RNA Interference Functions As An Anti-nodamura Virus Innate Immunity In Mammals

Hosts including plants,nematodes,fruit flies produce virus-derived small interfering RNAs(siRNAs)to direct antiviral immunity by RNA interference(RNAi).However,it remains uncertain whether the RNAi has the similar antiviral function in mammals.Here,we study on Nodamura virus,an arthropod-transmissible virus,and the infection of mammals,to elucidate the induction and suppression of RNAi following the infection,the involvement of pattern recognition receptors in anti-NoV innate immunity as well as the synergistic role in RNAi antiviral response to NoV.In order to detect NoV replication and accumulation after infection,we firstly prepared polyclonal antibodies specific to coat protein(CP)and RNA-dependent RNA polymerase(RdRp),respectively,which are necessary for our study on host antiviral functions.In two independent experiments of NoV infection on BHK-21 cell lines and BALB/c suckling mice,we found that the virus suppressed RNAi function via B2 protein.Host produced a large population of 22-nt vsRNAs,containing a 20-nt perfectly base-paired duplex region with 2-nt 3'overhangs following the infection of a B2-deficient mutant of NoV,NoV?B2.In contrast,the successive canonical siRNAs were not readily detected by NoV infection.These data demonstrated that effective RNAi was induced to protect infected mice against lethal viral infection in the absence of RNAi suppressor,B2.These results were consistent with previous studies of antiviral RNAi induced by flock house virus(FHV)in nematodes and fruit flies,which revealed that RNAi also played similar antiviral role in mammals.We generated a variety of mouse embryonic fibroblasts(MEFs)derived from wild type C57BL/6,RIG-?-/-?MDA5-/-?LGP2-/-?MAVS-/-?TLR3-/-and TLR7-/-mice.We determined that RIG-?-/-MEFs were more susceptible than other MEFs with NoV infection,suggesting the involvement of RIG-? in the host's defense against NoV.Of note,the expression of IFN-? was almost abrogated in infected cells knockout of RIG-?,and NoV replication was restrained in IFN-? pre-treated cells,with no expression of RNAi system-related genes significantly induced,indicating an IFN-dependent antiviral response mediated by RIG-?.Data presented here suggest that RIG-? directs a typical IFN-dependent antiviral response against an RNA virus capable of suppressing the RNAi response.Study on NoV?B2 infection of RIG-?-/-MEFs demonstrated that RNAi failed to inhibit virus replication without the function of RIG-1.No significant difference was detected between fibroblasts infected with NoV and NoV?B2 in the expression of RIG-?,IFN-? and STAT1,although they showed certain levels of induction.Although no expression of RNAi system-related genes was significantly induced in both wildtype and RIG-?-/-MEFs,we characterized NoV?B2 infection on C57?RIG-?-/-and MAVS-/-suckling mice and found that,without the suppression of RNAi,RIG-? conducted a MAVS-independent way to enhance the production of virus-derived siRNAs,indicating that RIG-? plays a synergistic role in RNAi antiviral functions.