Antiviral Stress Granules Research Articles

Chandipura Virus Forms Cytoplasmic Inclusion Bodies through Phase Separation and Proviral Association of Cellular Protein Kinase R and Stress Granule Protein TIA-1.

Negative-strand RNA viruses form cytoplasmic inclusion bodies (IBs) representing virus replication foci through phase separation or biomolecular condensation of viral and cellular proteins, as a hallmark of their infection. Alternatively, mammalian cells form stalled mRNA containing antiviral stress granules (SGs), as a consequence of phosphorylation of eukaryotic initiation factor 2α (eIF2α) through condensation of several RNA-binding proteins including TIA-1. Whether and how Chandipura virus (CHPV), an emerging human pathogen causing influenza-like illness, coma and death, forms IBs and evades antiviral SGs remain unknown. By confocal imaging on CHPV-infected Vero-E6 cells, we found that CHPV infection does not induce formation of distinct canonical SGs. Instead, CHPV proteins condense and co-localize together with SG proteins to form heterogeneous IBs, which ensued independent of the activation of eIF2α and eIF2α kinase, protein kinase R (PKR). Interestingly, siRNA-mediated depletion of PKR or TIA-1 significantly decreased viral transcription and virion production. Moreover, CHPV infection also caused condensation and recruitment of PKR to IBs. Compared to SGs, IBs exhibited significant rapidity in disassembly dynamics. Altogether, our study demonstrating that CHPV replication co-optimizes with SG proteins and revealing an unprecedented proviral role of TIA-1/PKR may have implications in understanding the mechanisms regulating CHPV-IB formation and designing antiviral therapeutics. Importance: CHPV is an emerging tropical pathogen reported to cause acute influenza-like illness and encephalitis in children with a very high mortality rate of ~70%. Lack of vaccines and an effective therapy against CHPV makes it a potent pathogen for causing an epidemic in tropical parts of globe. Given these forewarnings, it is of paramount importance that CHPV biology must be understood comprehensively. Targeting of host factors offers several advantages over targeting the viral components due to the generally higher mutation rate in the viral genome. In this study, we aimed at understanding the role of SGs forming cellular RNA-binding proteins in CHPV replication. Our study helps understand participation of cellular factors in CHPV replication and could help develop effective therapeutics against the virus.

TRIM25 predominately associates with anti-viral stress granules

Stress granules (SGs) are induced by various environmental stressors, resulting in their compositional and functional heterogeneity. SGs play a crucial role in the antiviral process, owing to their potent translational repressive effects and ability to trigger signal transduction; however, it is poorly understood how these antiviral SGs differ from SGs induced by other environmental stressors. Here we identify that TRIM25, a known driver of the ubiquitination-dependent antiviral innate immune response, is a potent and critical marker of the antiviral SGs. TRIM25 undergoes liquid-liquid phase separation (LLPS) and co-condenses with the SG core protein G3BP1 in a dsRNA-dependent manner. The co-condensation of TRIM25 and G3BP1 results in a significant enhancement of TRIM25’s ubiquitination activity towards multiple antiviral proteins, which are mainly located in SGs. This co-condensation is critical in activating the RIG-I signaling pathway, thus restraining RNA virus infection. Our studies provide a conceptual framework for better understanding the heterogeneity of stress granule components and their response to distinct environmental stressors.

Cellular Stress Responses against Coronavirus Infection: A Means of the Innate Antiviral Defense.

Cellular stress responses are crucial for maintaining cellular homeostasis. Stress granules (SGs), activated by eIF2α kinases in response to various stimuli, play a pivotal role in dealing with diverse stress conditions. Viral infection, as one kind of cellular stress, triggers specific cellular programs aimed at overcoming virus-induced stresses. Recent studies have revealed that virus-derived stress responses are tightly linked to the host's antiviral innate immunity. Virus infection-induced SGs act as platforms for antiviral sensors, facilitating the initiation of protective antiviral responses called "antiviral stress granules" (avSGs). However, many viruses, including coronaviruses, have evolved strategies to suppress avSG formation, thereby counteracting the host's immune responses. This review discusses the intricate relationship between cellular stress responses and antiviral innate immunity, with a specific focus on coronaviruses. Furthermore, the diverse mechanisms employed by viruses to counteract avSGs are described.

The Amino Acid at Position 95 in the Matrix Protein of Rabies Virus Is Involved in Antiviral Stress Granule Formation in Infected Cells.

Stress granules (SGs) are dynamic structures that store cytosolic messenger ribonucleoproteins. SGs have recently been shown to serve as a platform for activating antiviral innate immunity; however, several pathogenic viruses suppress SG formation to evade innate immunity. In this study, we investigated the relationship between rabies virus (RABV) virulence and SG formation, using viral strains with different levels of virulence. We found that the virulent Nishigahara strain did not induce SG formation, but its avirulent offshoot, the Ni-CE strain, strongly induced SG formation. Furthermore, we demonstrated that the amino acid at position 95 in the RABV matrix protein (M95), a pathogenic determinant for the Nishigahara strain, plays a key role in inhibiting SG formation, followed by protein kinase R (PKR)-dependent phosphorylation of the α subunit of eukaryotic initiation factor 2α (eIF2α). M95 was also implicated in the accumulation of RIG-I, a viral RNA sensor protein, in SGs and in the subsequent acceleration of interferon induction. Taken together, our findings strongly suggest that M95-related inhibition of SG formation contributes to the pathogenesis of RABV by allowing the virus to evade the innate immune responses of the host. IMPORTANCE Rabies virus (RABV) is a neglected zoonotic pathogen that causes lethal infections in almost all mammalian hosts, including humans. Recently, RABV has been reported to induce intracellular formation of stress granules (SGs), also known as platforms that activate innate immune responses. However, the relationship between SG formation capacity and pathogenicity of RABV has remained unclear. In this study, by comparing two RABV strains with completely different levels of virulence, we found that the amino acid mutation from valine to alanine at position 95 of matrix protein (M95), which is known to be one of the amino acid mutations that determine the difference in virulence between the strains, plays a major role in SG formation. Importantly, M95 was involved in the accumulation of RIG-I in SGs and in promoting interferon induction. These findings are the first report of the effect of a single amino acid substitution associated with SGs on viral virulence.

Bruton’s tyrosine kinase phosphorylates scaffolding and RNA-binding protein G3BP1 to induce stress granule aggregation during host sensing of foreign ribonucleic acids

The Ras-GTPase activating protein SH3 domain-binding protein 1 (G3BP1) plays a critical role in the formation of classical and antiviral stress granules in stressed and virus-infected eukaryotic cells, respectively. While G3BP1 is known to be phosphorylated at serine residues which could affect stress granule assembly, whether G3BP1 is phosphorylated at tyrosine residues and how this posttranslational modification might affect its functions is less clear. Here, we show using immunoprecipitation and immunoblotting studies with 4G10 antibody that G3BP1 is tyrosine-phosphorylated when cells are stimulated with the synthetic double-stranded RNA analog polyinosinic:polycytidylic acid to mimic viral infection. We further demonstrate via co-immunoprecipitation and inhibitor studies that Bruton’s tyrosine kinase (BTK) binds and phosphorylates G3BP1. The nuclear transport factor 2–like domain of G3BP1 was previously shown to be critical for its self-association to form stress granules. Our mass spectrometry, mutational and biochemical cross-linking analyses indicate that the tyrosine-40 residue in this domain is phosphorylated by BTK and critical for G3BP1 oligomerization. Furthermore, as visualized via confocal microscopy, pretreatment of cells with the BTK inhibitor LFM-A13 or genetic deletion of the btk gene or mutation of G3BP1-Y40 residue to alanine or phenylalanine all significantly attenuated the formation of antiviral stress granule aggregates upon polyinosinic:polycytidylic acid treatment. Taken together, our data indicate that BTK phosphorylation of G3BP1 induces G3BP1 oligomerization and facilitates the condensation of ribonucleoprotein complexes into macromolecular aggregates.

Repurposed antibiotic and antiviral drugs inhibit the immune evasive endoribonuclease of SARS-CoV-2 and restrict coronavirus infection in vitro

Abstract Despite the success of SARS-CoV-2 vaccines in curbing viral transmission and severe disease, infections in unvaccinated and immune deficient patients continue to drive significant morbidity and mortality globally. To address the needs of this patient population, multiple antiviral drugs must be developed to expand on the protection currently offered by convalescent plasma, monoclonal antibodies, remdesivir, and other next generation SARS-CoV-2 antivirals which may be contraindicated for some patient groups. To this end, we employed a machine-learning based virtual ligand screening algorithm, FRAGSITE, to screen a library of FDA approved compounds for binding to the SARS-CoV-2 endoribonuclease (nsp15), a structurally conserved and demonstrated coronavirus virulence factor in SARS-CoV-1, HCoV-229E, IBV, MHV, and PEDV. Using recombinant SARS-CoV-2 nsp15, we identified the inhibition of nuclease activity by 11 drugs in vitro; 6 also restrict infection by the related human OC43 coronavirus in human A549 airway epithelial cells as measured by a focus forming assay. Among these 6 candidates, pibrentasvir remained active against HCoV-OC43 at the lowest concentration (IC50 < 0.625μM). Consistent with previous findings, HCoV-OC43 infection of A549 cells did not provoke an innate response to the viral dsRNA as quantified by IFNB1 mRNA production, antiviral stress granule formation, or RNase L activation. Our preliminary results suggest that antiviral stress granule formation is partially restored by nsp15 inhibitor treatment in HCoV-OC43 infected cells, although IFNβ production and RNase L activation remain unchanged. Supported by grants from the Emory School of Medicine

SARS-CoV-2 NSP5 and N protein counteract the RIG-I signaling pathway by suppressing the formation of stress granules

As a highly pathogenic human coronavirus, SARS-CoV-2 has to counteract an intricate network of antiviral host responses to establish infection and spread. The nucleic acid-induced stress response is an essential component of antiviral defense and is closely related to antiviral innate immunity. However, whether SARS-CoV-2 regulates the stress response pathway to achieve immune evasion remains elusive. In this study, SARS-CoV-2 NSP5 and N protein were found to attenuate antiviral stress granule (avSG) formation. Moreover, NSP5 and N suppressed IFN expression induced by infection of Sendai virus or transfection of a synthetic mimic of dsRNA, poly (I:C), inhibiting TBK1 and IRF3 phosphorylation, and restraining the nuclear translocalization of IRF3. Furthermore, HEK293T cells with ectopic expression of NSP5 or N protein were less resistant to vesicular stomatitis virus infection. Mechanistically, NSP5 suppressed avSG formation and disrupted RIG-I–MAVS complex to attenuate the RIG-I–mediated antiviral immunity. In contrast to the multiple targets of NSP5, the N protein specifically targeted cofactors upstream of RIG-I. The N protein interacted with G3BP1 to prevent avSG formation and to keep the cofactors G3BP1 and PACT from activating RIG-I. Additionally, the N protein also affected the recognition of dsRNA by RIG-I. This study revealed the intimate correlation between SARS-CoV-2, the stress response, and innate antiviral immunity, shedding light on the pathogenic mechanism of COVID-19.

Inhibition of anti-viral stress granule formation by coronavirus endoribonuclease nsp15 ensures efficient virus replication.

Cytoplasmic stress granules (SGs) are generally triggered by stress-induced translation arrest for storing mRNAs. Recently, it has been shown that SGs exert anti-viral functions due to their involvement in protein synthesis shut off and recruitment of innate immune signaling intermediates. The largest RNA viruses, coronaviruses, impose great threat to public safety and animal health; however, the significance of SGs in coronavirus infection is largely unknown. Infectious Bronchitis Virus (IBV) is the first identified coronavirus in 1930s and has been prevalent in poultry farm for many years. In this study, we provided evidence that IBV overcomes the host antiviral response by inhibiting SGs formation via the virus-encoded endoribonuclease nsp15. By immunofluorescence analysis, we observed that IBV infection not only did not trigger SGs formation in approximately 80% of the infected cells, but also impaired the formation of SGs triggered by heat shock, sodium arsenite, or NaCl stimuli. We further demonstrated that the intrinsic endoribonuclease activity of nsp15 was responsible for the interference of SGs formation. In fact, nsp15-defective recombinant IBV (rIBV-nsp15-H238A) greatly induced the formation of SGs, along with accumulation of dsRNA and activation of PKR, whereas wild type IBV failed to do so. Consequently, infection with rIBV-nsp15-H238A strongly triggered transcription of IFN-β which in turn greatly affected rIBV-nsp15-H238A replication. Further analysis showed that SGs function as an antiviral hub, as demonstrated by the attenuated IRF3-IFN response and increased production of IBV in SG-defective cells. Additional evidence includes the aggregation of pattern recognition receptors (PRRs) and signaling intermediates to the IBV-induced SGs. Collectively, our data demonstrate that the endoribonuclease nsp15 of IBV interferes with the formation of antiviral hub SGs by regulating the accumulation of viral dsRNA and by antagonizing the activation of PKR, eventually ensuring productive virus replication. We further demonstrated that nsp15s from PEDV, TGEV, SARS-CoV, and SARS-CoV-2 harbor the conserved function to interfere with the formation of chemically-induced SGs. Thus, we speculate that coronaviruses employ similar nsp15-mediated mechanisms to antagonize the host anti-viral SGs formation to ensure efficient virus replication.

DDX3X coordinates host defense against influenza virus by activating the NLRP3 inflammasome and type I interferon response

Viruses and hosts have coevolved for millions of years, leading to the development of complex host–pathogen interactions. Influenza A virus (IAV) causes severe pulmonary pathology and is a recurrent threat to human health. Innate immune sensing of IAV triggers a complex chain of host responses. IAV has adapted to evade host defense mechanisms, and the host has coevolved to counteract these evasion strategies. However, the molecular mechanisms governing the balance between host defense and viral immune evasion is poorly understood. Here, we show that the host protein DEAD-box helicase 3 X-linked (DDX3X) is critical to orchestrate a multifaceted antiviral innate response during IAV infection, coordinating the activation of the nucleotide-binding oligomerization domain-like receptor with a pyrin domain 3 (NLRP3) inflammasome, assembly of stress granules, and type I interferon (IFN) responses. DDX3X activated the NLRP3 inflammasome in response to WT IAV, which carries the immune evasive nonstructural protein 1 (NS1). However, in the absence of NS1, DDX3X promoted the formation of stress granules that facilitated efficient activation of type I IFN signaling. Moreover, induction of DDX3X-containing stress granules by external stimuli after IAV infection led to increased type I IFN signaling, suggesting that NS1 actively inhibits stress granule–mediated host responses and DDX3X-mediated NLRP3 activation counteracts this action. Furthermore, the loss of DDX3X expression in myeloid cells caused severe pulmonary pathogenesis and morbidity in IAV-infected mice. Together, our findings show that DDX3X orchestrates alternate modes of innate host defense which are critical to fight against NS1-mediated immune evasion strategies during IAV infection.

RNase L Amplifies Interferon Signaling by Inducing Protein Kinase R-Mediated Antiviral Stress Granules.

Virus infection leads to activation of the interferon (IFN)-induced endoribonuclease RNase L, which results in degradation of viral and cellular RNAs. Both cellular and viral RNA cleavage products of RNase L bind pattern recognition receptors (PRRs), like retinoic acid-inducible I (Rig-I) and melanoma differentiation-associated protein 5 (MDA5), to further amplify IFN production and antiviral response. Although much is known about the mechanics of ligand binding and PRR activation, how cells coordinate RNA sensing with signaling response and interferon production remains unclear. We show that RNA cleavage products of RNase L activity induce the formation of antiviral stress granules (avSGs) by regulating activation of double-stranded RNA (dsRNA)-dependent protein kinase R (PKR) and recruit the antiviral proteins Rig-I, PKR, OAS, and RNase L to avSGs. Biochemical analysis of purified avSGs showed interaction of a key stress granule protein, G3BP1, with only PKR and Rig-I and not with OAS or RNase L. AvSG assembly during RNase L activation is required for IRF3-mediated IFN production, but not IFN signaling or proinflammatory cytokine induction. Consequently, cells lacking avSG formation or RNase L signaling produced less IFN and showed higher susceptibility during Sendai virus infection, demonstrating the importance of avSGs in RNase L-mediated host defense. We propose a role during viral infection for RNase L-cleaved RNAs in inducing avSGs containing antiviral proteins to provide a platform for efficient interaction of RNA ligands with pattern recognition receptors to enhance IFN production to mount an effective antiviral response.IMPORTANCE Double-stranded RNAs produced during viral infections serve as pathogen-associated molecular patterns (PAMPs) and bind pattern recognition receptors to stimulate IFN production. RNase L is an IFN-regulated endoribonuclease that is activated in virus-infected cells and cleaves single-stranded viral and cellular RNAs. The RNase L-cleaved dsRNAs signal to Rig-like helicases to amplify IFN production. This study identifies a novel role of antiviral stress granules induced by RNase L as an antiviral signaling hub to coordinate the RNA ligands with cognate receptors to mount an effective host response during viral infections.

Articles of Significant Interest in This Issue

Virus infection activates RNase L, an interferon (IFN)-regulated endoribonuclease, to cleave viral and cellular RNAs. The RNase L-cleaved, double-stranded RNA products induce formation of antiviral stress granules (avSGs) and recruit antiviral proteins RIG-I, PKR, OAS, and RNase L to avSG to amplify IFN production. Manivannan et al. (e00205-20) identified a novel role of avSGs induced by RNase L as an antiviral signaling hub to coordinate RNA ligands with cognate receptors to mount an effective host response during viral infection. These findings shed light on the importance of asSGs for host immunity.

Spatio-temporal characterization of the antiviral activity of the XRN1-DCP1/2 aggregation against cytoplasmic RNA viruses to prevent cell death

Host nucleases are implicated in antiviral response through the processing of pathogen-derived nucleic acids. Among many host RNases, decapping enzymes DCP1 and 2, and 5′→3′ exonuclease XRN1, which are components of the RNA decay machinery, have been extensively studied in prokaryotes, plants, and invertebrates but less so in mammalian systems. As a result, the implication of XRN1 and DCPs in viral replication, in particular, the spatio-temporal dynamics during RNA viral infections remains elusive. Here, we highlight that XRN1 and DCPs play a critical role in limiting several groups of RNA viral infections. This antiviral activity was not obvious in wild-type cells but clearly observed in type I interferon (IFN-I)-deficient cells. Mechanistically, infection with RNA viruses induced the enrichment of XRN1 and DCPs in viral replication complexes (vRCs), hence forming distinct cytoplasmic aggregates. These aggregates served as sites for direct interaction between XRN1, DCP1/2, and viral ribonucleoprotein that contains viral RNA (vRNA). Although these XRN1-DCP1/2-vRC-containing foci resemble antiviral stress granules (SGs) or P-body (PB), they did not colocalize with known SG markers and did not correlate with critical PB functions. Furthermore, the presence of 5′ mono- and 5′ triphosphate structures on vRNA was not required for the formation of XRN1-DCP1/2-vRC-containing foci. On the other hand, single-, double-stranded, and higher-ordered vRNA species play a role but are not deterministic for efficient formation of XRN1-DCP1/2 foci and consequent antiviral activity in a manner proportional to RNA length. These results highlight the mechanism behind the antiviral function of XRN1-DCP1/2 in RNA viral infections independent of IFN-I response, protein kinase R and PB function.

Inclusion bodies of human parainfluenza virus type 3 inhibit antiviral stress granule formation by shielding viral RNAs.

Viral invasion triggers the activation of the host antiviral response. Besides the innate immune response, stress granules (SGs) also act as an additional defense response to combat viral replication. However, many viruses have evolved various strategies to suppress SG formation to facilitate their own replication. Here, we show that viral mRNAs derived from human parainfluenza virus type 3 (HPIV3) infection induce SG formation in an eIF2α phosphorylation- and PKR-dependent manner in which viral mRNAs are sequestered and viral replication is inhibited independent of the interferon signaling pathway. Furthermore, we found that inclusion body (IB) formation by the interaction of the nucleoprotein (N) and phosphoprotein (P) of HPIV3 correlated with SG suppression. In addition, co-expression of P with NL478A (a point mutant of N, which is unable to form IBs with P) or with NΔN10 (lacking N-terminal 10 amino acids of N, which could form IBs with P but was unable to synthesize or shield viral RNAs) failed to inhibit SG formation, suggesting that inhibition of SG formation also correlates with the capacity of IBs to synthesize and shield viral RNAs. Therefore, we provide a model whereby viral IBs escape the antiviral effect of SGs by concealing their own newly synthesized viral RNAs and offer new insights into the emerging role of IBs in viral replication.

Impaired Antiviral Stress Granule and IFN-β Enhanceosome Formation Enhances Susceptibility to Influenza Infection in Chronic Obstructive Pulmonary Disease Epithelium.

Chronic obstructive pulmonary disease (COPD) is a serious lung disease that progressively worsens lung function. Those affected are highly susceptible to influenza virus infections that result in exacerbations with exaggerated symptoms with increased mortality. The mechanisms underpinning this increased susceptibility to infection in COPD are unclear. In this study, we show that primary bronchial epithelial cells (pBECs) from subjects with COPD have impaired induction of type I IFN (IFN-β) and lead to heightened viral replication after influenza viral infection. COPD pBECs have reduced protein levels of protein kinase (PK) R and decreased formation of PKR-mediated antiviral stress granules, which are critical in initiating type I IFN inductions. In addition, reduced protein expression of p300 resulted in decreased activation of IFN regulatory factor 3 and subsequent formation of IFN-β enhanceosome in COPD pBECs. The decreased p300 induction was the result of enhanced levels of microRNA (miR)-132. Ectopic expression of PKR or miR-132 antagomiR alone failed to restore IFN-β induction, whereas cotreatment increased antiviral stress granule formation, induction of p300, and IFN-β in COPD pBECs. This study reveals that decreased induction of both PKR and p300 proteins contribute to impaired induction of IFN-β in COPD pBECs upon influenza infection.

Ubiquitin-like modifier FAT10 attenuates RIG-I mediated antiviral signaling by segregating activated RIG-I from its signaling platform.

RIG-I is a key cytosolic RNA sensor that mediates innate immune defense against RNA virus. Aberrant RIG-I activity leads to severe pathological states such as autosomal dominant multi-system disorder, inflammatory myophathies and dermatomyositis. Therefore, identification of regulators that ensure efficient defense without harmful immune-pathology is particularly critical to deal with RIG-I-associated diseases. Here, we presented the inflammatory inducible FAT10 as a novel negative regulator of RIG-I-mediated inflammatory response. In various cell lines, FAT10 protein is undetectable unless it is induced by pro-inflammatory cytokines. FAT10 non-covalently associated with the 2CARD domain of RIG-I, and inhibited viral RNA-induced IRF3 and NF-kB activation through modulating the RIG-I protein solubility. We further demonstrated that FAT10 was recruited to RIG-I-TRIM25 to form an inhibitory complex where FAT10 was stabilized by E3 ligase TRIM25. As the result, FAT10 inhibited the antiviral stress granules formation contains RIG-I and sequestered the active RIG-I away from the mitochondria. Our study presented a novel mechanism to dampen RIG-I activity. Highly accumulated FAT10 is observed in various cancers with pro-inflammatory environment, therefore, our finding which uncovered the suppressive effect of the accumulated FAT10 during virus-mediated inflammatory response may also provide molecular clue to understand the carcinogenesis related with infection and inflammation.

Leader-Containing Uncapped Viral Transcript Activates RIG-I in Antiviral Stress Granules.

RIG-I triggers antiviral responses by recognizing viral RNA (vRNA) in the cytoplasm. However, the spatio-temporal dynamics of vRNA sensing and signal transduction remain elusive. We investigated the time course of events in cells infected with Newcastle disease virus (NDV), a non-segmented negative-strand RNA virus. RIG-I was recruited to viral replication complexes (vRC) and triggered minimal primary type I interferon (IFN) production. RIG-I subsequently localized to antiviral stress granules (avSG) induced after vRC formation. The inhibition of avSG attenuated secondary IFN production, suggesting avSG as a platform for efficient vRNA detection. avSG selectively captured positive-strand vRNA, and poly(A)+ RNA induced IFN production. Further investigations suggested that uncapped vRNA derived from read-through transcription was sensed by RIG-I in avSG. These results highlight how viral infections stimulate host stress responses, thereby selectively recruiting uncapped vRNA to avSG, in which RIG-I and other components cooperate in an efficient antiviral program.

ID: 70: A novel function of human Pumilio proteins in antiviral innate immunity

RIG-I-like receptor (RLR) plays a pivotal role in the detection of invading pathogens to initiate type I interferon (IFN) gene transcription. Since aberrant IFN production is harmful, RLR signaling is strictly regulated. However, the regulatory mechanisms are not fully understood. By expression cloning, we identified Pumilio proteins, PUM1 and PUM2, as candidate positive regulators of RIG-I signaling. Overexpression of Pumilio proteins and their knockdown augmented and diminished IFN-β promoter activity induced by Newcastle disease virus (NDV), respectively. Both proteins showed a specific association with LGP2, but not with RIG-I or MDA5. Furthermore, all of these components were recruited to NDV-induced antiviral stress granules. Interestingly, biochemical analyses revealed that Pumilio increased double-stranded (ds) RNA binding affinity of LGP2; however, Pumilio was absent in the dsRNA-LGP2 complex, suggesting that Pumilio facilitates viral RNA recognition by LGP2 through its chaperon-like function. Collectively, our results demonstrate an unknown function of Pumilio in viral recognition by LGP2.

IFN-β-inducing, unusual viral RNA species produced by paramyxovirus infection accumulated into distinct cytoplasmic structures in an RNA-type-dependent manner.

The interferon (IFN) system is one of the most important defensive responses of mammals against viruses, and is rapidly evoked when the pathogen-associated molecular patterns (PAMPs) of viruses are sensed. Non-self, virus-derived RNA species have been identified as the PAMPs of RNA viruses. In the present study, we compared different types of IFN-β-inducing and -non-inducing viruses in the context of Sendai virus infection. We found that some types of unusual viral RNA species were produced by infections with IFN-β-inducing viruses and accumulated into distinct cytoplasmic structures in an RNA-type-dependent manner. One of these structures was similar to the so-called antiviral stress granules (avSGs) formed by an infection with IFN-inducing viruses whose C proteins were knocked-out or mutated. Non-encapsidated, unusual viral RNA harboring the 5′-terminal region of the viral genome as well as RIG-I and typical SG markers accumulated in these granules. Another was a non-SG-like inclusion formed by an infection with the Cantell strain; a copyback-type DI genome, but not an authentic viral genome, specifically accumulated in the inclusion, whereas RIG-I and SG markers did not. The induction of IFN-β was closely associated with the production of these unusual RNAs as well as the formation of the cytoplasmic structures.

A novel function of human Pumilio proteins in cytoplasmic sensing of viral infection.

RIG-I-like receptor (RLR) plays a pivotal role in the detection of invading pathogens to initiate type I interferon (IFN) gene transcription. Since aberrant IFN production is harmful, RLR signaling is strictly regulated. However, the regulatory mechanisms are not fully understood. By expression cloning, we identified Pumilio proteins, PUM1 and PUM2, as candidate positive regulators of RIG-I signaling. Overexpression of Pumilio proteins and their knockdown augmented and diminished IFN-β promoter activity induced by Newcastle disease virus (NDV), respectively. Both proteins showed a specific association with LGP2, but not with RIG-I or MDA5. Furthermore, all of these components were recruited to NDV-induced antiviral stress granules. Interestingly, biochemical analyses revealed that Pumilio increased double-stranded (ds) RNA binding affinity of LGP2; however, Pumilio was absent in the dsRNA-LGP2 complex, suggesting that Pumilio facilitates viral RNA recognition by LGP2 through its chaperon-like function. Collectively, our results demonstrate an unknown function of Pumilio in viral recognition by LGP2.

81: Critical role of PKR in antiviral stress granule and IFN-β enhanceosome formation, and is impaired in chronic obstructive pulmonary disease

Aim PKR is important in the formation of antiviral stress granules (AvSGs) and subsequent induction of IFN- β , however it is unclear if PKR or AvSG is important in the formation of the transcription factor complex IFN- β enhanceosome. People with chronic obstructive pulmonary disease (COPD) are more susceptible to influenza infection, the mechanisms of which remain unclear. In this study we aim to investigate if PKR is essential in AvSG, IFN- β enhanceosome formation, and subsequent IFN- β protein induction. We also aimed to determine if these responses are impaired in people with COPD, leading to deficient antiviral responses to influenza infection. Methods Primary bronchial epithelial cells (pBECs) from healthy controls and subjects with COPD were infected with human influenza A/H1N1 at MOI of 5. AvSG proteins PKR, TIAR, G3BP were detected by confocal microscopy, and by western blotting. IFN- β enhanceosome proteins pIRF3, pATF2, and p300 were also measured. IFN- β protein was measured by western blotting. Viral replication was measured by plaque assay. PKR was silenced by specific siRNA 24 before infection. Co-localisation test was performed using ImageJ according to instructions. Results Influenza infection resulted in reduced AvSG component PKR, TIAR, and G3BP expression and decreased formation of AvSG in COPD pBECs. The formation of IFN- β enhanceosome was also significantly decreased in COPD pBECs with reduced expression of enhanceosome components pIRF3, pATF2, and p300. We then determined that PKR was important in the formation of AvSG, as PKR inhibition by siRNA led to impaired AvSG formation and also IFN- β enhanceosome. This then led to reduced IFN- β induction and increased influenza viral replication in COPD pBECs. Conclusion PKR is important in the formation of AvSGs and IFN- β enhanceosome, and was impaired in COPD pBECs, leading to reduced antiviral responses and heightened viral replication to influenza infection.