The Mechanism Of HSP60 And IFI16 In Inhibiting Porcine Reproductive And Respiratory Syndrome Virus Replication

Porcine reproductive and respiratory syndrome(PRRS)is one of the most destructive animal viral diseases in the world.The causative agent of PRRS is PRRSV(PRRS virus).PRRSV is a member of the Arteriviridae family and consists of an enveloped single-stranded positive-sense RNA genome.PRRSV has genetic diversity and PRRSV infection could cause immunosuppression and persistent infection,resulting in enormous burden and challenge for controlling PRRS.It has been reported that type ? interferons(?-IFNs),IFN-stimulated genes(ISGs)and some host proteins have antiviral functions against PRRSV.In this study,we screened HSP60 and IFI16 that could inhibit PRRSV replication by mi R-382-5p,and then focused on the mechanism of HSP60 and IFI16 in inhibiting PRRSV replication,which mainly includes the following three parts.1.Screening of anti-PRRSV genes based on mi R-382-5pPrevious report has shown that mi R-382-5p was a differentially expressed host mi RNA by the high-throughput sequencing of small RNAs from MARC-145 cells infected with PRRSV.Mi RNAs are a class of conserved non-coding RNAs that are widely found in eukaryotic cells,and function as posttranscriptional regulators of cellular processes such as cell proliferation,differentiation,apoptosis and immune response.In this study,we found that mi R-382-5p could promote PRRSV replication.And mi R-382-5p could negatively regulate ploy I:C-induced the production of IFN-?.In addition,at the post-transcriptional level,mi RNAs could regulate gene expression by typically binding to complementary sequences of target m RNAs to induce m RNA degradation or suppress translation of the target gene.Thus,we found that DDX3 X,HSP60,IFI16,USP8,FADD and SKP1 may be the target genes of mi R-382-5p through prediction and verification.And then,further study demonstrated that HSP60 and IFI16 have the ability to repress PRRSV replication.2.The mechanism of HSP60 in inhibiting PRRSV replicationThe chaperone protein,HSP60,is a node in intercellular immune networks that could regulate the immune response and virus replication.In this study,the m RNA levels of PRRSV ORF7,the virus titers and the N protein expression levels were significantly increased in MARC-145 and PAM cells transfected with si HSP60.And overexpression of HSP60 could markedly inhibit PRRSV replication.These data indicated that HSP60 acts as an antiviral protein against PRRSV.Then,we determined that HSP60 could promote ploy I:C-mediated production of IFN-? by dual-luciferase reporter assay and q PCR.And further studies showed that HSP60 enhanced MAVS-mediated IFN-? promoter activity and the transcription levels of IFN-?,ISG15 and ISG56 in a dose-dependent manner.Besides,HSP60 could not promote poly I:C-mediated IFN-? promoter activity and the m RNA levels of IFN-? upon silencing of MAVS.However,HSP60 could increase poly I:C-mediated the production of I-IFN in cells transfected with nontargeting control si RNAs,which indicated that HSP60 promotes the production of I-IFN in a MAVS-dependent manner.The co-immunoprecipitation assays(Co-IP)assay and confocal microscopy analyses showed that HSP60 specifically interacted and co-localized with MAVS.Additionally,the N-terminal CARD,PRO and C-terminal TM domain of MAVS were all required for its interaction with HSP60.Next,we found that HSP60 could not suppress PRRSV replication in MARC-145 cells transfected with si MAVS.These data suggested that the anti-PRRSV activity of HSP60 required the involvement of MAVS.3.The mechanism of IFI16 in inhibiting PRRSV replicationIFI16 is the interferon ?-inducible protein and has been reported to have a broader role in the regulation of the ?-IFN response to RNA and DNA viruses in antiviral immunity.In this study,IFI16 was demonstrated to be an antiviral protein that could inhibit PRRSV replication.Besides,IFI16 can be upregulated upon IFN-? and PRRSV infection,and IFI16 could promote PRRSV or poly I:C-induced production of IFN-?.Then,further studies showed that IFI16 could significantly enhance MAVS-mediated IFN-? promoter activity and the m RNA levels of IFN-?,ISG15 and ISG56.And the IFN-? promoter activity and the m RNA levels of IFN-?,ISG15 and ISG56 mediated by MAVS increased greatly with the increasing doses of IFI16.Additionally,IFI16 could not promote poly I:C-mediated the production of IFN-? upon silencing of MAVS.The data suggested that IFI16 could positively regulate MAVS-mediated ?-IFN signaling.In addition,Co-IP assays showed that IFI16 could specifically interact with MAVS,and the N-terminal CARD,PRO and C-terminal TM domain of MAVS were sufficient and efficient for the binding with IFI16.More importantly,we showed that IFI16 exerts antiviral effects in an MAVS-dependent manner.In conclusion,the study demonstrated that HSP60 and IFI16 are the antiviral proteins and could inhibit PRRSV replication.Besides,HSP60 and IFI16 could positively regulate MAVS-mediated ?-IFN signaling and the antiviral effects of HSP60 and IFI16 depend on MAVS.The study illustrated the roles of HSP60 and IFI16 in the MAVS-mediated signaling pathway,and revealed a novel mechanism of HSP60 and IFI16 in inhibiting PRRSV replication,which may contribute to understanding the roles of HSP60 and IFI16 in the regulation of RLR signaling pathway and virus replication,and may provide new ideas for anti-PRRSV.