Subcellular Localizations of RIG-I, TRIM25, and MAVS Complexes.
The retinoic acid-inducible gene 1 (RIG-I) signaling pathway is essential for the recognition of viruses and the initiation of host interferon (IFN)-mediated antiviral responses. Once activated, RIG-I interacts with polyubiquitin chains generated by TRIM25 and binds mitochondrial antiviral signaling protein (MAVS), leading to the production of type I IFN. We now show specific interactions among these key partners in the RLR pathway through the use of bimolecular fluorescence complementation (BiFC) and super-resolution microscopy. Dimers of RIG-I, TRIM25, and MAVS localize into different compartments. Upon activation, we show that TRIM25 is redistributed into cytoplasmic dots associated with stress granules, while RIG-I associates with TRIM25/stress granules and with mitochondrial MAVS. In addition, MAVS competes with TRIM25 for RIG-I binding, and this suggests that upon TRIM25-mediated activation of RIG-I, RIG-I moves away from TRIM25 to interact with MAVS at the mitochondria. For the first time, the distribution of these three proteins was analyzed at the same time in virus-infected cells. We also investigated how specific viral proteins modify some of the protein complexes in the pathway. The protease NS3/4A from hepatitis C virus redistributes the complexes RIG-I/MAVS and MAVS/MAVS but not RIG-I/TRIM25. In contrast, the influenza A virus NS1 protein interacts with RIG-I and TRIM25 in specific areas in the cell cytoplasm and inhibits the formation of TRIM25 homocomplexes but not the formation of RIG-I/TRIM25 heterocomplexes, preventing the formation of RIG-I/MAVS complexes. Thus, we have localized spatially in the cell different complexes formed between RIG-I, TRIM25, and MAVS, in the presence or absence of two viral IFN antagonistic proteins.


OBJECTIVE
The first line of defense against viral infections is the innate immune response. Viruses are recognized by pathogen recognition receptors, such as the RIG-I like receptor family, that activate a signaling cascade that induces IFN production. In the present study, we visualized, for the first time in cells, both in overexpression and endogenous levels, complexes formed among key proteins involved in this innate immune signaling pathway. Through different techniques we were able to analyze how these proteins are distributed and reorganized spatially within the cell in order to transmit the signal, leading to an efficient antiviral state. In addition, this work presents a new means by how, when, and where viral proteins can target these pathways and act against the host immune system in order to counteract the activation of the immune response.
Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, New York, USA.|Global Health and Emerging Pathogens Institute, Icahn School of Medicine at Mount Sinai, New York, New York, USA.
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Sánchez-Aparicio
Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, New York, USA.|Global Health and Emerging Pathogens Institute, Icahn School of Medicine at Mount Sinai, New York, New York, USA.
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Ayllón
The Leon H. Charney Division of Cardiology, New York University School of Medicine (NYU-SoM), New York, New York, USA.
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Leo-Macias
Division of Influenza Viruses and Other Respiratory Viruses, Robert Koch Institute, Berlin, Germany.
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Wolff
Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, New York, USA adolfo.garcia-sastre@mssm.edu.|Global Health and Emerging Pathogens Institute, Icahn School of Medicine at Mount Sinai, New York, New York, USA.|Department of Medicine, Division of Infectious Diseases, Icahn School of Medicine at Mount Sinai, New York, New York, USA.
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García-Sastre
D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry / Q000378:metabolism*; D002460:Cell Line; D000071457:DEAD Box Protein 58 / Q000737:chemistry / Q000378:metabolism*; D054884:Host-Pathogen Interactions; D006801:Humans; D007113:Immunity, Innate; D042541:Intracellular Space; D046912:Multiprotein Complexes / Q000378:metabolism*; D011485:Protein Binding; D055503:Protein Multimerization; D021381:Protein Transport; D015398:Signal Transduction; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism*; D000070584:Tripartite Motif Proteins / Q000737:chemistry / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry / Q000378:metabolism*; D017361:Viral Nonstructural Proteins / Q000378:metabolism; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
D048868:Adaptor Proteins, Signal Transducing; D000071457:DEAD Box Protein 58; D046912:Multiprotein Complexes; D014157:Transcription Factors; D000070584:Tripartite Motif Proteins; D044767:Ubiquitin-Protein Ligases; D017361:Viral Nonstructural Proteins; C085820:INS1 protein, influenza virus; C505371:MAVS protein, human; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; C093542:NS4 protein, hepatitis C virus; C084523:TRIM25 protein, human
RIG-I; influenza; innate immunity; microscopy; pathogen recognition receptors; virus