Mitochondrial-associated endoplasmic reticulum membranes (MAM) form innate immune synapses and are targeted by hepatitis C virus

Abstract

RIG-I is a cytosolic pathogen recognition receptor that engages viral RNA in infected cells to trigger innate immune defenses through its adaptor protein MAVS. MAVS resides on mitochondria and peroxisomes, but how its signaling is coordinated among these organelles has not been defined. Here we show that a major site of MAVS signaling is the mitochondrial-associated membrane (MAM), a distinct membrane compartment that links the endoplasmic reticulum to mitochondria. During RNA virus infection, RIG-I is recruited to the MAM to bind MAVS. Dynamic MAM tethering to mitochondria and peroxisomes then coordinates MAVS localization to form a signaling synapse between membranes. Importantly, the hepatitis C virus NS3/4A protease, which cleaves MAVS to support persistent infection, targets this synapse for MAVS proteolysis from the MAM, but not from mitochondria, to ablate RIG-I signaling of immune defenses. Thus, the MAM mediates an intracellular immune synapse that directs antiviral innate immunity.

Fig. 1.

MAVS is localized to MAM, mitochondria, and peroxisomes. (A) Confocal microscopy analysis of immunostained Huh7 cells expressing MAVS-mEGFP for PMP70 (peroxisomes) and TOM20 (OMM). (B) Immunoblot analysis of fractionated Huh7 cells. F1, containing MAM and peroxisomes, is labeled as MAM & Pex. Fractionation markers: calnexin, ER; Cox-1, mitochondria; FACL4, MAM; Pex19, peroxisomes. (C) Confocal microscopy analysis of endogenous MAVS and FACL4 Huh7 cells preloaded with Mitotracker. In A and C, arrows indicate MAVS on MAM or between peroxisomes and mitochondria. Single-cell images are representative of at least 10 cells analyzed. Histograms display measured fluorescence intensity along the line in the merge panels, with gray lines indicating local peaks of MAVS intensity. (Scale bar: 10 μm.)

Fig. 2.

NS3/4A localizes to junctions between MAM and mitochondria. (A) Immunoblot analysis of subcellular fractions from HeLa cells stably expressing mEGFP-PSS-1 (MAM) and transiently expressing NS3/4A. (B–E) Confocal micrographs of cells transfected with mEGFP-PSS-1 or left untransfected, preloaded with Mitotracker, and immunostained. (B) Huh7-HCV K2040 cells. White arrows indicate NS3 and NS4A adjacent to mitochondria. Histograms display the measured fluorescence intensity along the line in the merge panels, with gray lines marking local peaks of HCV protein intensity. (C) Huh7 cells transiently expressing HCV NS3/4A. (D and E) Huh7.5 cells infected with HCV for 48 h. Large arrows indicate NS3 and NS4A near mitochondria, and small arrows indicate membranous areas devoid of mitochondria. Single-cell images are representative of at least 10 cells analyzed. (Scale bars: 10 μm in B–D; 2 μm in E.)

Fig. 3.

The MAM is an innate immune signaling platform. (A and B) Immunoblot analysis of fractionated Huh7 (−) and Huh7-HCV K2040 (+) cells. Arrows indicate full-length (FL) and cleaved (C) MAVS. (C) Immunoblot analysis of fractions immunoprecipitated with anti–RIG-I from mock- or SenV-infected (for 8 h) PH5CH8 cells and input. *Nonspecific band. Fractionation markers: calnexin, ER; Cox-1, mitochondria; FACL4, MAM; tubulin, cytosol; Pex19, peroxisomes.

Fig. 4.

Mitochondrial–ER contacts regulate MAVS localization and signaling. (A) Immunostain of endogenous MAVS and TOM20 (OMM). Arrows indicate MAVS localized independent of TOM20. (C) Immunostain of cells expressing MAVS-mEGFP for PMP70 (peroxisomes) and TOM20. (B, D, and E) Values (mean ± SD; n = 3–4 cells) of MAVS localization to mitochondria (B) or peroxisomes (D) (object-based overlap approach) (26), or overlap between peroxisomes and mitochondria (E) (Pearson's coefficient) (26). *P ≤ 0.05; **P ≤0.005; ***P ≤0.0005, by unpaired Student t test. Single-cell images are representative of two experiments and >10 cells analyzed per experiment. (Scale bar: 10 μm.)

Fig. 5.

Mitochondrial–ER contacts regulate RIG-I pathway antiviral signaling. (A and B) Immunoblot analysis and IFN-β promoter luciferase expression of Huh7 cells treated with nontargeting control (CTRL), MFN1, or MFN2 siRNA pools or methyl-β cyclodextrin and then mock- or SenV-infected. Values are mean ± SD (n = 3) of one of three representative experiments. *P ≤ 0.05; **P ≤0.005; ***P ≤0.0005, by unpaired Student t test. (C) HCV copy number (mean ± SD; n = 3). (Inset) Immunoblot analysis of MAVS; full-length (FL) and cleaved (C). (D) (Upper) HCV-positive cells after 72 h of infection (mean ± SD; n = 5–6). (Lower) Percent VSV-positive cells after 16 h of VSV infection (mean ± SD; n = 3–5, >1000+ cells counted). (E and F) Confocal micrographs of immunostained SenV-infected Huh7 cells expressing mEGFP-PSS-1 (MAM) (E) or HCV NS4A-mEGFP (F) with PMP70 (peroxisomes) and TOM20 (OMM) and 3D reconstruction of Z stacks. Arrows mark synapses. Single-cell images are representative of two experiments and >10 cells analyzed per experiment. (Scale bar: 10 μm.)