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G3BP1, G3BP2 and CAPRIN1 are required for translation of interferon stimulated mRNAs and are targeted by a dengue virus non-coding RNA.

ABSTRACT: Viral RNA-host protein interactions are critical for replication of flaviviruses, a genus of positive-strand RNA viruses comprising major vector-borne human pathogens including dengue viruses (DENV). We examined three conserved host RNA-binding proteins (RBPs) G3BP1, G3BP2 and CAPRIN1 in dengue virus (DENV-2) infection and found them to be novel regulators of the interferon (IFN) response against DENV-2. The three RBPs were required for the accumulation of the protein products of several interferon stimulated genes (ISGs), and for efficient translation of PKR and IFITM2 mRNAs. This identifies G3BP1, G3BP2 and CAPRIN1 as novel regulators of the antiviral state. Their antiviral activity was antagonized by the abundant DENV-2 non-coding subgenomic flaviviral RNA (sfRNA), which bound to G3BP1, G3BP2 and CAPRIN1, inhibited their activity and lead to profound inhibition of ISG mRNA translation. This work describes a new and unexpected level of regulation for interferon stimulated gene expression and presents the first mechanism of action for an sfRNA as a molecular sponge of anti-viral effectors in human cells.

SUBMITTER: Bidet K

PROVIDER: S-EPMC4081823 | biostudies-literature | 2014 Jul

REPOSITORIES: biostudies-literature

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G3BP1, G3BP2 and CAPRIN1 are required for translation of interferon stimulated mRNAs and are targeted by a dengue virus non-coding RNA.

Bidet Katell K Dadlani Dhivya D Garcia-Blanco Mariano A MA

PLoS pathogens 20140703 7

Viral RNA-host protein interactions are critical for replication of flaviviruses, a genus of positive-strand RNA viruses comprising major vector-borne human pathogens including dengue viruses (DENV). We examined three conserved host RNA-binding proteins (RBPs) G3BP1, G3BP2 and CAPRIN1 in dengue virus (DENV-2) infection and found them to be novel regulators of the interferon (IFN) response against DENV-2. The three RBPs were required for the accumulation of the protein products of several interfe ...[more]

PMID: 24992036

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Project description:UnlabelledStress granules (SGs) are protein-mRNA aggregates that are formed in response to environmental stresses, resulting in translational inhibition. SGs are generally believed to play an antiviral role and are manipulated by many viruses, including various alphaviruses. GTPase-activating protein (SH3 domain)-binding protein 1 (G3BP1) is a key component and commonly used marker of SGs. Its homolog G3BP2 is a less extensively studied SG component. Here, we demonstrate that Chikungunya virus (CHIKV) infection induces cytoplasmic G3BP1- and G3BP2-containing granules that differ from bona fide SGs in terms of morphology, composition, and behavior. For several Old World alphaviruses it has been shown that nonstructural protein 3 (nsP3) interacts with G3BPs, presumably to inhibit SG formation, and we have confirmed this interaction in CHIKV-infected cells. Surprisingly, CHIKV also relied on G3BPs for efficient replication, as simultaneous depletion of G3BP1 and G3BP2 reduced viral RNA levels, CHIKV protein expression, and viral progeny titers. The G3BPs colocalized with CHIKV nsP2 and nsP3 in cytoplasmic foci, but no colocalization with nsP1, nsP4, or dsRNA was observed. Furthermore, G3BPs could not be detected in a cellular fraction enriched for CHIKV replication/transcription complexes, suggesting that they are not directly involved in CHIKV RNA synthesis. Depletion of G3BPs did not affect viral entry, translation of incoming genomes, or nonstructural polyprotein processing but resulted in severely reduced levels of negative-stranded (and consequently also positive-stranded) RNA. This suggests a role for the G3BPs in the switch from translation to genome amplification, although the exact mechanism by which they act remains to be explored.ImportanceChikungunya virus (CHIKV) causes a severe polyarthritis that has affected millions of people since its reemergence in 2004. The lack of approved vaccines or therapeutic options and the ongoing explosive outbreak in the Caribbean underline the importance of better understanding CHIKV replication. Stress granules (SGs) are cytoplasmic protein-mRNA aggregates formed in response to various stresses, including viral infection. The RNA-binding proteins G3BP1 and G3BP2 are essential SG components. SG formation and the resulting translational inhibition are generally considered an antiviral response, and many viruses manipulate or block this process. Late in infection, we and others have observed CHIKV nonstructural protein 3 in cytoplasmic G3BP1- and G3BP2-containing granules. These virally induced foci differed from true SGs and did not appear to represent replication complexes. Surprisingly, we found that G3BP1 and G3BP2 were also needed for efficient CHIKV replication, likely by facilitating the switch from translation to genome amplification early in infection.

Project description:Like other viruses, the picornavirus foot-and-mouth disease virus (FMDV; genus Aphthovirus), one of the most notorious pathogens in the global livestock industry, needs to navigate antiviral host responses to establish an infection. There is substantial insight into how FMDV suppresses the type I interferon (IFN) response, but it is largely unknown whether and how FMDV modulates the integrated stress response. Here, we show that the stress response is suppressed during FMDV infection. Using a chimeric recombinant encephalomyocarditis virus (EMCV), in which we functionally replaced the endogenous stress response antagonist by FMDV leader protease (Lpro) or 3Cpro, we demonstrate an essential role for Lpro in suppressing stress granule (SG) formation. Consistently, infection with a recombinant FMDV lacking Lpro resulted in SG formation. Additionally, we show that Lpro cleaves the known SG scaffold proteins G3BP1 and G3BP2 but not TIA-1. We demonstrate that the closely related equine rhinitis A virus (ERAV) Lpro also cleaves G3BP1 and G3BP2 and also suppresses SG formation, indicating that these abilities are conserved among aphthoviruses. Neither FMDV nor ERAV Lpro interfered with phosphorylation of RNA-dependent protein kinase (PKR) or eIF2?, indicating that Lpro does not affect SG formation by inhibiting the PKR-triggered signaling cascade. Taken together, our data suggest that aphthoviruses actively target scaffolding proteins G3BP1 and G3BP2 and antagonize SG formation to modulate the integrated stress response.IMPORTANCE The picornavirus foot-and-mouth disease virus (FMDV) is a notorious animal pathogen that puts a major economic burden on the global livestock industry. Outbreaks have significant consequences for animal health and product safety. Like many other viruses, FMDV must manipulate antiviral host responses to establish infection. Upon infection, viral double-stranded RNA (dsRNA) is detected, which results in the activation of the RNA-dependent protein kinase (PKR)-mediated stress response, leading to a stop in cellular and viral translation and the formation of stress granules (SG), which are thought to have antiviral properties. Here, we show that FMDV can suppress SG formation via its leader protease (Lpro). Simultaneously, we observed that Lpro can cleave the SG scaffolding proteins G3BP1 and G3BP2. Understanding the molecular mechanisms of the antiviral host response evasion strategies of FMDV may help to develop countermeasures to control FMDV infections in the future.

Project description:UnlabelledStress granules (SGs) are dynamic cytoplasmic repositories containing translationally silenced mRNAs that assemble upon cellular stress. We recently reported that the SG nucleating protein G3BP1 promotes antiviral activity and is essential in double-stranded RNA-dependent protein kinase (PKR) recruitment to stress granules, thereby driving phosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF2α). Here, we delineate the mechanism for SG-dependent PKR activation. We show that G3BP1 and inactive PKR directly interact with each other, dependent on both the NTF2-like and PXXP domains of G3BP1. The G3BP1-interacting protein Caprin1 also directly interacts with PKR, regulates efficient PKR activation at the stress granule, and is also integral for the release of active PKR into the cytoplasm to engage in substrate recognition. The G3BP1-Caprin1-PKR complex represents a new mode of PKR activation and is important for antiviral activity of G3BP1 and PKR during infection with mengovirus. Our data links stress responses and their resultant SGs with innate immune activation through PKR without a requirement for foreign double-stranded RNA (dsRNA) pattern recognition.ImportanceOur previous work indicates that stress granules have antiviral activity and mediate innate immunity through functions of G3BP1; however, the mechanistic details of these functions were not resolved. We show that much of the antiviral activity of stress granules is contingent on the function of PKR in a complex with G3BP1 and Caprin1. The PKR-G3BP1-Caprin1 complex undergoes dynamic transitioning within and outside stress granules to accomplish PKR activation and translational repression. This mechanism appears to function distinctly from canonical pattern recognition of double-stranded RNA by PKR. Therefore, this mechanism bridges the stress response with innate immunity, allowing the cell to respond to many cellular stressors and amplify the pathogen pattern recognition systems of innate immunity.

Dataset Information

G3BP1, G3BP2 and CAPRIN1 are required for translation of interferon stimulated mRNAs and are targeted by a dengue virus non-coding RNA.

Publications

G3BP1, G3BP2 and CAPRIN1 are required for translation of interferon stimulated mRNAs and are targeted by a dengue virus non-coding RNA.

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