Inhibition of cytoplasmic mRNA stress granule formation by a viral proteinase

Mammalian cells form dynamic cytoplasmic mRNA stress granules (SGs) in response to environmental stresses including viral infections. SGs are involved in regulating host mRNA function and metabolism, although their precise role during viral infection is unknown. SGs are thought to assemble based on functions of the RNA-binding proteins TIA-1/TIAR or Ras-GAP SH3 domain-binding protein (G3BP). Here, we investigated the relationship between a prototypical plus-strand RNA virus and SGs. Early during poliovirus infection, SG formation is induced, but as infection proceeds this ability is lost, and SGs disperse. Infection resulted in cleavage of G3BP, but not TIA-1 or TIAR, by poliovirus 3C proteinase. Expression of a cleavage-resistant G3BP restored SG formation during poliovirus infection and significantly inhibited virus replication. These results elucidate a mechanism for viral interference with mRNP metabolism and gene regulation and support a critical role of G3BP in SG formation and restriction of virus replication.     

Encephalomyocarditis Virus Disrupts Stress Granules,the Critical Platform for Triggering Antiviral Innate Immune Responses

In response to stress, cells induce ribonucleoprotein aggregates, termed stress granules (SGs). SGs are transient loci containing translation-stalled mRNA, which is eventually degraded or recycled for translation. Infection of some viruses, including influenza A virus with a deletion of nonstructural protein 1 (IAVΔNS1), induces SG-like protein aggregates. Previously, we showed that IAVΔNS1-induced SGs are required for efficient induction of type I interferon (IFN). Here, we investigated SG formation by different viruses using green fluorescent protein (GFP)-tagged Ras-Gap SH3 domain binding protein 1 (GFP-G3BP1) as an SG probe. HeLa cells stably expressing GFP-G3BP1 were infected with different viruses, and GFP fluorescence was monitored live with time-lapse microscopy. SG formations by different viruses was classified into 4 different patterns: no SG formation, stable SG formation, transient SG formation, and alternate SG formation. We focused on encephalomyocarditis virus (EMCV) infection, which exhibited transient SG formation. We found that EMCV disrupts SGs by cleavage of G3BP1 at late stages of infection (>8 h) through a mechanism similar to that used by poliovirus. Expression of a G3BP1 mutant that is resistant to the cleavage conferred persistent formation of SGs as well as an enhanced induction of IFN and other cytokines at late stages of infection. Additionally, knockdown of endogenous G3BP1 blocked SG formation with an attenuated induction of IFN and potentiated viral replication. Taken together, our findings suggest a critical role of SGs as an antiviral platform and shed light on one of the mechanisms by which a virus interferes with host stress and subsequent antiviral responses.     

Production of a Dominant-Negative Fragment Due to G3BP1 Cleavage Contributes to the Disruption of Mitochondria-Associated Protective Stress Granules during CVB3 Infection

Stress granules (SGs) are dynamic cytosolic aggregates containing messenger ribonucleoproteins and target poly-adenylated (A)-mRNA. A key component of SGs is Ras-GAP SH3 domain binding protein-1 (G3BP1), which in part mediates protein-protein and protein-RNA interactions. SGs are modulated during infection by several viruses, however, the function and significance of this process remains poorly understood. In this study, we investigated the interplay between SGs and Coxsackievirus type B3 (CVB3), a member of the Picornaviridae family. Our studies demonstrated that SGs were formed early during CVB3 infection; however, G3BP1-positive SGs were actively disassembled at 5 hrs post-infection, while poly(A)-positive RNA granules persisted. Furthermore, we confirmed G3BP1 cleavage by 3C^pro at Q325. We also demonstrated that overexpression of G3BP1-SGs negatively impacted viral replication at the RNA, protein, and viral progeny levels. Using electron microscopy techniques, we showed that G3BP1-positive SGs localized near mitochondrial surfaces. Finally, we provided evidence that the C-terminal cleavage product of G3BP1 inhibited SG formation and promoted CVB3 replication. Taken together, we conclude that CVB3 infection selectively targets G3BP1-SGs by cleaving G3BP1 to produce a dominant-negative fragment that further inhibits G3BP1-SG formation and facilitates viral replication.     

Hepatitis C virus (HCV) induces formation of stress granules whose proteins regulate HCV RNA replication and virus assembly and egress

Stress granules (SGs) are cytoplasmic structures that are induced in response to environmental stress, including viral infections. Here we report that hepatitis C virus (HCV) triggers the appearance of SGs in a PKR- and interferon (IFN)-dependent manner. Moreover, we show an inverse correlation between the presence of stress granules and the induction of IFN-stimulated proteins, i.e., MxA and USP18, in HCV-infected cells despite high-level expression of the corresponding MxA and USP18 mRNAs, suggesting that interferon-stimulated gene translation is inhibited in stress granule-containing HCV-infected cells. Finally, in short hairpin RNA (shRNA) knockdown experiments, we found that the stress granule proteins T-cell-restricted intracellular antigen 1 (TIA-1), TIA1-related protein (TIAR), and RasGAP-SH3 domain binding protein 1 (G3BP1) are required for efficient HCV RNA and protein accumulation at early time points in the infection and that G3BP1 and TIA-1 are required for intracellular and extracellular infectious virus production late in the infection, suggesting that they are required for virus assembly. In contrast, TIAR downregulation decreases extracellular infectious virus titers with little effect on intracellular RNA content or infectivity late in the infection, suggesting that it is required for infectious particle release. Collectively, these results illustrate that HCV exploits the stress granule machinery at least two ways: by inducing the formation of SGs by triggering PKR phosphorylation, thereby downregulating the translation of antiviral interferon-stimulated genes, and by co-opting SG proteins for its replication, assembly, and egress.     

Tracking and elucidating alphavirus-host protein interactions

Viral infections cause profound alterations in host cells. Here, we explore the interactions between proteins of the Alphavirus Sindbis and host factors during the course of mammalian cell infection. Using a mutant virus expressing the viral nsP3 protein tagged with green fluorescent protein (GFP) we directly observed nsP3 localization and isolated nsP3-interacting proteins at various times after infection. These results revealed that host factor recruitment to nsP3-containing complexes was time dependent, with a specific early and persistent recruitment of G3BP and a later recruitment of 14-3-3 proteins. Expression of GFP-tagged G3BP allowed reciprocal isolation of nsP3 in Sindbis infected cells, as well as the identification of novel G3BP-interacting proteins in both uninfected and infected cells. Note-worthy interactions include nuclear pore complex components whose interactions with G3BP were reduced upon Sindbis infection. This suggests that G3BP is a nuclear transport factor, as hypothesized previously, and that viral infection may alter RNA transport. Immunoelectron microscopy showed that a portion of Sindbis nsP3 is localized at the nuclear envelope, suggesting a possible site of G3BP recruitment to nsP3-containing complexes. Our results demonstrate the utility of using a standard GFP tag to both track viral protein localization and elucidate specific viral-host interactions over time in infected mammalian cells.     

Mammalian orthoreovirus escape from host translational shutoff correlates with stress granule disruption and is independent of eIF2alpha phosphorylation and PKR

In response to mammalian orthoreovirus (MRV) infection, cells initiate a stress response that includes eIF2α phosphorylation and protein synthesis inhibition. We have previously shown that early in infection, MRV activation of eIF2α phosphorylation results in the formation of cellular stress granules (SGs). In this work, we show that as infection proceeds, MRV disrupts SGs despite sustained levels of phosphorylated eIF2α and, further, interferes with the induction of SGs by other stress inducers. MRV interference with SG formation occurs downstream of eIF2α phosphorylation, suggesting the virus uncouples the cellular stress signaling machinery from SG formation. We additionally examined mRNA translation in the presence of SGs induced by eIF2α phosphorylation-dependent and -independent mechanisms. We found that irrespective of eIF2α phosphorylation status, the presence of SGs in cells correlated with inhibition of viral and cellular translation. In contrast, MRV disruption of SGs correlated with the release of viral mRNAs from translational inhibition, even in the presence of phosphorylated eIF2α. Viral mRNAs were also translated in the presence of phosphorylated eIF2α in PKR(-/-) cells. These results suggest that MRV escape from host cell translational shutoff correlates with virus-induced SG disruption and occurs in the presence of phosphorylated eIF2α in a PKR-independent manner.     

Modulation of stress granules and P bodies during dicistrovirus infection

Stress granules (SGs) are dynamic cytosolic aggregates composed of ribonucleoproteins that are induced during cellular stress when protein synthesis is inhibited. The function of SGs is poorly understood, but they are thought to be sites for reorganizing mRNA and protein. Several viruses can modulate SG formation, suggesting that SGs have an impact on virus infection. In this study, we have investigated the relationship of SG formation in Drosophila S2 cells infected by cricket paralysis virus (CrPV), a member of the Dicistroviridae family. Despite a rapid shutoff of host translation during CrPV infection, several hallmark SG markers such as the Drosophila TIA-1 and G3BP (RasGAP-SH3-binding protein) homologs, Rox8 and Rin, respectively, do not aggregate in CrPV-infected cells, even when challenged with potent SG inducers such as heat shock, oxidative stress, and pateamine A treatment. Furthermore, we demonstrate that a subset of P body markers become moderately dispersed at late times of infection. In contrast, as shown by fluorescent in situ hybridization, poly(A)(+) RNA granules still form at late times of infection. These poly(A)(+) RNA granules do not contain viral RNA nor do they colocalize with P body markers. Finally, our results demonstrate that the CrPV viral 3C protease is sequestered to SGs under cellular stress but not during virus infection. In summary, we propose that dicistrovirus infection leads to the selective inhibition of distinct SGs so that viral proteins are available for viral processing.     

The C-Terminal Repeat Domains of nsP3 from the Old World Alphaviruses Bind Directly to G3BP

The Old World alphaviruses block stress granule assembly by sequestration of RasGAP SH3-domain binding protein (G3BP). Here, we show that the proline-rich sequences in the hypervariable domain of nonstructural protein 3 (nsP3) of both Semliki Forest virus and Chikungunya virus were dispensable for binding to G3BP. nsP3 variants with or without this domain colocalized with G3BP. Furthermore, we show that the C-terminal repeat motifs of nsP3 were sufficient for G3BP binding.     

Influenza A Virus Host Shutoff Disables Antiviral Stress-Induced Translation Arrest

Influenza A virus (IAV) polymerase complexes function in the nucleus of infected cells, generating mRNAs that bear 5′ caps and poly(A) tails, and which are exported to the cytoplasm and translated by host machinery. Host antiviral defences include mechanisms that detect the stress of virus infection and arrest cap-dependent mRNA translation, which normally results in the formation of cytoplasmic aggregates of translationally stalled mRNA-protein complexes known as stress granules (SGs). It remains unclear how IAV ensures preferential translation of viral gene products while evading stress-induced translation arrest. Here, we demonstrate that at early stages of infection both viral and host mRNAs are sensitive to drug-induced translation arrest and SG formation. By contrast, at later stages of infection, IAV becomes partially resistant to stress-induced translation arrest, thereby maintaining ongoing translation of viral gene products. To this end, the virus deploys multiple proteins that block stress-induced SG formation: 1) non-structural protein 1 (NS1) inactivates the antiviral double-stranded RNA (dsRNA)-activated kinase PKR, thereby preventing eIF2α phosphorylation and SG formation; 2) nucleoprotein (NP) inhibits SG formation without affecting eIF2α phosphorylation; 3) host-shutoff protein polymerase-acidic protein-X (PA-X) strongly inhibits SG formation concomitant with dramatic depletion of cytoplasmic poly(A) RNA and nuclear accumulation of poly(A)-binding protein. Recombinant viruses with disrupted PA-X host shutoff function fail to effectively inhibit stress-induced SG formation. The existence of three distinct mechanisms of IAV-mediated SG blockade reveals the magnitude of the threat of stress-induced translation arrest during viral replication.     

Chikungunya Virus nsP3 Blocks Stress Granule Assembly by Recruitment of G3BP into Cytoplasmic Foci

Chikungunya virus nonstructural protein nsP3 has an essential but unknown role in alphavirus replication and interacts with Ras-GAP SH3 domain-binding protein (G3BP). Here we describe the first known function of nsP3, to inhibit stress granule assembly by recruiting G3BP into cytoplasmic foci. A conserved SH3 domain-binding motif in nsP3 is essential for both nsP3-G3BP interactions and viral RNA replication. This study reveals a novel role for nsP3 as a regulator of the cellular stress response.     

In silico identification of MicroRNAs targeting the key nucleator of stress granules,G3BP: Promising therapeutics for SARS-CoV-2 infection

Stress granules (SGs) are non-membrane ribonucleoprotein condensates formed in response to environmental stress conditions via liquid–liquid phase separation (LLPS). SGs are involved in the pathogenesis of aging and aging-associated diseases, cancers, viral infection, and several other diseases. GTPase-activating protein (SH3 domain)-binding protein 1 and 2 (G3BP1/2) is a key component and commonly used marker of SGs. Recent studies have shown that SARS-CoV-2 nucleocapsid protein via sequestration of G3BPs inhibits SGs formation in the host cells. In this study, we have identified putative miRNAs targeting G3BP in search of modulators of the G3BP expression. These miRNAs could be considered as new therapeutic targets against COVID-19 infection via the regulation of SG assembly and dynamics.     

Stress Granule Formation is One of the Early Antiviral Mechanisms for Host Cells Against Coxsackievirus B Infection

Stress granules (SGs) are intracellular granules formed when cellular translation is blocked and have been reported to be involved in a variety of viral infections. Our previous studies revealed that SGs are involved in the coxsackievirus B (CVB) infection process, but the role of SGs in CVB infection has not been fully explored. In this study, we found that CVB type 3 (CVB3) could induce SG formation in the early phase of infection. Results showed that levels of CVB3 RNA and protein were significantly inhibited during the early stage of CVB3 infection by the elevated formation of SGs, while viral RNA and protein synthesis were significantly promoted when SG formation was blocked. Our findings suggest that SG formation is one of the early antiviral mechanisms for host cells against CVB infection.     

Importance of eIF2alpha phosphorylation and stress granule assembly in alphavirus translation regulation

Alphavirus infection results in the shutoff of host protein synthesis in favor of viral translation. Here, we show that during Semliki Forest virus (SFV) infection, the translation inhibition is largely due to the activation of the cellular stress response via phosphorylation of eukaryotic translation initiation factor 2alpha subunit (eIF2alpha). Infection of mouse embryo fibroblasts (MEFs) expressing a nonphosphorylatable mutant of eIF2alpha does not result in efficient shutoff, despite efficient viral protein production. Furthermore, we show that the SFV translation enhancer element counteracts the translation inhibition imposed by eIF2alpha phosphorylation. In wild-type MEFs, viral infection induces the transient formation of stress granules (SGs) containing the cellular TIA-1/R proteins. These SGs are disassembled in the vicinity of viral RNA replication, synchronously with the switch from cellular to viral gene expression. We propose that phosphorylation of eIF2alpha and the consequent SG assembly is important for shutoff to occur and that the localized SG disassembly and the presence of the enhancer aid the SFV mRNAs to elude general translational arrest.     

The RasGAP-associated endoribonuclease G3BP assembles stress granules

Stress granules (SGs) are formed in the cytoplasm in response to various toxic agents, and are believed to play a critical role in the regulation of mRNA metabolism during stress. In SGs, mRNAs are stored in an abortive translation initiation complex that can be routed to either translation initiation or degradation. Here, we show that G3BP, a phosphorylation-dependent endoribonuclease that interacts with RasGAP, is recruited to SGs in cells exposed to arsenite. G3BP may thus determine the fate of mRNAs during cellular stress. Remarkably, SG assembly can be either dominantly induced by G3BP overexpression, or on the contrary, inhibited by expressing a central domain of G3BP. This region binds RasGAP and contains serine 149, whose dephosphorylation is induced by arsenite treatment. Critically, a phosphomimetic mutant (S149E) fails to oligomerize and to assemble SGs, whereas a nonphosphorylatable G3BP mutant (S149A) does both. These results suggest that G3BP is an effector of SG assembly, and that Ras signaling contributes to this process by regulating G3BP dephosphorylation.     

Herpes simplex virus 2 infection impacts stress granule accumulation

Interference with stress granule (SG) accumulation is gaining increased appreciation as a common strategy used by diverse viruses to facilitate their replication and to cope with translational arrest. Here, we examined the impact of infection by herpes simplex virus 2 (HSV-2) on SG accumulation by monitoring the localization of the SG components T cell internal antigen 1 (TIA-1), Ras-GTPase-activating SH3-domain-binding protein (G3BP), and poly(A)-binding protein (PABP). Our results indicate that SGs do not accumulate in HSV-2-infected cells and that HSV-2 can interfere with arsenite-induced SG accumulation early after infection. Surprisingly, SG accumulation was inhibited despite increased phosphorylation of eukaryotic translation initiation factor 2α (eIF2α), implying that HSV-2 encodes previously unrecognized activities designed to maintain translation initiation downstream of eIF2α. SG accumulation was not inhibited in HSV-2-infected cells treated with pateamine A, an inducer that works independently of eIF2α phosphorylation. The SGs that accumulated following pateamine A treatment of infected cells contained G3BP and PABP but were largely devoid of TIA-1. We also identified novel nuclear structures containing TIA-1 that form late in infection. These structures contain the RNA binding protein 68-kDa Src-associated in mitosis (Sam68) and were noticeably absent in infected cells treated with inhibitors of viral DNA replication, suggesting that they arise as a result of late events in the virus replicative cycle.     

Stress granules are dispensable for mRNA stabilization during cellular stress

During cellular stress, protein synthesis is severely reduced and bulk mRNA is recruited to stress granules (SGs). Previously, we showed that the SG-recruited IGF2 mRNA-binding protein 1 (IGF2BP1) interferes with target mRNA degradation during cellular stress. Whether this requires the formation of SGs remained elusive. Here, we demonstrate that the sustained inhibition of visible SGs requires the concomitant knockdown of TIA1, TIAR and G3BP1. FRAP and photo-conversion studies, however, indicate that these proteins only transiently associate with SGs. This suggests that instead of forming a rigid scaffold for mRNP recruitment, TIA proteins and G3BP1 promote SG-formation by constantly replenishing mRNPs. In contrast, RNA-binding proteins like IGF2BP1 or HUR, which are dispensable for SG-assembly, are stably associated with SGs and the IGF2BP1/HUR-G3BP1 association is increased during stress. The depletion of IGF2BP1 enhances the degradation of target mRNAs irrespective of inhibiting SG-formation, whereas the turnover of bulk mRNA remains unaffected when SG-formation is impaired. Together these findings indicate that the stabilization of mRNAs during cellular stress is facilitated by the formation of stable mRNPs, which are recruited to SGs by TIA proteins and/or G3BP1. Importantly, however, the aggregation of mRNPs to visible SGs is dispensable for preventing mRNA degradation.