Mechanism Of SARS-CoV-2 Nsp15 Protein Inhibiting RLRs Signaling Pathway

The rapid outbreak and spread of SARS-CoV-2 around the world has had a huge impact on public health.Various structural,non-structural,and accessory proteins encoded in the SARS-CoV-2 genome play a crucial role in mediating viral invasion,recombination,and evasion of host immune functions.The wide spread of SARS-CoV-2 is largely due to the mechanism of viral infection and immune escape.Type I interferon is an important part of the innate antiviral immune response.Some studies have shown that SARS-CoV-2 can significantly inhibit the production of type I interferon,but the molecular mechanism of its immune escape is not fully understood.In this study,we found that SARS-CoV-2 encoded nonstructural protein 15(Nsp15)can effectively inhibit the production of type I interferon through previous screening.We further investigated the molecular regulation mechanism of this protein in the process of antagonizing host innate immunity.The main research contents and results are as follows:(1)To verify the antagonistic effect of the Nsp15 protein of SARS-CoV-2 on the RLRs signaling pathway,we used the dual luciferase reporter gene detection system of human IFN-β promoter luciferase reporter plasmid(p GL3-h IFN-β-luc)and renaline luciferase reporter plasmid(p RL-TK).Nsp15 inhibited the RIG-IN(the constitutively active N-terminal domains of RIG-I)/MDA5-induced IFN-β promoter activity.Furthermore,Nsp15 inhibited the m RNA transcription of IFN-β,CXCL10 and ISG56 genes induced by RIG-IN and Poly(I:C).It also restored the replication capacity of vesicular stomatitis virus(VSV)in HEK-293 T cells transfected with RIG-IN.Western Blot results showed that overexpression of Nsp15 inhibited IRF3 phosphorylation induced by RIG-I,but did not affect total IRF3 expression.The immunofluorescence assay revealed that Nsp15 expression hindered the IRF3 nuclear translocation induced by RIG-IN and Sendai virus(Se V).These results indicate that SARS-CoV-2 Nsp15 is an important protein that antagonizes type I interferon production.(2)In order to further explore the molecular mechanism of Nsp15 antagonization against the RLRs signaling pathway,we transfected key signal molecules in the RLRs pathway to stimulate the activation of IFN-β luciferase reporter gene.Nsp15 coexpression obviously suppressed the IFN-β production induced by the key components of the RLRs pathway in a dose-dependent manner.To elucidate the molecular regulatory mechanism,we used Co-IP,GST pull-down assay,and laser scanning confocal microscopy to confirm protein interactions.The findings revealed TBK1 as a crucial protein that interacts with Nsp15.In addition,the TBK1 kinase domain(KD)was identified as the key Nsp15 binding region of SARS-CoV-2 by coimmunoprecipitation experiments with TBK1 truncated mutants.(3)Nsp15 is a conserved uridine-specific endoribonuclease in the coronavirus family.To explore the role of the Nsp15 active domain in the regulation of the RLRs pathway,we constructed a truncated mutant of Nsp15 and co-immunoprecipitation with TBK1.The results showed that Nsp15’s Endo U domain was the key region for TBK1 binding.Dual luciferase reporter revealed the active domain of Nsp15 inhibited TBK1-induced interferon production,but the inhibitory effect was weaker than that of intact Nsp15 protein.Previous studies have shown that the hexamer form of Nsp15 is the key to maintaining its activity.These results indicate that the integrity of Nsp15 is essential for SARS-CoV-2 to play a regulatory role.In addition,the effect of Nsp15 binding to TBK1 on TBK1-IRF3 interaction was further investigated.Coimmunoprecipitation showed that with the increase of Nsp15 expression,the amount of IRF3 protein bound to TBK1 gradually decreased.These results indicated that Nsp15 could competitively bind TBK1 with IRF3.(4)To investigate the mechanism by which Nsp15 inhibits IRF3 nuclear translocation,Nsp15 was co-transfected with karyopherin α1/α2(KPNA1/2),two key nuclear import proteins that mediate IRF3 nuclear translocation.Coimmunoprecipitation showed that Nsp15 could interact with KPNA1.Further studies showed that SARS-CoV-2 Nsp15 inhibited IRF3 nuclear translocation by binding to and reducing KPNA1 protein expression.In summary,we identified two distinct ways in which Nsp15 inhibits type I interferon production:(1)Nsp15 attenuates the interaction between TBK1 and IRF3 by competitively binding to TBK1,thereby reducing IRF3 phosphorylation;(2)Nsp15inhibited the nuclear translocation of phosphorylated IRF3 by binding to and degrading nuclear pore transporter α1.This study not only helps to reveal the mechanism of SARS-CoV-2 Nsp15 antagonizing the RLRs signaling pathway of innate immunity,but also provides a new intervention target for the treatment of SARS-CoV-2.