In this study, we show that a protein encoded by SARS-CoV designated as open reading frame-9b (ORF-9b) localizes to mitochondria and causes mitochondrial elongation by triggering ubiquitination and proteasomal degradation of dynamin-like protein 1, a host protein involved in mitochondrial fission. Also, acting on mitochondria, ORF-9b targets the mitochondrial-associated adaptor molecule MAVS signalosome by usurping PCBP2 and the HECT domain E3 ligase AIP4 to trigger the degradation of MAVS, TRAF3, and TRAF 6. This severely limits host cell IFN responses. Reducing either PCBP2 or AIP4 expression substantially reversed the ORF-9b–mediated reduction of MAVS and the suppression of antiviral transcriptional responses. Finally, transient ORF-9b expression led to a strong induction of autophagy in cells. The induction of autophagy depended upon ATG5, a critical autophagy regulator, but the inhibition of MAVS signaling did not. These results indicate that SARS-CoV ORF-9b manipulates host cell mitochondria and mitochondrial function to help evade host innate immunity. This study has uncovered an important clue to the pathogenesis of SARS-CoV infection and illustrates the havoc that a small ORF can cause in cells.
Our observations indicate the SARS-CoV ORF-9b provides a receptive intracellular environment for viral replication by targeting the mitochondrial MAVS signalosome. ORF-9b localizes to mitochondria, where it promotes mitochondrial elongation by enhancing DRP1 degradation. In the presence of ORF-9b, MAVS becomes concentrated into small puncta and subject to PCBP2 and AIP4 ubiquitination. The degradation of MAVS is accompanied by a loss of TRAF3 and TRAF6, two key signaling intermediaries in antiviral defenses. In addition, acute ORF-9b expression results in ATG5-dependent autophagosome formation.
The expression of ORF-9b in A549, HEK 293, and THP-1 cells resulted in a rather striking set of cellular changes. ORF-9b localized to mitochondria and led to mitochondrial elongation. This is in contrast to the usual mitochondrial fragmentation that occurs following viral infection and, to our knowledge, has not been reported with another viral ORF. Mitochondrial elongation has been reported following infection of cells with a defective strain of Sendai virus. The mitochondrial elongation was linked to RLR activation and enhanced MAVS signaling . This differed from ORF-9b expression, which also caused mitochondrial elongation, but severely limited MAVS signaling. In contradistinction to the defective virus, the wild-type Sendai virus triggered mitochondrial fragmentation and aggregation . Similarly, hepatitis C virus infection caused mitochondrial perinuclear clustering, the translocation of Parkin to mitochondria, and mitophagy . Perhaps ORF-9b counteracts the impact of other cellular stresses during SARS-CoV infection, which fragment and aggregate mitochondria, thereby helping to promote cell survival during viral replication. Mitochondrial fusion is known to protect mitochondria from starvation-induced autophagosomal degradation .
Our localization results differ from those previously reported localization results of ORF-9b in Vero cells. We observed a similar localization of ORF-9b in three different human cell types using both Flag- and GFP-tagged versions. We show mitochondrial localization in all three cell types and failed to see a visible accumulation of ORF-9b in the nucleus of any of three cell types. The mitochondrial localization reported in this work was confirmed by life cell imaging of cells coexpressing Mito-tracker and by immunostaining fixed cells for Tomm20 and MAVS. The two other studies did not look for mitochondria localization. As mitochondria are often closely associated with the endoplasmic reticulum, the colocalization of ORF-9b with an endoplasmic marker may have obscured its true localization. Less likely is that the localization of ORF-9b has a cell type–dependent distribution pattern.
In the ORF-9b–expressing cells, DRP1 levels declined as a consequence of ubiquitination and proteasomal degradation, most likely explaining the change in mitochondria morphology. Parkin is an E3 ligase that is known to ubiquitinate DRP1 for proteasome-dependent degradation. Reducing Parkin expression in HeLa cells enhanced DRP1 levels and caused mitochondrial fragmentation. To test whether ORF-9b used Parkin to ubiquitinate DRP1, we reduced its expression and examined the mitochondrial morphology in the knockdown cells. The reduced Parkin expression did not alter the mitochondrial morphology changes triggered by ORF-9b expression, arguing that another E3 ligase ubiquitinates DRP1 following ORF-9b expression (C. Shi, unpublished result). We also considered whether autophagosomal degradation contributed to the reduced DRP1 levels noted in ORF-9b–expressing cells. Inhibitors of autophagy increased DRP1 levels in HEK 293T cells, and increasing autophagy reduced DRP1 levels in neurons; however, an autophagy inhibitor did not reverse the lowered DRP1 levels observed in the ORF-9b–expressing cells.
SARS-CoV ORF-9b most likely has little role in directly supporting viral replication, but rather its primary function is to inactivate the RLR pathway by triggering degradation of the MAVS/TRAF3/TFAF6 signalosome. As mentioned in the introductory section, RNA viruses have evolved several different mechanisms to disable MAVS signaling, including cleaving MAVS from the mitochondria , assembling a degradasome, and usurping a host cell-negative regulatory system, the AIP4-PCBP2-MAVS axis, as we describe in this work. PCBP2 serves as an adapter molecule recruited to mitochondrial MAVS following viral infection or MAVS overexpression. The C-terminal portion of MAVS is needed to recruit PCBP2 and ORF-9b. Following ORF-9b expression, we found that PCBP2 immunoprecipitates contained both MAVS and ORF-9b. This suggests the localization of ORF-9b to the mitochondria had led to the translocation of PCBP2 from the nucleus to the mitochondria. Rescue of the ORF-9b phenotype by knockdown of PCBP2 also indicates that ORF-9b expression had led to PCBP2 recruitment to mitochondria. Although the physiologic signal that triggers PCBP2 nuclear export and its localization to mitochondria following viral infection is unknown, mitochondria are a logical source. Pathogenic viral infections cause mitochondrial stress. Stressed mitochondria release reactive oxygen species and a variety of other mediators and affect intracellular calcium levels. However, mitochondrial stress typically causes mitochondrial fission, not mitochondria fusion, as we noted in the ORF-9b–expressing cells.
Not only did we observe a strong reduction in MAVS levels following ORF-9b expression, but also in two key signaling intermediates, TRAF3 and TRAF6. Because reducing either AIP4 or PCBP2 expression largely rescued the ORF-9b–induced signaling defect, the TRAF3 and TRAF6 levels are likely to have been restored. It is unlikely that TRAF3 and TRAF6 are direct targets of the PCBP2/AIP4, but rather are indirectly targeted for destruction by their association with MAVS. Whereas TRAF3 is directly targeted by the E3 ligase Triad3, which also negatively regulates MAVS signaling, Triad3 does not cause MAVS degradation. The viral degradasome induced by the expression of respiratory syncytial virus NS1 proteins results in the degradation of a number of proteins in the IFN induction and response pathway, including RIG-I, TRAF3, and IRF3, but MAVS was not one of them. Our current studies are aimed at understanding how ORF-9b expression recruits PCBP2/AIP4 and determining how TRAF3 and TRAF6 are also degraded. Our working hypothesis is that the ORF-9b/MAVS interaction triggered a partial assembly of the MAVS/TRAF3/TRAF6 signalosome, which leads to a signal that recruits PCBP2/AIP4. As a consequence, MAVS and perhaps other proteins are ubiquitinated and delivered to the proteasome for degradation.
Although the observed decreases in DRP1 and MAVS/TRAF3/TRAF6 can explain the mitochondrial elongation and reduced antiviral signaling in the ORF-9b expressing, we have not found an immediately proximal cause of the increase autophagy in HEK293 and A549 cells. Given its localization to mitochondria, mitochondria involvement in the enhanced autophagy noted in the ORF-9b–expressing cells seems likely. However, whereas mitochondrial damage and increased mitochondrial reactive oxygen species production are known triggers for autophagosome formation, they had no clearly defined role in this work. Because the mitochondrial potential in control and ORF-9b–expressing cells did not differ, mitochondrial reactive oxygen species production is an unlikely culprit. We also examined autophagy as assessed by LC3 immunoblotting in the THP-1 cells permanently transfected with ORF-9b. Although we observed a small increase in LC3 processing in the ORF-9b–expressing cells, the difference was less marked than what we had observed following the acute expression of ORF-9b in A549 and HEK 293 cells. Further studies will be needed to delineate the mechanisms by which ORF-9b triggers marked autophagosome formation in some cell types and not others.
We also considered whether the enhanced autophagy in A549 and HEK 293 cells might contribute to the reduced MAVS activity that we observed. Previous studies have shown that some viruses activate the autophagy pathway to reduce MAVS signaling and impair antiviral responses. Furthermore, the ATG5–ATG12 conjugate has been shown to negatively regulate the type I IFN production pathway by direct association with RIG-I and IFN-β promoter stimulator 1. However, in our hands, ORF-9b–mediated suppression of MAVS signaling did not depend upon the autophagy factor ATG5, as reducing its expression did not impact the suppression in antiviral signaling. In addition, the pharmacological inhibition of autophagy did not reverse the loss of MAVS following ORF-9b expression. The modest increase in autophagy in the 9b-GFP–expressing THP-1 despite a potent reduction in MAVS expression also argues that the induction of autophagy did not contribute to the loss of MAVS and MAVS signaling that we noted.
Finally, the ORF-9b–mediated loss of MAVS may further limit host innate responses by reducing NLRP3 inflammasome activity, as MAVS promotes NLRP3 mitochondrial localization and inflammasome activation. We are currently testing whether the expression of ORF-9b also affects inflammasome activity by targeting NLRP3 levels.
In conclusion, SARS-CoV uses ORF-9b to target host cell mitochondria to disable MAVS signaling. Our data support the use of IFN-α therapy as a prophylactic treatment against SARS-CoV, but suggest that it will be less effective when administered to patients with an established infection.
Reference & source information: https://www.jimmunol.org/
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