DHX36 Enhances RIG-I Signaling by Facilitating PKR-Mediated Antiviral Stress Granule Formation

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.

Section: Discussionsupporting

confidence: 87%

Section: Analysis Of Endogenous Rig-i Trim25 and Mavs Localization mentioning

confidence: 99%

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

Sánchez-Aparicio

Ayllón

Leo‐Macias

et al. 2017

“…A significant fraction of transfected cells in our system contain G3BP1-induced stress granules, and we previously showed that assembly of large G3BP1-induced SGs triggers PKR activation. Therefore, we considered whether G3BP1-induced SGs could recruit PKR, similar to findings reported for virus-induced SGs (12,33). When G3BP1 was expressed in HeLa cells, G3BP1 KO MEFs, and U2OS cells and stained for PKR, we found that PKR strongly colocalizes with G3BP1-induced stress granules in all cell types (Fig.…”

Section: G3bp1 and G3bp1-induced Sgs Restrict Enterovirus Infectionsupporting

confidence: 76%

The Stress Granule Protein G3BP1 Recruits Protein Kinase R To Promote Multiple Innate Immune Antiviral Responses

Reineke

Lloyd

2015

181

189

Stress granules (SGs) are cytoplasmic storage sites containing translationally silenced mRNPs that can be released to resume translation after stress subsides. We previously showed that poliovirus 3C proteinase cleaves the SG-nucleating protein G3BP1, blocking the ability of cells to form SGs late in infection. Many other viruses also target G3BP1 and inhibit SG formation, but the reasons why these functions evolved are unclear. Previously, we also showed a link between G3BP1-induced SGs and protein kinase R (PKR)-mediated translational control, but the mechanism of PKR interplay with SG and the antiviral consequences are unknown. Here, we show that G3BP1 exhibits antiviral activity against several enteroviruses, whereas truncated G3BP1 that cannot form SGs does not. G3BP1-induced SGs are linked to activation of innate immune transcriptional responses through NF-B and JNK. The G3BP1-induced SGs also recruit PKR and other antiviral proteins. We show that the PXXP domain within G3BP1 is essential for the recruitment of PKR to SGs, for eIF2␣ phosphorylation driven by PKR, and for nucleating SGs of normal composition. We also show that deletion of the PXXP domain in G3BP1 compromises its antiviral activity. These findings tie PKR activation to its recruitment to SGs by G3BP1 and indicate that G3BP1 promotes innate immune responses at both the transcriptional and translational levels and integrates cellular stress responses and innate immunity. IMPORTANCEStress granules appear during virus infection, and their importance is not well understood. Previously, it was assumed that they were nonfunctional artifacts associated with cellular stress. PKR is a well-known antiviral protein; however, its regulation in cells is not well understood. Our work links cellular stress granules with activation of PKR and other innate immune pathways through the activity of G3BP1, a critical stress granule component. The ability of stress granules and G3BP1 to activate PKR and other innate immune transcriptional responses indicates that G3BP1 is an antiviral protein. This work helps to refine a longstanding paradigm indicating stress granules are inert structures and explains why G3BP1 is subverted by many viruses to promote a productive infection. Stress granules (SGs) are macromolecular triage centers for mRNAs that contain translationally silenced messenger RNPs (mRNPs). Stress granules contain many translation initiation factors, 40S ribosomal subunits, mRNAs, and RNA-binding proteins, which form in response to cellular stresses that inhibit protein synthesis (1, 2). Stress responses are induced during virus infection and are countered by viruses to maximize replication efficiency. Indeed, several examples of viral SG disruption and viral subversion of SG proteins have been described (3-6), indicating stress granules may play antiviral roles against these viruses. Ras-GTPase-activating protein (SH3 domain) binding protein 1 (G3BP1) is a stress granule-resident protein that nucleates stress granule assembly and is also inactiv...

“…Rather than being inert structures, recent reports suggest SGs have antiviral properties; the granules concentrate and promote interactions between a number of innate immune activators, their downstream interferon-stimulated genes (RIG-I, PKR, MDA5, and OAS), and their targets (5,41,43). For example, pattern recognition receptors like RIG-I are activated upon binding to viral RNAs in SGs, which leads to the stimulation of interferon-related genes (42,43,82). Alternatively, recent studies suggest that in the absence of virus infection, SGs can activate innate immune signaling (11,83,84).…”

Section: Discussionmentioning

confidence: 99%

Disruption of Stress Granule Formation by the Multifunctional Cricket Paralysis Virus 1A Protein

Khong

Kerr

Yeung

et al. 2017

Stress granules (SGs) are cytosolic ribonucleoprotein aggregates that are induced during cellular stress. Several viruses modulate SG formation, suggesting that SGs have an impact on virus infection. However, the mechanisms and impact of modulating SG assembly in infected cells are not completely understood. In this study, we identify the dicistrovirus cricket paralysis virus 1A (CrPV-1A) protein that functions to inhibit SG assembly during infection. Moreover, besides inhibiting RNA interference, CrPV-1A also inhibits host transcription, which indirectly modulates SG assembly. Thus, CrPV-1A is a multifunctional protein. We identify a key R146A residue that is responsible for these effects, and mutant CrPV(R146A) virus infection is attenuated in Drosophila melanogaster S2 cells and adult fruit flies and results in increased SG formation. Treatment of CrPV(R146A)-infected cells with actinomycin D, which represses transcription, restores SG assembly suppression and viral yield. In summary, CrPV-1A modulates several cellular processes to generate a cellular environment that promotes viral translation and replication.IMPORTANCE RNA viruses encode a limited set of viral proteins to modulate an array of cellular processes in order to facilitate viral replication and inhibit antiviral defenses. In this study, we identified a viral protein, called CrPV-1A, within the dicistrovirus cricket paralysis virus that can inhibit host transcription, modulate viral translation, and block a cellular process called stress granule assembly. We also identified a specific amino acid within CrPV-1A that is important for these cellular processes and that mutant viruses containing mutations of CrPV-1A attenuate virus infection. We also demonstrate that the CrPV-1A protein can also modulate cellular processes in human cells, suggesting that the mode of action of CrPV-1A is conserved. We propose that CrPV-1A is a multifunctional, versatile protein that creates a cellular environment in virus-infected cells that permits productive virus infection.