Influenza A virus NS1 effector domain is required for PA-X-mediated host shutoff in infected cells.

Many viruses inhibit general host gene expression to limit innate immune responses and gain preferential access to the cellular translational apparatus for their protein synthesis. This process is known as host shutoff. Influenza A viruses (IAVs) encode two host shutoff proteins: nonstructural protein 1 (NS1) and polymerase acidic X (PA-X). NS1 inhibits host nuclear pre-messenger RNA maturation and export, and PA-X is an endoribonuclease that preferentially cleaves host spliced nuclear and cytoplasmic messenger RNAs. Emerging evidence suggests that in circulating human IAVs NS1 and PA-X co-evolve to ensure optimal magnitude of general host shutoff without compromising viral replication that relies on host cell metabolism. However, the functional interplay between PA-X and NS1 remains unexplored. In this study, we sought to determine whether NS1 function has a direct effect on PA-X activity by analyzing host shutoff in A549 cells infected with wild-type or mutant IAVs with NS1 effector domain deletion. This was done using conventional quantitative reverse transcription polymerase chain reaction techniques and direct RNA sequencing using nanopore technology. Our previous research on the molecular mechanisms of PA-X function identified two prominent features of IAV-infected cells: nuclear accumulation of cytoplasmic poly(A) binding protein (PABPC1) and increase in nuclear poly(A) RNA abundance relative to the cytoplasm. Here we demonstrate that NS1 effector domain function augments PA-X host shutoff and is necessary for nuclear PABPC1 accumulation. By contrast, nuclear poly(A) RNA accumulation is not dependent on either NS1 or PA-X-mediated host shutoff and is accompanied by nuclear retention of viral transcripts. Our study demonstrates for the first time that NS1 and PA-X may functionally interact in mediating host shutoff.IMPORTANCERespiratory viruses including the influenza A virus continue to cause annual epidemics with high morbidity and mortality due to the limited effectiveness of vaccines and antiviral drugs. Among the strategies evolved by viruses to evade immune responses is host shutoff-a general blockade of host messenger RNA and protein synthesis. Disabling influenza A virus host shutoff is being explored in live attenuated vaccine development as an attractive strategy for increasing their effectiveness by boosting antiviral responses. Influenza A virus encodes two proteins that function in host shutoff: the nonstructural protein 1 (NS1) and the polymerase acidic X (PA-X). We and others have characterized some of the NS1 and PA-X mechanisms of action and the additive effects that these viral proteins may have in ensuring the blockade of host gene expression. In this work, we examined whether NS1 and PA-X functionally interact and discovered that NS1 is required for PA-X to function effectively. This work significantly advances our understanding of influenza A virus host shutoff and identifies new potential targets for therapeutic interventions against influenza and further informs the development of improved live attenuated vaccines.

Nsp1 proteins of human coronaviruses HCoV-OC43 and SARS-CoV2 inhibit stress granule formation.

Stress granules (SGs) are cytoplasmic condensates that often form as part of the cellular antiviral response. Despite the growing interest in understanding the interplay between SGs and other biological condensates and viral replication, the role of SG formation during coronavirus infection remains poorly understood. Several proteins from different coronaviruses have been shown to suppress SG formation upon overexpression, but there are only a handful of studies analyzing SG formation in coronavirus-infected cells. To better understand SG inhibition by coronaviruses, we analyzed SG formation during infection with the human common cold coronavirus OC43 (HCoV-OC43) and the pandemic SARS-CoV2. We did not observe SG induction in infected cells and both viruses inhibited eukaryotic translation initiation factor 2α (eIF2α) phosphorylation and SG formation induced by exogenous stress. Furthermore, in SARS-CoV2 infected cells we observed a sharp decrease in the levels of SG-nucleating protein G3BP1. Ectopic overexpression of nucleocapsid (N) and non-structural protein 1 (Nsp1) from both HCoV-OC43 and SARS-CoV2 inhibited SG formation. The Nsp1 proteins of both viruses inhibited arsenite-induced eIF2α phosphorylation, and the Nsp1 of SARS-CoV2 alone was sufficient to cause a decrease in G3BP1 levels. This phenotype was dependent on the depletion of cytoplasmic mRNA mediated by Nsp1 and associated with nuclear accumulation of the SG-nucleating protein TIAR. To test the role of G3BP1 in coronavirus replication, we infected cells overexpressing EGFP-tagged G3BP1 with HCoV-OC43 and observed a significant decrease in virus replication compared to control cells expressing EGFP. The antiviral role of G3BP1 and the existence of multiple SG suppression mechanisms that are conserved between HCoV-OC43 and SARS-CoV2 suggest that SG formation may represent an important antiviral host defense that coronaviruses target to ensure efficient replication.

Thiopurines inhibit coronavirus Spike protein processing and incorporation into progeny virions.

There is an outstanding need for broadly acting antiviral drugs to combat emerging viral diseases. Here, we report that thiopurines inhibit the replication of the betacoronaviruses HCoV-OC43 and SARS-CoV-2. 6-Thioguanine (6-TG) disrupted early stages of infection, limiting accumulation of full-length viral genomes, subgenomic RNAs and structural proteins. In ectopic expression models, we observed that 6-TG increased the electrophoretic mobility of Spike from diverse betacoronaviruses, matching the effects of enzymatic removal of N-linked oligosaccharides from Spike in vitro. SARS-CoV-2 virus-like particles (VLPs) harvested from 6-TG-treated cells were deficient in Spike. 6-TG treatment had a similar effect on production of lentiviruses pseudotyped with SARS-CoV-2 Spike, yielding pseudoviruses deficient in Spike and unable to infect ACE2-expressing cells. Together, these findings from complementary ectopic expression and infection models strongly indicate that defective Spike trafficking and processing is an outcome of 6-TG treatment. Using biochemical and genetic approaches we demonstrated that 6-TG is a pro-drug that must be converted to the nucleotide form by hypoxanthine phosphoribosyltransferase 1 (HPRT1) to achieve antiviral activity. This nucleotide form has been shown to inhibit small GTPases Rac1, RhoA, and CDC42; however, we observed that selective chemical inhibitors of these GTPases had no effect on Spike processing or accumulation. By contrast, the broad GTPase agonist ML099 countered the effects of 6-TG, suggesting that the antiviral activity of 6-TG requires the targeting of an unknown GTPase. Overall, these findings suggest that small GTPases are promising targets for host-targeted antivirals.

UV damage induces G3BP1-dependent stress granule formation that is not driven by mTOR inhibition-mediated translation arrest.

Translation arrest is a part of the cellular stress response that decreases energy consumption and enables rapid reprioritisation of gene expression. Often translation arrest leads to condensation of untranslated messenger ribonucleoproteins (mRNPs) into stress granules (SGs). Studies into mechanisms of SG formation and functions are complicated because various types of stress cause formation of SGs with different properties and composition. In this work, we focused on the mechanism of SG formation triggered by UV damage. We demonstrate that UV-induced inhibition of translation does not involve inhibition of the mechanistic target of rapamycin (mTOR) signaling or dissociation of the 48S preinitiation complexes. The general control non-derepressible 2 (GCN2; also known as EIF2AK4) kinase contributes to UV-induced SG formation, which is independent of the phosphorylation of the eukaryotic translation initiation factor 2α. Like many other types of SGs, condensation of UV-induced granules requires the Ras-GTPase-activating protein SH3-domain-binding protein 1 (G3BP1). Our work reveals that, in UV-treated cells, the mechanisms of translation arrest and SG formation may be unlinked, resulting in SGs that do not contain the major type of polysome-free preinitiation complexes that accumulate in the cytoplasm.This article has an associated First Person interview with the first author of the paper.

Thiopurines activate an antiviral unfolded protein response that blocks influenza A virus glycoprotein accumulation.

Influenza A viruses (IAVs) utilize host shutoff mechanisms to limit antiviral gene expression and redirect translation machinery to the synthesis of viral proteins. Previously, we showed that IAV replication is sensitive to protein synthesis inhibitors that block translation initiation and induce formation of cytoplasmic condensates of untranslated messenger ribonucleoprotein complexes called stress granules (SGs). In this study, using an image-based high-content screen, we identified two thiopurines, 6-thioguanine (6-TG) and 6-thioguanosine (6-TGo), that triggered SG formation in IAV-infected cells and blocked IAV replication in a dose-dependent manner without eliciting SG formation in uninfected cells. 6-TG and 6-TGo selectively disrupted the synthesis and maturation of IAV glycoproteins hemagglutinin (HA) and neuraminidase (NA) without affecting the levels of the viral RNAs that encode them. By contrast, these thiopurines had minimal effect on other IAV proteins or the global host protein synthesis. Disruption of IAV glycoprotein accumulation by 6-TG and 6-TGo correlated with activation of unfolded protein response (UPR) sensors activating transcription factor-6 (ATF6), inositol requiring enzyme-1 (IRE1) and PKR-like endoplasmic reticulum kinase (PERK), leading to downstream UPR gene expression. Treatment of infected cells with the chemical chaperone 4-phenylbutyric acid diminished thiopurine-induced UPR activation and partially restored the processing and accumulation of HA and NA. By contrast, chemical inhibition of the integrated stress response downstream of PERK restored accumulation of NA monomers but did not restore processing of viral glycoproteins. Genetic deletion of PERK enhanced the antiviral effect of 6-TG without causing overt cytotoxicity, suggesting that while UPR activation correlates with diminished viral glycoprotein accumulation, PERK could limit the antiviral effects of drug-induced ER stress. Taken together, these data indicate that 6-TG and 6-TGo are effective host-targeted antivirals that trigger the UPR and selectively disrupt accumulation of viral glycoproteins.IMPORTANCESecreted and transmembrane proteins are synthesized in the endoplasmic reticulum (ER), where they are folded and modified prior to transport. Many viruses rely on the ER for the synthesis and processing of viral glycoproteins that will ultimately be incorporated into viral envelopes. Viral burden on the ER can trigger the unfolded protein response (UPR). Much remains to be learned about how viruses co-opt the UPR to ensure efficient synthesis of viral glycoproteins. Here, we show that two FDA-approved thiopurine drugs, 6-TG and 6-TGo, induce the UPR, which represents a previously unrecognized effect of these drugs on cell physiology. This thiopurine-mediated UPR activation blocks influenza virus replication by impeding viral glycoprotein accumulation. Our findings suggest that 6-TG and 6-TGo may have broad antiviral effect against enveloped viruses that require precise tuning of the UPR to support viral glycoprotein synthesis.

Defective Influenza A Virus RNA Products Mediate MAVS-Dependent Upregulation of Human Leukocyte Antigen Class I Proteins.

Influenza A virus (IAV) increases the presentation of class I human leukocyte antigen (HLA) proteins that limit antiviral responses mediated by natural killer (NK) cells, but molecular mechanisms for these processes have not yet been fully elucidated. We observed that infection with A/Fort Monmouth/1/1947(H1N1) IAV significantly increased the presentation of HLA-B, -C, and -E on lung epithelial cells. Virus entry was not sufficient to induce HLA upregulation because UV-inactivated virus had no effect. Aberrant internally deleted viral RNAs (vRNAs) known as mini viral RNAs (mvRNAs) and defective interfering RNAs (DI RNAs) expressed from an IAV minireplicon were sufficient for inducing HLA upregulation. These defective RNAs bind to retinoic acid-inducible gene I (RIG-I) and initiate mitochondrial antiviral signaling (MAVS) protein-dependent antiviral interferon (IFN) responses. Indeed, MAVS was required for HLA upregulation in response to IAV infection or ectopic mvRNA/DI RNA expression. The effect was partially due to paracrine signaling, as we observed that IAV infection or mvRNA/DI RNA-expression stimulated production of IFN-ß and IFN-λ1 and conditioned media from these cells elicited a modest increase in HLA surface levels in naive epithelial cells. HLA upregulation in response to aberrant viral RNAs could be prevented by the Janus kinase (JAK) inhibitor ruxolitinib. While HLA upregulation would seem to be advantageous to the virus, it is kept in check by the viral nonstructural 1 (NS1) protein; we determined that NS1 limits cell-intrinsic and paracrine mechanisms of HLA upregulation. Taken together, our findings indicate that aberrant IAV RNAs stimulate HLA presentation, which may aid viral evasion of innate immunity.IMPORTANCE Human leukocyte antigens (HLAs) are cell surface proteins that regulate innate and adaptive immune responses to viral infection by engaging with receptors on immune cells. Many viruses have evolved ways to evade host immune responses by modulating HLA expression and/or processing. Here, we provide evidence that aberrant RNA products of influenza virus genome replication can trigger retinoic acid-inducible gene I (RIG-I)/mitochondrial antiviral signaling (MAVS)-dependent remodeling of the cell surface, increasing surface presentation of HLA proteins known to inhibit the activation of an immune cell known as a natural killer (NK) cell. While this HLA upregulation would seem to be advantageous to the virus, it is kept in check by the viral nonstructural 1 (NS1) protein, which limits RIG-I activation and interferon production by the infected cell.

Selective Degradation of Host RNA Polymerase II Transcripts by Influenza A Virus PA-X Host Shutoff Protein.

Influenza A viruses (IAVs) inhibit host gene expression by a process known as host shutoff. Host shutoff limits host innate immune responses and may also redirect the translation apparatus to the production of viral proteins. Multiple IAV proteins regulate host shutoff, including PA-X, a ribonuclease that remains incompletely characterized. We report that PA-X selectively targets host RNA polymerase II (Pol II) transcribed mRNAs, while sparing products of Pol I and Pol III. Interestingly, we show that PA-X can also target Pol II-transcribed RNAs in the nucleus, including non-coding RNAs that are not destined to be translated, and reporter transcripts with RNA hairpin structures that block ribosome loading. Transcript degradation likely occurs in the nucleus, as PA-X is enriched in the nucleus and its nuclear localization correlates with reduction in target RNA levels. Complete degradation of host mRNAs following PA-X-mediated endonucleolytic cleavage is dependent on the host 5'->3'-exonuclease Xrn1. IAV mRNAs are structurally similar to host mRNAs, but are synthesized and modified at the 3' end by the action of the viral RNA-dependent RNA polymerase complex. Infection of cells with wild-type IAV or a recombinant PA-X-deficient virus revealed that IAV mRNAs resist PA-X-mediated degradation during infection. At the same time, loss of PA-X resulted in changes in the synthesis of select viral mRNAs and a decrease in viral protein accumulation. Collectively, these results significantly advance our understanding of IAV host shutoff, and suggest that the PA-X causes selective degradation of host mRNAs by discriminating some aspect of Pol II-dependent RNA biogenesis in the nucleus.

Timing Is Everything: Coordinated Control of Host Shutoff by Influenza A Virus NS1 and PA-X Proteins.

Like all viruses, influenza viruses (IAVs) use host translation machinery to decode viral mRNAs. IAVs ensure efficient translation of viral mRNAs through host shutoff, a process whereby viral proteins limit the accumulation of host proteins through subversion of their biogenesis. Despite its small genome, the virus deploys multiple host shutoff mechanisms at different stages of infection, thereby ensuring successful replication while limiting the communication of host antiviral responses. In this Gem, we review recent data on IAV host shutoff proteins, frame the outstanding questions in the field, and propose a temporally coordinated model of IAV host shutoff.

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.

Human Betacoronavirus OC43 Interferes with the Integrated Stress Response Pathway in Infected Cells.

Viruses evolve many strategies to ensure the efficient synthesis of their proteins. One such strategy is the inhibition of the integrated stress response-the mechanism through which infected cells arrest translation through the phosphorylation of the alpha subunit of the eukaryotic translation initiation factor 2 (eIF2α). We have recently shown that the human common cold betacoronavirus OC43 actively inhibits eIF2α phosphorylation in response to sodium arsenite, a potent inducer of oxidative stress. In this work, we examined the modulation of integrated stress responses by OC43 and demonstrated that the negative feedback regulator of eIF2α phosphorylation GADD34 is strongly induced in infected cells. However, the upregulation of GADD34 expression induced by OC43 was independent from the activation of the integrated stress response and was not required for the inhibition of eIF2α phosphorylation in virus-infected cells. Our work reveals a complex interplay between the common cold coronavirus and the integrated stress response, in which efficient viral protein synthesis is ensured by the inhibition of eIF2α phosphorylation but the GADD34 negative feedback loop is disrupted.

Kaposi's sarcoma-associated herpesvirus G-protein-coupled receptor prevents AU-rich-element-mediated mRNA decay.

During lytic Kaposi's sarcoma-associated herpesvirus (KSHV) infection, host gene expression is severely restricted by a process of global mRNA degradation known as host shutoff, which rededicates translational machinery to the expression of viral proteins. A subset of host mRNAs is spared from shutoff, and a number of these contain cis-acting AU-rich elements (AREs) in their 3' untranslated regions. AREs are found in labile mRNAs encoding cytokines, growth factors, and proto-oncogenes. Activation of the p38/MK2 signal transduction pathway reverses constitutive decay of ARE-mRNAs, resulting in increased protein production. The viral G-protein-coupled receptor (vGPCR) is thought to play an important role in promoting the secretion of angiogenic molecules from KSHV-infected cells during lytic replication, but to date it has not been clear how vGPCR circumvents host shutoff. Here, we demonstrate that vGPCR activates the p38/MK2 pathway and stabilizes ARE-mRNAs, augmenting the levels of their protein products. Using MK2-deficient cells, we demonstrate that MK2 is essential for maximal vGPCR-mediated ARE-mRNA stabilization. ARE-mRNAs are normally delivered to cytoplasmic ribonucleoprotein granules known as processing bodies (PBs) for translational silencing and decay. We demonstrate that PB formation is prevented during KSHV lytic replication or in response to vGPCR-mediated activation of RhoA subfamily GTPases. Together, these data show for the first time that vGPCR impacts gene expression at the posttranscriptional level, coordinating an attack on the host mRNA degradation machinery. By suppressing ARE-mRNA turnover, vGPCR may facilitate escape of certain target mRNAs from host shutoff and allow secretion of angiogenic factors from lytically infected cells.

Influenza A virus inhibits cytoplasmic stress granule formation.

An important component of the mammalian stress response is the reprogramming of translation. A variety of stresses trigger abrupt polysome disassembly and the accumulation of stalled translation preinitiation complexes. These complexes nucleate cytoplasmic stress granules (SGs), sites of mRNA triage in which mRNAs from disassembling polysomes are sorted and the fates of individual transcripts are determined. Here, we demonstrate that influenza A virus (IAV) actively suppresses SG formation during infection, thereby allowing translation of viral mRNAs. Complete inhibition of SG formation is dependent on the function of the viral nonstructural protein 1 (NS1); at late times postinfection, cells infected with NS1-mutant viruses formed SGs in a double-stranded RNA-activated protein kinase (PKR)-dependent fashion. In these cells, SG formation correlated with inhibited viral protein synthesis. Together, these experiments demonstrate the antiviral potential of SGs and reveal a viral countermeasure that limits SG formation.

Assays for monitoring viral manipulation of host ARE-mRNA turnover.

Early host responses to viral infection rapidly induce an antiviral gene expression program that limits viral replication and recruits sentinel cells of the innate immune system. These responses are mediated by cytokines. The mRNAs that encode cytokines typically harbor destabilizing adenine- and uridine-rich elements (AREs) that direct their constitutive degradation in the cytoplasm. In response to a variety of signals, including viral infection, small pools of cytoplasmic ARE-mRNAs are rapidly stabilized and translated. Thus, mRNA stability plays a key role in antiviral gene expression. Intriguingly, recent studies have identified viral proteins that specifically target ARE-mRNAs for stabilization, suggesting that certain proteins encoded by ARE-mRNAs may be advantageous for infection. Here, we discuss the development of a suite of sensitive and complementary assays to monitor ARE-mRNA turnover. These include luciferase- and destabilized-GFP-based assays that can be adapted for high-throughput screening applications.

Improved methods for expression and purification of Saccharomyces cerevisiae TFIIF and TFIIH; identification of a functional Escherichia coli promoter and internal translation initiation within the N-terminal coding region of the TFIIF TFG1 subunit.

The basal RNA polymerase II (RNAPII) transcription machinery is composed of RNAPII and the general transcription factors (TF) TATA binding protein (TBP), TFIIB, TFIIE, TFIIF and TFIIH. Due to the powerful genetic and molecular approaches that can be utilized, the budding yeast Saccharomyces cerevisiae has proven to be an invaluable model system for studies of the mechanisms of RNAPII transcription. Complementary biochemical studies of the S. cerevisiae basal transcription machinery, however, have been hampered by difficulties in the purification of TFIIF and TFIIH, most notably due to the severe toxicity of the TFIIF Tfg1 subunit in Escherichia coli and the complexity of the purification scheme for native TFIIH. Here, we report the elimination of TFG1-associated toxicity in E. coli, the identification and removal of a functional E. coli promoter and internal translation initiation within the N-terminal coding region of TFG1, and the efficient production and two-step purification of recombinant TFIIF complexes. We also report conditions for the efficient two-step tandem affinity purification (TAP) of holo-TFIIH, core TFIIH and TFIIK complexes from yeast whole cell extracts.

The Influenza A Virus Endoribonuclease PA-X Usurps Host mRNA Processing Machinery to Limit Host Gene Expression.

Many viruses shut off host gene expression to inhibit antiviral responses. Viral proteins and host proteins required for viral replication are typically spared in this process, but the mechanisms of target selectivity during host shutoff remain poorly understood. Using transcriptome-wide and targeted reporter experiments, we demonstrate that the influenza A virus endoribonuclease PA-X usurps RNA splicing to selectively target host RNAs for destruction. Proximity-labeling proteomics reveals that PA-X interacts with cellular RNA processing proteins, some of which are partially required for host shutoff. Thus, PA-X taps into host nuclear pre-mRNA processing mechanisms to destroy nascent mRNAs shortly after their synthesis. This mechanism sets PA-X apart from other viral host shutoff proteins that target actively translating mRNAs in the cytoplasm. Our study reveals a unique mechanism of host shutoff that helps us understand how influenza viruses suppress host gene expression.

Adaptive Mutations in Influenza A/California/07/2009 Enhance Polymerase Activity and Infectious Virion Production.

Mice are not natural hosts for influenza A viruses (IAVs), but they are useful models for studying antiviral immune responses and pathogenesis. Serial passage of IAV in mice invariably causes the emergence of adaptive mutations and increased virulence. Here, we report the adaptation of IAV reference strain A/California/07/2009(H1N1) (also known as CA/07) in outbred Swiss Webster mice. Serial passage led to increased virulence and lung titers, and dissemination of the virus to brains. We adapted a deep-sequencing protocol to identify and enumerate adaptive mutations across all genome segments. Among mutations that emerged during mouse-adaptation, we focused on amino acid substitutions in polymerase subunits: polymerase basic-1 (PB1) T156A and F740L and polymerase acidic (PA) E349G. These mutations were evaluated singly and in combination in minigenome replicon assays, which revealed that PA E349G increased polymerase activity. By selectively engineering three PB1 and PA mutations into the parental CA/07 strain, we demonstrated that these mutations in polymerase subunits decreased the production of defective viral genome segments with internal deletions and dramatically increased the release of infectious virions from mouse cells. Together, these findings increase our understanding of the contribution of polymerase subunits to successful host adaptation.

Translation inhibition and stress granules in the antiviral immune response.

Efficient viral gene expression is threatened by cellular stress response programmes that rapidly reprioritize the translation machinery in response to varied environmental assaults, including virus infection. This results in inhibition of bulk synthesis of housekeeping proteins and causes the aggregation of messenger ribonucleoprotein complexes into cytoplasmic foci that are known as stress granules, which can entrap viral mRNAs. There is accumulating evidence for the antiviral nature of stress granules, which is supported by the discovery of many viral factors that interfere with stress granule formation and/or function. This Review focuses on recent advances in our understanding of the role of translation inhibition and stress granules in antiviral immune responses.

Stress Granule-Inducing Eukaryotic Translation Initiation Factor 4A Inhibitors Block Influenza A Virus Replication.

Eukaryotic translation initiation factor 4A (eIF4A) is a helicase that facilitates assembly of the translation preinitiation complex by unwinding structured mRNA 5' untranslated regions. Pateamine A (PatA) and silvestrol are natural products that disrupt eIF4A function and arrest translation, thereby triggering the formation of cytoplasmic aggregates of stalled preinitiation complexes known as stress granules (SGs). Here we examined the effects of eIF4A inhibition by PatA and silvestrol on influenza A virus (IAV) protein synthesis and replication in cell culture. Treatment of infected cells with either PatA or silvestrol at early times post-infection resulted in SG formation, arrest of viral protein synthesis and failure to replicate the viral genome. PatA, which irreversibly binds to eIF4A, sustained long-term blockade of IAV replication following drug withdrawal, and inhibited IAV replication at concentrations that had minimal cytotoxicity. By contrast, the antiviral effects of silvestrol were fully reversible; drug withdrawal caused rapid SG dissolution and resumption of viral protein synthesis. IAV inhibition by silvestrol was invariably associated with cytotoxicity. PatA blocked replication of genetically divergent IAV strains, suggesting common dependence on host eIF4A activity. This study demonstrates that the core host protein synthesis machinery can be targeted to block viral replication.

Functions of Saccharomyces cerevisiae TFIIF during transcription start site utilization.

Previous studies have shown that substitutions in the Tfg1 or Tfg2 subunits of Saccharomyces cerevisiae transcription factor IIF (TFIIF) can cause upstream shifts in start site utilization, resulting in initiation patterns that more closely resemble those of higher eukaryotes. In this study, we report the results from multiple biochemical assays analyzing the activities of wild-type yeast TFIIF and the TFIIF Tfg1 mutant containing the E346A substitution (Tfg1-E346A). We demonstrate that TFIIF stimulates formation of the first two phosphodiester bonds and dramatically stabilizes a short RNA-DNA hybrid in the RNA polymerase II (RNAPII) active center and, importantly, that the Tfg1-E346A substitution coordinately enhances early bond formation and the processivity of early elongation in vitro. These results are discussed within a proposed model for the role of yeast TFIIF in modulating conformational changes in the RNAPII active center during initiation and early elongation.