An Insect Viral Protein Disrupts Stress Granule Formation in Mammalian Cells

Stress granules (SGs) are cytosolic RNA-protein aggregates assembled during stress-induced translation arrest. Virus infection, in general, modulates and blocks SG formation. We previously showed that the model dicistrovirus Cricket paralysis virus (CrPV) 1A protein blocks stress granule formation in insect cells, which is dependent on a specific arginine 146 residue. CrPV-1A also inhibits SG formation in mammalian cells suggesting that this insect viral protein may be acting on a fundamental process that regulates SG formation. The mechanism underlying this process is not fully understood. Here, we show that overexpression of wild-type CrPV-1A, but not the CrPV-1A(R146A) mutant protein, inhibits distinct SG assembly pathways in HeLa cells. CrPV-1A mediated SG inhibition is independent of the Argonaute-2 (Ago-2) binding domain and the E3 ubiquitin ligase recruitment domain. CrPV-1A expression leads to nuclear poly(A)+ RNA accumulation and is correlated with the localization of CrPV-1A to the nuclear periphery. Finally, we show that the overexpression of CrPV-1A blocks FUS and TDP-43 granules, which are pathological hallmarks of neurodegenerative diseases. We propose a model whereby CrPV-1A expression in mammalian cells blocks SG formation by depleting cytoplasmic mRNA scaffolds via mRNA export inhibition. CrPV-1A provides a new molecular tool to study RNA-protein aggregates and potentially uncouple SG functions.     

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.     

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.     

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.     

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.     

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.     

Sequestration of G3BP coupled with efficient translation inhibits stress granules in Semliki Forest virus infection

Dynamic, mRNA-containing stress granules (SGs) form in the cytoplasm of cells under environmental stresses, including viral infection. Many viruses appear to employ mechanisms to disrupt the formation of SGs on their mRNAs, suggesting that they represent a cellular defense against infection. Here, we report that early in Semliki Forest virus infection, the C-terminal domain of the viral nonstructural protein 3 (nsP3) forms a complex with Ras-GAP SH3-domain–binding protein (G3BP) and sequesters it into viral RNA replication complexes in a manner that inhibits the formation of SGs on viral mRNAs. A viral mutant carrying a C-terminal truncation of nsP3 induces more persistent SGs and is attenuated for propagation in cell culture. Of importance, we also show that the efficient translation of viral mRNAs containing a translation enhancer sequence also contributes to the disassembly of SGs in infected cells. Furthermore, we show that the nsP3/G3BP interaction also blocks SGs induced by other stresses than virus infection. This is one of few described viral mechanisms for SG disruption and underlines the role of SGs in antiviral defense.     

Induction of Stress Granule-Like Structures in Vesicular Stomatitis Virus-Infected Cells

Previous studies from our laboratory revealed that cellular poly(C) binding protein 2 (PCBP2) downregulates vesicular stomatitis virus (VSV) gene expression. We show here that VSV infection induces the formation of granular structures in the cytoplasm containing cellular RNA-binding proteins, including PCBP2, T-cell-restricted intracellular antigen 1 (TIA1), and TIA1-related protein (TIAR). Depletion of TIA1 via small interfering RNAs (siRNAs), but not depletion of TIAR, results in enhanced VSV growth and gene expression. The VSV-induced granules appear to be similar to the stress granules (SGs) generated in cells triggered by heat shock or oxidative stress but do not contain some of the bona fide SG markers, such as eukaryotic initiation factor 3 (eIF3) or eIF4A, or the processing body (PB) markers, such as mRNA-decapping enzyme 1A (DCP1a), and thus may not represent canonical SGs or PBs. Our results revealed that the VSV-induced granules, called SG-like structures here, contain the viral replicative proteins and RNAs. The formation and maintenance of the SG-like structures required viral replication and ongoing protein synthesis, but an intact cytoskeletal network was not necessary. These results suggest that cells respond to VSV infection by aggregating the antiviral proteins, such as PCBP2 and TIA1, to form SG-like structures. The functional significance of these SG-like structures in VSV-infected cells is currently under investigation.     

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.     

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.     

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.     

Dynamic oscillation of translation and stress granule formation mark the cellular response to virus infection

Virus infection-induced global protein synthesis suppression is linked to assembly of stress granules (SGs), cytosolic aggregates of stalled translation preinitiation complexes. To study long-term stress responses, we developed an imaging approach for extended observation and analysis of SG dynamics during persistent hepatitis C virus (HCV) infection. In combination with type 1 interferon, HCV infection induces highly dynamic assembly/disassembly of cytoplasmic SGs, concomitant with phases of active and stalled translation, delayed cell division, and prolonged cell survival. Double-stranded RNA (dsRNA), independent of viral replication, is sufficient to trigger these oscillations. Translation initiation factor eIF2α phosphorylation by protein kinase R mediates SG formation and translation arrest. This is antagonized by the upregulation of GADD34, the regulatory subunit of protein phosphatase 1 dephosphorylating eIF2α. Stress response oscillation is a general mechanism to prevent long-lasting translation repression and a conserved host cell reaction to multiple RNA viruses, which HCV may exploit to establish persistence.     

Poliovirus unlinks TIA1 aggregation and mRNA stress granule formation

In response to environmental stress and viral infection, mammalian cells form foci containing translationally silenced mRNPs termed stress granules (SGs). As aggregates of stalled initiation complexes, SGs are defined by the presence of translation initiation machinery in addition to mRNA binding proteins. Here, we report that cells infected with poliovirus (PV) can form SGs early that contain T-cell-restricted intracellular antigen 1 (TIA1), translation initiation factors, RNA binding proteins, and mRNA. However, this response is blocked as infection progresses, and a type of pseudo-stress granule remains at late times postinfection and contains TIA but lacks translation initiation factors, mRNA binding proteins, and most polyadenylated mRNA. This result was observed using multiple stressors, including viral infection, oxidative stress, heat shock, and endoplasmic reticulum stress. Multiple proteins required for efficient viral internal ribosome entry site-dependent translation are localized to SGs under stress conditions, providing a potential rationale for the evolution and maintenance of the SG inhibition phenotype. Further, the expression of a noncleavable form of the RasGAP-SH3 domain binding protein in PV-infected cells enables SGs whose constituents are consistent with the presence of stalled 48S translation preinitiation complexes to persist throughout infection. These results indicate that in poliovirus-infected cells, the functions of TIA self-aggregation and aggregation of stalled translation initiation complexes into stress granules are severed, leading to novel foci that contain TIA1 but lack other stress granule-defining components.     

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.     

The leader protein of cardioviruses inhibits stress granule assembly

Stress granules (SG) are cytoplasmic aggregates of stalled translation preinitiation complexes that form in cells exposed to various environmental stresses. Here, we show that stress granules assemble in cells infected with Theiler's murine encephalomyelitis virus (TMEV) mutants carrying alterations in the leader (L) protein, but not in cells infected with wild-type TMEV. Stress granules also formed in STAT1-deficient cells, suggesting that SG formation was not a consequence of increased type I interferon (IFN) production when cells were infected with the mutant virus. Ectopic expression of the wild-type L protein was sufficient to inhibit stress granule formation induced by sodium arsenite or thapsigargin treatment. In conclusion, TMEV infection induces stress granule assembly, but this process is inhibited by the L protein. Unlike poliovirus-induced stress granules, TMEV-induced stress granules did not contain the nuclear protein Sam68 but contained polypyrimidine tract binding protein (PTB), an internal ribosome entry site (IRES)-interacting protein. Moreover, G3BP was not degraded and was found in SG after TMEV infection, suggesting that SG content could be virus specific. Despite the colocalization of PTB with SG and the known interaction of PTB with viral RNA, in situ hybridization and immunofluorescence assays failed to detect viral RNA trapped in infection-induced SG. Recombinant Theiler's viruses expressing the L protein of Saffold virus 2 (SAFV-2), a closely related human theilovirus, or the L protein of mengovirus, an encephalomyocarditis virus (EMCV) strain, also inhibited infection-induced stress granule assembly, suggesting that stress granule antagonism is a common feature of cardiovirus L proteins.     

RNA-binding proteins TIA-1 and TIAR link the phosphorylation of eIF-2 alpha to the assembly of mammalian stress granules

In response to environmental stress, the related RNA-binding proteins TIA-1 and TIAR colocalize with poly(A)(+) RNA at cytoplasmic foci that resemble the stress granules (SGs) that harbor untranslated mRNAs in heat shocked plant cells (Nover et al. 1989; Nover et al. 1983; Scharf et al. 1998). The accumulation of untranslated mRNA at SGs is reversible in cells that recover from a sublethal stress, but irreversible in cells subjected to a lethal stress. We have found that the assembly of TIA-1/R(+) SGs is initiated by the phosphorylation of eIF-2alpha. A phosphomimetic eIF-2alpha mutant (S51D) induces the assembly of SGs, whereas a nonphosphorylatable eIF-2alpha mutant (S51A) prevents the assembly of SGs. The ability of a TIA-1 mutant lacking its RNA-binding domains to function as a transdominant inhibitor of SG formation suggests that this RNA-binding protein acts downstream of the phosphorylation of eIF-2alpha to promote the sequestration of untranslated mRNAs at SGs. The assembly and disassembly of SGs could regulate the duration of stress- induced translational arrest in cells recovering from environmental stress.     

West nile virus infections suppress early viral RNA synthesis and avoid inducing the cell stress granule response

West Nile virus (WNV) recently became endemic in the United States and is a significant cause of human morbidity and mortality. Natural WNV strain infections do not induce stress granules (SGs), while W956IC (a lineage 2/1 chimeric WNV infectious clone) virus infections produce high levels of early viral RNA and efficiently induce SGs through protein kinase R (PKR) activation. Additional WNV chimeric viruses made by replacing one or more W956IC genes with the lineage 1 Eg101 equivalent in the W956IC backbone were analyzed. The Eg-NS4b+5, Eg-NS1+3+4a, and Eg-NS1+4b+5 chimeras produced low levels of viral RNA at early times of infection and inefficiently induced SGs, suggesting the possibility that interactions between viral nonstructural proteins and/or between viral nonstructural proteins and cell proteins are involved in suppressing early viral RNA synthesis and membrane remodeling during natural WNV strain infections. Detection of exposed viral double-stranded RNA (dsRNA) in W956IC-infected cells suggested that the enhanced early viral RNA synthesis surpassed the available virus-induced membrane protection and allowed viral dsRNA to activate PKR.