Identification Of A Novel Porcine OASL Variant Exhibiting Antiviral Activity

2',5'-oligoadenylate synthetase(2',5'-Oligoadenylate Synthesis,OAS)is an interferon(IFN)induced antiviral protein that acts in the innate immune process,belongs to the interferon regulatory factor(ISGs),and is categorized as the OAS family because of its ability to synthesize 2-5 oligoadenylate(2-5A).Members of the group,by activating RNase L,degrade virus RNA to play an antiviral role.OASs are also involved in biological processes such as cell apoptosis,cell growth and differentiation,tumor occurrence and regulation.OAS is widely distributed in various mammalian tissues,and its biological function has been further reported.The porcine OAS family consists of OAS1(OAS1a and OAS1b),OAS2 and OASL,locateing on the chromosome 15.Notably OAS3 is absent.The amino acid sequences of porcine OASs are highly homologous to those expressed in humans and mice,and all of them can be induced by IFNs.The pOASL gene was observed to contain a premature stop codon,resulting in a truncated protein lacking the typical C-terminal double ubiquitin domains.This suggests that pOASL does not take the same pathway as hOASL to act antiviral activity.Gene structure analysis demonstrates that pOASL and hOASL get high homology in their OAS-Like domain.OAS-Like domain in hOASL enhanced dsRNA binding,leading to an enhancement of RIG-I signaling.However,the function of OAS-Like domain in pOASL is unclear.Japanese encephalitis virus is a single stranded RNA virus belonging to the flaviviridae flavivirus.In Asia,JEV is an important pathogen leading to human neurologic diseases.About 70000 cases of JEV are infected each year,and nearly 10000 cases die.Like other members of the Flaviviridae,JEV transmitted by mosquitoes,the natural transmission cycle including birds and livestock.The widespread transmission of livestock leads to continuous diseases of livestock,from the infection symptoms of some animals to other acute neurological symptoms of other animals.The effect of JEV infection on pigs is especially serious.Although the infection of adult pigs does not have obvious symptoms,it poses a serious threat to its reproduction,such as sow abortion,stillbirth and deformities,and the infected piglets can have a fatal neurological disease.Therefore,in-depth study of host's resistance mechanism to JEV is of great significance for developing effective anti JEV biological products.1.Discovery and identification of a novel porcine OASL variant2',5'-oligonucleotides synthetases(OASs)is a kind of IFN-induced antiviral proteins,which are important components of the mammalian immune system.Here,a novel variant of porcine OASL(pOASL2)was identified through RT-PCR amplification.This gene is distinguishable from the previously described wild-type porcine OASL(pOASL1).The gene appears to be derived from a truncation of exon 4 plus 8 nucleotides of exon 5 with a premature termination,measuring only 633 bp in length,although its position corresponds to that of pOASLl.Preparing rabbit anti-pOASL polyclonal antibody was specific to the common part of pOASL1 and pOASL2 that could detect pOASL0 Western blotting showed that IFN-?stimulated PK-15 cells could produce pOASL1 and pOASL2.2.Impact of the new porcine OASL variant on viral infection2',5'-Oligoadenylate synthetase-lilke(OASL)protein is an atypical oligoadenylate synthetase(OAS)family member,which possesses antiviral activity but lacks 2',5'-oligoadenylate synthetase activity.Studies described in chapter three identified a novel variant of porcine OASL(pOASL2)through RT-PCR amplification.Given this novel gene appears to be a variant of pOASL,we assayed for antiviral activity of the protein.We demonstrated that pOASL2 could inhibit Japanese encephalitis virus(JEV)proliferation as well as pOASLl in a transient overexpression assay of pOASL1 and pOASL2 in PK-15 and Vero cells.In addition to JEV,pOASL1 and pOASL2 also decreased the proliferations of Porcine reproductive and respiratory syndrome virus(PRRSV)and vesicular stomatitis virus(VSV),but did not exhibit antiviral activity against pseudorabies virus(PRV).Structural analysis showed that the pOASL2 gene retained only the first three exons at the 5'-.To investigate the role of the aN4 helix in pOASL in antiviral responses like that in hOASL,we mutated key residues in the anchor domain of the aN4 helix in pOASL2,based on the domain's location in hOASL.However,the antiviral activity of pOASL2 was not affected.Thus,the aN4 helix of pOASL likely does not play a significant role in its antiviral activity.In conclusion,pOASL2 acts as a new splice isoform of pOASL that plays a role in resistance to infection of several kinds of RNA viruses.