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During viral infection, cytosolic viral RNA triggers activation of mitochondrial antiviral-signaling protein (MAVS) and the formation of MAVS signalosome (Kawai T et al. 2005; Seth RB et al. 2005; Xu LG et al. 2005). MAVS localizes on the outer membrane of mitochondria through its C-terminal transmembrane (TM) domain. Activated MAVS recruits TANK-binding kinase 1 (TBK1) and interferon regulatory factor 3 (IRF3) to mitochondria leading to the activation of IRF3 and subsequent production of type I interferons.
Immunoprecipitation assays coupled to mass spectrometry analysis revealed that TOMM70 interacted with exogenously expressed MAVS in Sendai virus (SeV)-stimulated human embryonic kidney (HEK293) cells (Liu XY et al. 2010). The TM domains of both MAVS and TOMM70 were required for their interaction. In addition, TOMM70 interacted strongly with the C-terminal motif (EEVD) of HSP90 (Liu XY et al. 2010; Gava LM et al. 2011). TOMM70 also co-immunoprecipitated with TBK1 and IRF3 in HEK293 cells (Liu XY et al. 2010). Further, both TBK1 and IRF3 were found to associate with HSP90, which facilitated signal transduction from TBK1 to IRF3 in SeV-infected HEK293 cells (Yang K et l. 2006). Moreover, SeV infection enhanced the interaction between IRF3 and apoptosis regulator BAX (BAX) in HEK293T cells (Wei B et al. 2015). In SeV-stimulated HEK293 cells, cytosolic BAX translocated to the mitochondrial outer membrane and induced apoptosis in the IRF3-dependent manner via the formation of the TOMM70:HSP90:IRF3:BAX protein complex (Wei B et al. 2015). Knockdown of HSP90 by small interfering RNA (siRNA) decreased the association of TOMM70 with TBK1 and IRF3 (Liu XY et al. 2010). Overexpression of TOMM70 enhanced mRNA levels of IRF3-responsive genes (including IFNB, IFIT1 and RANTES) in HEK293 cells during SeV infection or poly(I:C) stimulation, whereas knockdown of TOMM70 by siRNA showed an inhibitory effect. Similar results were obtained in murine bone marrow-derived macrophages and bone marrow-derived dendritic cells (Liu XY et al. 2010). Thus, the association of MAVS with TOMM70 is thought to potentiate the HSP90-mediated recruitment of TBK1and IRF3 to mitochondria during viral infection thereby inducing IRF3-mediated host antiviral responses. In addition, binding of MAVS to TOMM70 can also trigger BAX-dependent apoptosis (Wei B et al. 2015). TOMM70 also associated with TRADD, TRAF6 and STING in HEK293 cells, further indicating that TOMM70 is a component of MAVS signal complex on mitochondria (Liu XY et al. 2010).
The viral orf9b (9b) proteins derived from SARS‑CoV-1 and SARS-CoV-2 inhibit the MAVS-mediated production of type I IFNs by targeting TOMM70 on the mitochondria (Jiang HW et al. 2020).
This Reactome event shows the association of MAVS with TOMM70.
TOMM70 is a member of the TOM complex that transports cytosolic pre-proteins destined for the mitochondrial compartments (reviewed in Sokol AM et al. 2014; Kreimendahl S & Rassow J 2020). The C-terminal tetratricopeptide repeat (TPR) motifs in TOMM70 recognizes the internal mitochondrial targeting signals (MTSs) of mitochondrial protein precursors in the cytosol and mediates their translocation to the mitochondria (reviewed in Sokol AM et al. 2014; Kreimendahl S & Rassow J 2020). The molecular chaperone complexes of heat shock protein 90 kDa (HSP90) and HSP70 bind to the N-terminal TPR clamp domain of TOMM70 and thus facilitate delivery of precursor proteins to TOMM70 (Young JC et al. 2003; Zanphorlin LM et al. 2016). Further, TOMM70 is involved in the activation of antiviral immunity by interacting with the mitochondrial antiviral signaling protein (MAVS) (Liu XY et al. 2010). Association of TOMM70 with MAVS is thought to promote the recruitment and activation of TBK1 and IRF3 to MAVS upon viral infection (Liu XY et al. 2010). Cryo–electron microscopy (cryo-EM) structure of the TOMM70:SARS-CoV-2 9b complex suggests that viral 9b binds to the substrate recognition site of TOMM70 thus competing with substrate proteins involved in the induction of type I IFN synthesis (Jiang HW et al. 2020; Gordon DE et al. 2020a; reviewed in Kreimendahl S & Joachim Rassow J 2020). Structural and biochemical analysis revealed that Ser53 of the viral 9b protein and Glu477 of TOMM70 are crucial for the association of both proteins and recruitment of HSP90 (Gao X et al. 2021; Brandherm L et al. 2021). Circular dichroism experiments and differential scanning calorimetry showed that the 9b:TOMM70 complex is more thermally stable than free TOMM70 (Ayinde KS etal. 2022). Isothermal titration calorimetry (ITC) showed that a synthetic peptide harboring a central segment of 9b binds TOMM70 with nanomolar KD (Gao X et al. 2021). Further, the binding affinity between the C-terminal EEVD motif of HSP90 and the N-terminal TPR clamp domain TOMM70 was decreased when TOMM70 was associated with viral 9b (Gao X et al. 2021). These data suggest that SARS-CoV-2 9b (ORF9b) interaction with TOMM70 inhibits binding of HSP90 to TOMM70.
This Reactome event shows binding of SARS-CoV-2 9b to TOMM70.
SARS-CoV-1 9b is highly homologous to SARS-CoV-2 9b (Jiang HW et al. 2020; Gordon DE et al. 2020a). Both SARS-CoV-1 and SARS-CoV-2 9b proteins localize to the membrane of mitochondria to suppress host innate immunity (Shi CS et al. 2014; Jiang HW et al. 2020; Gordon DE et al. 2020a). Affinity purification using HEK 293T cells coupled with mass spectrometry (AP-MS) analysis identified direct interaction between the mitochondrial import receptor subunit TOM70 (TOMM70) protein and tagged 9b of SARS-CoV-1 and SARS-CoV-2 (Gordon DE et al. 2020a, b). Further, endogenous TOMM70 co-precipitated with Strep-tagged 9b from SARS-CoV-1 and SARS-CoV-2 expressed in both HEK293T and human lung epithelial A549 cells (Gordon DE et al. 2020a). Immunostaining of tagged SARS-CoV-1 9b and SARS-CoV-2 9b revealed that viral 9b co-localized with TOMM70 upon expression in HEK 293T cells or in a human epithelial cell line derived from cervical cancer (HeLaM) (Jiang HW et al. 2020; Gordon DE et al. 2020a). Similar findings were observed in SARS-CoV-2-infected human colorectal adenocarcinoma (Caco-2) cells (Gordon DE et al. 2020a). Thus, viral 9b:TOMM70 interaction is conserved between SARS-CoV-1 and SARS-CoV-2 (Jiang HW et al. 2020; Gordon DE et al. 2020a).
TOMM70 is a member of the TOM complex that transports cytosolic pre-proteins destined for the mitochondrial compartments (reviewed in Sokol AM et al. 2014; Kreimendahl S & Rassow J 2020). The C-terminal tetratricopeptide repeat (TPR) motifs in TOMM70 recognizes the internal mitochondrial targeting signals (MTSs) of mitochondrial protein precursors in the cytosol and mediates their translocation to the mitochondria (reviewed in Sokol AM et al. 2014; Kreimendahl S & Rassow J 2020). The molecular chaperone complexes of heat shock protein 90 kDa (HSP90) and HSP70 bind to the N-terminal TPR clamp domain of TOMM70 and thus facilitate delivery of precursor proteins to TOMM70 (Young JC et al. 2003; Zanphorlin LM et al. 2016). Further, TOMM70 is involved in the activation of antiviral immunity by interacting with the mitochondrial antiviral signaling protein (MAVS) (Liu XY et al. 2010). Association of TOMM70 with MAVS is thought to promote the recruitment of TBK1 and IRF3 to MAVS upon viral infection (Liu XY et al. 2010). Activated IRF3 translocates into the nucleus to induce the transcription of genes encoding type I interferons (IFN). SARS‑CoV-1 and SARS-CoV-2 9b localized to mitochondria and inhibited the MAVS-mediated production of type I IFNs by targeting TOMM70 (Jiang HW et al. 2020). Cryo–electron microscopy (cryo-EM) structure of the TOMM70:SARS-CoV-2 9b complex suggests that viral 9b binds to the substrate recognition site of TOMM70 thus competing with substrate proteins involved in the induction of type I IFN synthesis (Jiang HW et al. 2020; Gordon DE et al. 2020a; reviewed in Kreimendahl S & Joachim Rassow J 2020). Moreover, SARS-CoV-1 9b suppressed the MAVS-mediated signaling by recruiting poly(rC) binding protein 2 (PCBP2) and the HECT domain E3 ligase AIP4 (ITCH) which triggered the K48-linked polyubiquitination and subsequent degradation of MAVS via the proteasomal pathway (Shi CS et al. 2014).
This Reactome event shows SARS-CoV-1 9b binding to TOMM70.
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