SARS-CoV-2 ORF10 Suppresses The Antiviral Innate Immune Response By Degrading MAVS Through Mitophagy

SARS-CoV-2 is the pathogen of COVID-19.SARS-CoV-2 is highly infectious,the number of patients infected with novel coronavirus and death has exceeded 490 million and 6 million,respectively,posing a serious threat to human health and life safety,and influencing the development of global politics and economy deeply.Compared with SARS-CoV-1 caused disasters to humans,SARS-CoV-2 encodes an ORF10 protein that SARS-CoV-1 does not possess,and whether it plays a role in the replication and pathogenesis of SARS-CoV-2 has not been reported.Our preliminary study revealed that ORF10 induced autophagy and downregulated the expression of MAVS,and then inhibited type Ⅰ interferon signaling responses.It has been reported that viruses such as human parainfluenza virus type 3 degraded MAVS by inducing mitophagy and blocked the host type Ⅰ interferon signaling pathway.Although SARS-CoV-2 could cause mitochondrial depolarization in host cells,altered mitochondrial permeability,and caused mitochondrial damage,it is not clear whether and how mitophagy was induced.This study mainly focused on the downregulated the expression of MAVS protein by ORF10,and focusing on whether ORF10 induces mitophagy and degrading MAVS through mitophagy,and deeply explored the biological function of ORF10 and its molecular mechanism of inhibiting type Ⅰ interferon response,and the following results are obtained:(1)SARS-CoV-2 ORF10 inhibited the type Ⅰ interferon signaling pathway by degrading MAVS.The overexpressed ORF10 plasmid was transfected into HeLa-ACE2 cells,and treated with poly(I:C)or infected with SARS-CoV-2 at 24 hours after transfection,and then the cells and culture supernatant were collected after 12 h of the above treatment.It was found that ORF10 significantly down-regulated the mRNA expression of IFN-α1 and IFN-β by qRT-PCR.At the same time,it was found that ORF10 significantly down-regulated the protein expression of IFN-β by ELISA detection.At the same time,it was also found that ORF10 significantly down-regulated the mRNA and protein expressions of interferon-stimulated gene expression ISG15 and OAS1 by qRT-PCR and Western Blot.The results suggested that ORF10 inhibited the anti-SARS-CoV-2 type Ⅰ interferon signaling pathway.The innate immune molecular plasmid,ORF10 plasmid,firefly luciferase plasmid IFN-β-Luc and Renilla luciferase plasmid TK were co-transfected into HEK293T cells,then cells were collected at 24 hours after transfection.It was found that MAVS,RIG-1 and MDA5 could inhibit the IFN-β promoter activity activated by Dual-Luciferase Reporter Assay assay.The ORF10 plasmid was transfected into HeLa cells,and treated with poly(I:C)at 24 h after transfection,and cells were collected at 12 h after the above treatment.It was found that ORF10 down-regulated the expression of MAVS,and the phosphorylation of TBK1 and IRF3,but did not affect the expression of RIG-I and MDA5 by Western Blot.In addition,we transfected 0.4μg,0.8 μg and 1.6 μg of ORF10 plasmids,and treated them with poly(I:C)at 24 hours after transfection,and cells were collected after 12 hours.The results showed that ORF10 down-regulated the expression of MAVS in a dose-dependent manner by Western Blot.Cells transfected with ORF10 were treated with proteasome inhibitor MG132 and autophagolysosomal inhibitors Baf A1 and CQ,and it was found that ORF10 degraded MAVS through the autophagy pathway.Collectively,these results suggest that ORF10 inhibits the typeⅠ interferon signaling pathway by targeting the degradation of MAVS.(2)SARS-CoV-2 ORF10 interacted with NIX to induce mitophagy.The ORF10 plasmid was transfected into HeLa cells,and the cells were collected,and it was found that ORF10 upregulated the expression of LC3B protein and downregulated the expression of P62 protein by Western Blot.At the same time,the mCherry-GFP-LC3 and ORF10 plasmids were co-transfected into HeLa cells,and indirect immunofluorescence assay were performed,and showed that ORF10 promoted LC3 puncta and aggregated,and green fluorescence quenched by confocal immunofluorescence microscopy.The above results demonstrated that ORF10 induced autophagy.The ORF10 plasmids,mitochondria marker mito plasmids and LC3B plasmids were co-transfected into HeLa cells,and cells were used to indirect immunofluorescence assays,and the result showed that ORF10 co-localized with mitochondria and LC3B by confocal immunofluorescence microscopy.The ORF10 plasmid was transfected into HeLa cells,and the cells were collected at different time of transfection,and it was discovered that the expression gradient of LC3 increased and the mitochondrial outer membrane protein TOMM20 was gradient decreased by Western Blot.HeLa cells were pretreated with the autophagolysosomal inhibitor Baf A1,and then transfected with ORF10 plasmid,and it was found that the autophagy-lysosomal inhibitor Baf A1 blocked that ORF10 inhibited the expression of TOMM20 by Western Blot.The above results suggest that the SARS-CoV-2 ORF10 protein induces mitophagy.The ORF10 plasmid was transfected into HeLa cells,and the cells were collected,and then used to mitochondrial isolation experiment and Western Blot,and results showed that ORF10 upregulated the expression of NIX protein and increased the aggregation of LC3B on mitochondria.The ORF10 and NIX plasmids were co-transfected into HEK293T cells for co-immunoprecipitation experiments and Western Blot,and it was found that ORF10 interacted with exogenous NIX.At the same time,the ORF10 plasmid was transfected into HeLa cells,and the cells were collected for co-immunoprecipitation experiments and Western Blot.and the results of Co-IP assay showed that ORF10 interacts with endogenous NIX.The ORF10 and NIX were co-localizated by confocal immunofluorescence microscopy when the ORF10 and NIX plasmids were co-transfected into HeLa cells for indirect immunofluorescence assays.In addition,ORF10 and mitochondrial marker mito plasmid were co-transfected into NIX-KO cells or HeLa cells for indirect immunofluorescence assays.It was indicated that knocking out NIX attenuated ORF10 colocalizes with mitochondria by confocal immunofluorescence microscopy.At the same time,ORF10 plasmid was transfected into NIX-KO cells or HeLa cells for mitochondrial isolation experiment and Western Blot,and it was demonstrated that knocking out NIX reduced the aggregation of LC3B on mitochondria.Furthermore,the ORF10 plasmid was transfected into HeLa cells for co-immunoprecipitation experiments,and it was found that ORF10 interacted with LC3B by Western Blot.Taken together,these results suggest that SARS-CoV-2 ORF10 induced mitophagy by interacting with NIX and LC3B.(3)SARS-CoV-2 ORF10 degraded MAVS to promote virus replication.The ORF10,MAVS and NIX plasmids or siNIX were co-transfected into HEK293T cells for Dual-Luciferase Reporter Assay,and the results indicated that NIX enhanced the ability of ORF10 to inhibit IFN-β promoter activity.At the same time,the ORF10 and MAVS plasmids were co-transfected into NIX-KO cells or control cells for qRT-PCR,and showed that NIX enhanced the ability of ORF10 to inhibit the mRNA expression of IFN-β.The ORF10 plasmid was transfected into the NIX-KO cells,and the cells were collected at.It was found that ORF10 downregulated the expression of MAVS dependented on NIX protein by Western Blot.The mitophagy inhibitor Mdivi-1 was used to treat HeLa cells transfected with ORF10 and poly(I:C),and qRT-PCR showed that Mdivi-1 significantly blocked ORF10 and inhibited the mRNA expression of IFN-β.These results suggest that ORF10 inhibited type Ⅰ interferon response through mitophagy.The ORF10 plasmids or the silenced ORF10 plasmids were transfected into HeLa-ACE2 cells,and infected with SARS-CoV-2.It was found that overexpression of ORF10 protein significantly degraded MAVS and promoted the expression of N protein,whereas silencing ORF10 blocked the degradation of MAVS protein and inhibited the expression of N protein by Western Blot and qRT-PCR.In addition,it was also found that silencing of ORF10 significantly inhibited the virus titer of SARS-CoV-2.These results suggest that ORF10 promoted SARS-CoV-2 replication by degrading MAVS.In summary,this study is the first to discover the biological function of ORF10 protein in the interaction between SARS-CoV-2 and host cells,which promotes virus replication by inhibiting the type Ⅰ interferon signaling pathway.More importantly,ORF10 induced mitophagy by binding with NIX,and degraded MAVS protein through mitophagy,thereby inhibited the type Ⅰ interferon response,and finally promoted SARS-CoV-2 replication.This study was the first to reveal that ORF10 protein inhibited the signal transduction pathway of type Ⅰ interferon response through the mitophagy pathway,and enriched the molecular mechanism of SARS-CoV-2 inhibiting the innate immune response of host cells,which provides new ideas and target molecules for further development of anti-SARS-CoV-2 drug.