Molecular Mechanisms Of Avian And Mammalian 2'-5'-oligoadenylate Synthetase Like(OASL) Genes Against Influenza A Viruses

Virus infection of host cells prompts the innate immune system to initiate an antiviral response.The 2'-5' oligoadenylate synthetase(OAS)proteins associated with endoribonuclease RNase L is a viral RNA decay pathway known to play an important role in the innate antiviral immunity.The OAS repertoire is classed into four subfamilies that encode proteins of different isoforms in Metazoa.OAS-like(OASL)gene is an atypical member of the OAS family in the sense that it contains two tandem ubiquitin-like(UBL)domains in the C-terminus and performs the divergent OAS activity in mammalian cells,thereby activating the OAS/RNase L or OASL/RIG-I system to defense against viral infection.Compared to mammals,birds have a contractive OAS family,which contains one member(OASL)in most birds belonging to the Carinatae group(e.g.,ducks and chickens)and two members(OASL and OAS1)in a few birds belonging to the Ratitae group(e.g.,ostriches and white-throated tinamous).However,the functions of avian OASLs are largely unknown.To determine the role of divergent avian(duck and ostrich)OASL in the immune response,we compared viral replication of a highly pathogenic(A/duck/Hubei/49/05,DK/49)and a weakly pathogenic(A/goose/Hubei/65/05,GS/65)H5N1 virus in DF1 cells that stably expressed duck OASL(dOASL)to the corresponding in duck RIG-I recovery-expression DF1 cells.DF1dOASL+/+ and PF1dRIG-I+/+ cells showed comparatively lower levels of DK/49 and GS/65 virus titers compared to DF1 cells expressing empty vectors(P<0.05).We then generated OASL-deficient DF1 cells(DF1oASL-/-)using the CRISPR/Cas9 method.As expected,DF1dOASL+/+ cells had a significantly lower level of the CK/0513(A/chicken/huabei/0513/2007)H5N1 virus,whereas DF1oASL-/-cells showed a significantly higher level of the CK/0513 virus(P<0.05).Similarly,we found that ostrich OASL(oOASL)also significantly inhibited the viral replication CK/0513 in DF1 cells(P<0.05).Additionally,the expression of either dOASL or oOASL in DF1 cells significantly reduced the replication of another negative single-stranded RNA virus(NDV/La Sota)and two double-stranded RNA viruses(IBDV/B87 and REOV/Z97/C10)(P<0.05).By contrast,the absence of OASL in DF1 cells significantly enhanced the viral replication of the above three viruses(P<0.05).Further analysis indicated that dOASL inhibited the replication of a positive single-stranded RNA virus(O/BY/CHA/2010)in porcine kidney cells(IBRS2)(P<0.05).However,neither dOASL nor oOASL reduced replication of a double-stranded DNA virus in DF1 cells(FPV/CVCC/AV1003)and in PK15 cells(PRV/Henan/2014).These observations suggest that avian OASLs show antiviral activity against a wide range of RNA viruses but not against DNA viruses,similar to some mammalian OASLs.To investigate whether avian OASLs activate the OAS/RNase L pathway to decay viral RNA,we next examined the OAS activities of duck and ostrich OASL proteins.Like mouse Oasl2(mOasl2),both dOASL and oOASL synthesized dimeric(pppApA),trimeric(pppApApA)and longer oligomers using poly(I:C)as an activator,thereby activating RNase L.Duck and ostrich OASL reversibly switched off their OAS activities similarly to human OASL and mouse Oasl2 when mutations were introduced at three conserved D residues(homologous to Asp75-Asp77-Asp148 in hOASl).Additionally,OASLs with effective OAS activities significantly upregulated the mRNA level of RNaseL and multiple genes related to IFN signaling(e.g.,IFN?,IFN?,STAT1 and MX1)after infection with influenza A virus(IAV)(P<0.05).Duck and ostrich OASL activate and magnify the RNase L pathway in a UBL-dependent manner.This is in sharp contrast to mammalian enzymatic OASL,which activates and magnifies the OAS/RNase L pathway in a UBL-independent manner.Our data show that,unlike OLDs in mammals,duck and ostrich ones efficiently activate the OAS/RNaseL pathway to block viral replication by cooperating with their UBLs upon bounding dsRNA together.Duck and ostrich OASL mutants(OASL-3D*)enhanced RIG-? signaling in a similar manner to human OASL and mouse Oasl2-3D*.However,inactive OAS activity was necessary,but not sufficient,to enhance RIG-? signaling.The dOASL-3K*and hOASL-R*K*K*mutants activated and magnified neither the RNase L nor the RIG-? pathway.In addition,both dOASL and dOASL-3D*interacted with dRIG-? in DF1 cells before and after infection with the CK/0513 virus.Similarly,we observed that hOASL,mOasl2 and their mutants(hOASL-3D and mOasl2-3D*)co-precipitated with the corresponding human or mouse RIG-?.Thus,the mutation of the three conserved D residues did not affect the interaction between OASL and RIG-?.This observation,combined with the finding that truncations of avian and mammalian nonenzymatic OASLs did not prevent against virus infection,indicating that the OLD domain of OASL was sufficient to mediate the RIG-? interaction but insufficient to enhance RIG-? signaling.Furthermore,enhancing the RIG-? signaling by inactive avian and mammalian OASL proteins needs the two tandem UBLs.We found that,unlike mOasl1,duck OASL did not suppress IRF7 translation through binding the 5'UTR.Moreover,manually query the MBD1 repertoire against the non-redundant database in NCBI and examining avian assemblies in Ensembl(release 87)indicated that MBD1 appears to be absent in birds.Thus,avian OASLs may not directly interact with MBD1 protein similar to human OASL.Taken together,our findings show molecular mechanisms for the switching of the OAS/RNase L and OASL/RIG-? pathways.This study provides a theoretical basis for further research on avian and mammalian OASL molecules in response to RNA virus infections(especially for influenza A viruses).