previous study.The aim of this study was to assess the impact of genetic variants of porcine OAS1 single polymorphisms(SNPs)on viral infection.In this study,18 nonsynonymous coding SNPs were isolated and analyzed from the variety of porcine tissues.Based on each SNP site,the artificial mononucleotide mutagenic OAS1 gene was overexpressed in PK-15 cells individually,of which.M13(Q174K)(87.1%JEV genome in control group)and M16(D277N)(97.2%.JEV genome in control group),were absent of the antiviral activities probably due to the replacement of OAS1 single amino-acid residue in the site following by the protein structural change.Although the OAS activity was deficiency in M13 and M16 protein,the correlation with the susceptibility to JEV in pigs was not significant in backtracking analysis.The conclusion might be not exactly for the small sample size without efficient and convincing representativeness.In addition,in BHK-21 and Vero cells,OAS1 and its mutants overexpression could not inhibit viral replication effectively(80%-90%JEV genome in control group)because of the incomplete IFN system in these cell lines.In conclusion,the M13(Q174K)and M16(D277N)were proofed as key sites about OAS antiviral activity preliminarily,and the potential to be genetic markers needed to be further verified.