Subcellular Localizations of RIG-I, TRIM25, and MAVS Complexes

Abstract

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.IMPORTANCE 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.

MAVS polymers smaller than 80 nm induce mitochondrial membrane remodeling and interferon signaling

“…However, MDA5 staining remained predominantly cytosolic and no significant increase in colocalization was detected with poly(I:C) treatment based on the Pearson correlation. Similarly, RIG‐I was recently shown to partition into a MAVS‐associated mitochondrial fraction and a cytosolic stress granule fraction . In the absence of MAVS, mitochondria retained their filamentous morphology and failed to move toward the nucleus upon transfection with poly(I:C) (Fig.…”

Paramyxovirus V Proteins Interact with the RIG-I/TRIM25 Regulatory Complex and Inhibit RIG-I Signaling

“…V colocalizes with the RIG-I/TRIM25 complex. To explore the localization of the V/TRIM25 and V/RIG-I complexes, we employed a yellow fluorescent protein (YFP) reconstitution assay called bimolecular fluorescence complementation (BiFC) (46). BiFC, depicted in Fig.…”

Innate Immune Evasion by Human Respiratory RNA Viruses

“…Recent insights suggest that stress granules may form a platform for innate immune responses, since the accumulation of (viral) RNA species there provides a pool of substrates for cellular sensors such as RIG-I and MDA5 [92][93][94]. Indeed, these sensors have been shown to be recruited to stress granules, supporting this view [94][95][96]. In the last decade, it has become clear that many viruses manipulate stress granule formation to benefit their replication, for example, RSV, as was also mentioned above.…”