COX5B Regulates MAVS-mediated Antiviral Signaling through Interaction with ATG5 and Repressing ROS Production

<div><p>Innate antiviral immunity is the first line of the host defense system that rapidly detects invading viruses. Mitochondria function as platforms for innate antiviral signal transduction in mammals through the adaptor protein, MAVS. Excessive activation of MAVS-mediated antiviral signaling leads to dysfunction of mitochondria and cell apoptosis that likely causes the pathogenesis of autoimmunity. However, the mechanism of how MAVS is regulated at mitochondria remains unknown. Here we show that the Cytochrome c Oxidase (CcO) complex subunit COX5B physically interacts with MAVS and negatively regulates the MAVS-mediated antiviral pathway. Mechanistically, we find that while activation of MAVS leads to increased ROS production and COX5B expression, COX5B down-regulated MAVS signaling by repressing ROS production. Importantly, our study reveals that COX5B coordinates with the autophagy pathway to control MAVS aggregation, thereby balancing the antiviral signaling activity. Thus, our study provides novel insights into the link between mitochondrial electron transport system and the autophagy pathway in regulating innate antiviral immunity.</p> </div

COX5B mediates MAVS signaling by repressing ROS production.

<p>(A) HEK293 cells were pretreated with 10 ug/ml antimycin A for 2 h, then transfected with the indicated plasmids. Twenty-four hours after transfection, cells were lysed to measure the IFNβ induction. (B) HEK293 cells were pretreated with 250 or 500 µM Mito-TEMPO for 4 h, and then transfected with IFNβ reporter and pRSV/LacZ vectors together with empty vector or MAVS, or infected with VSVΔM51-GFP (MOI = 0.1). Cells were lysed to measure IFNβ activity after 24 h transfection or 10 h infection. (C and D) COX5B RNAi oligos or NC were transfected into HEK293 cells, after 20 h transfection, empty vector or MAVS plasmids were transfected again, 30 h after the second transfection, cells were collected for FACS analysis to check cellular or mitochondrial ROS production by staining with DCF (C) or MitoSOX (D), respectively. Results are presented relative to the FACS mean fluorescence intensity over control cells. (E) HEK293 cells were transfected with COX5B RNAi oligo or NC, 36 h after transfection, cells were pretreated with 250 µM Mito-TEMPO for 4 h, and transfected again with indicated plasmids. Twenty-four hours after second transfection, cells were lysed to measure the IFNβ induction. (F) After first transfection and treatment as described in (E), cells were transfected with IFNβ reporter and pRSV/LacZ vectors, followed by 16 h Sendai virus infection, cells were harvested for luciferase assays. (G) The experiments were carried out as in (F) except that cells were pretreated with 100 µM PDTC. (H) HEK293 cells were transfected with the indicated plasmids for 30 h, and then stained with MitoSOX followed by FACS analysis. Cells were treated with 0.1 µM H₂O₂ for 30 min as positive control. Results are presented relative to the FACS mean fluorescence intensity in control cells. (I) HEK293 cells were transfected with increasing amounts of MAVS expression plasmids, and empty vector was used to balance the total DNA amount. Total protein was prepared and subjected to immunoblot analysis after 24 h transfection. Data from A–B, E–G are representative of at least three independent experiments, (mean and s.d. of duplicate assays), and data from C, D and H are presented as mean ± SD from at least three independent experiments. *, P<0.05; **, P<0.01; *** P<0.001 versus control groups.</p

Overexpression of COX5B suppresses MAVS-mediated antiviral signaling.

<p>(A–C) MAVS and COX5B, or empty expression vectors were transfected in HEK293 cells together with luciferase reporter constructs driven by promoters of genes encoding IFNβ (A), NF-κB (B) or ISRE(C), as well as pRSV/LacZ as an internal control. Twenty-four hours after transfection, the luciferase activity was measured and normalized based on β-galactosidase activity. Results are presented relative to the luciferase activity in control cells. (D–F) RIG-I(N) and COX5B or empty expression vectors were transfected in HEK293 cells together with IFNβ-luc (D), NF-κB-luc (E) or IRSE-luc (F) along with pRSV/LacZ. Subsequently, cells were lysed for luciferase assays. (G) HEK293 cells were transfected with empty expression vector or COX5B for 24 h. The cells were then untreated or infected with Sendai virus (50HA units/ml) for 12 h, total RNA was isolated to check the expression of IFNβ mRNA by real-time PCR. (H) Hela cells were transfected with COX5B-GFP or COX5BΔTP-GFP and Flag-MAVS, the cells were then stained with the anti-Flag antibody and imaged by confocal microscopy. (I) HEK293 cells were transfected with the indicated constructs together with IFNβ reporter plasmids. Cells were lysed for luciferase assays after 24 h transfection. (J) HEK293 cells were transfected similarly as in (I), except that RIG-I(N) construct was used instead of MAVS. Data from A–G, I and J are representative of at least three independent experiments (mean and s.d. of duplicate or triplicate assays). *, P<0.05; **, P<0.01 versus the control groups.</p

COX5B interacts with MAVS.

<p>(A and B) HEK293 cells were transfected with combinations of DNA constructs as indicated. Twenty-four hours after transfection, cell lysates were prepared, immunoprecipitated with anti-Flag beads (A) or with anti-Myc antibody (B), followed by immunoblot analysis with the indicated antibodies. WCL (bottom), expression of transfected proteins in whole-cell lysates. (C) HEK293 cell lysates were prepared, immunoprecipitated with anti-MAVS antibody or control IgG, followed by immunoblot analysis. (D) Hela cells were transfected with COX5B-GFP and control vector or YFP-MAVS for 36 h. Cells were fixed, then stained with anti-MAVS antibody (Bottom) or mounted onto slides directly (Top), and imaged by confocal microscopy. (E) Schematic diagram of MAVS and truncated mutants. (F) HEK293 cells were transfected with the indicated plasmids, cell lysates were immunoprecipitated with anti-Flag beads, followed by immunoblot analysis.</p

COX5B and ATG5 suppress MAVS aggregation.

<p>(A) HEK293 cells were transfected with the indicated constructs for 36 h, and crude mitochondrial P5 extracts were isolated, then aliquots of the P5 extracts were subjected to SDD-AGE and SDS-PAGE assays using indicated antibodies respectively. (B) HEK293 cells were transfected with NC or ATG5 RNAi oligos. Thirty-six hours after transfection, Flag-MAVS or empty vectors were transfected into the RNAi cells for 24 h. Crude mitochondrial P5 extracts were prepared, followed by SDD-AGE and SDS-PAGE assays using MAVS or Flag antibody. (C) HEK293 cells were transfected with NC or different doses of ATG5 RNAi oligos as indicated. Thirty-six hours after transfection, knockdown cells were then infected with Sendai virus, 9 h after infection, crude mitochondrial P5 extracts were analyzed by SDD-AGE or SDS-PAGE assays using a MAVS antibody. (D) Cell transfection was performed with indicated plasmids, Thirty-six hours after transfection, crude mitochondrial P5 extracts were isolated, and then aliquots of the P5 extracts were subjected to SDD-AGE and SDS-PAGE assays using indicated antibodies respectively. (E) The experiments were performed as in (B), except that COX5B RNAi oligos were used in lieu of ATG5 RNAi oligos. (F) HEK293 cells were transfected with NC or COX5B RNAi oligos as indicated. Thirty-six hours after transfection, knockdown cells were then infected with Sendai virus, six or nine hours after infection, cell lysates were analyzed by SDD-AGE or SDS-PAGE assays using a MAVS antibody.</p

COX5B negatively controls the virus amplification.

<p>(A–C) HEK293 cells were transfected with COX5B RNAi oligos or NC. After 48 h transfection, cells were infected by Sendai virus for 12 h, and then lysed to isolate RNA to check the transcription levels of IFNβ (A), RANTES (B) and Viperin (C) by real-time PCR. (D–F) The transfection were performed as in (A–C), except that cells were infected by VSVΔM51-GFP (MOI = 0.1) for 9 h. (G) The RNAi oligo transfection was carried out as in (A), RNAi cells were infected by Sendai virus for 16 h, and the supernatant was collected for measurement of IFNβ by ELISA. (H–J) HEK293 cells were transfected with COX5B RNAi oligos for 48 h, then cells were infected by VSV-GFP (H) or VSVΔM51-GFP (I) at the MOI of 0.01 for 12 h, subsequently the culture supernatants were collected to measure virus titer by plaque assay, or cells were imaged by fluorescence microscopy (J). Data from A–I are representative of at least three independent experiments, (A–F, mean and s.d. of triplicate assays, G–I using duplicate assays). *, P<0.05; **, P<0.01 versus the control groups.</p