RESUMO
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.
Assuntos
Vírus da Raiva , Grânulos de Estresse , Proteínas da Matriz Viral , Aminoácidos/metabolismo , Animais , Fator de Iniciação 2 em Eucariotos/metabolismo , Humanos , Interferons/imunologia , Proteínas Quinases/imunologia , RNA Viral/metabolismo , Vírus da Raiva/genética , Vírus da Raiva/patogenicidade , Ribonucleoproteínas/metabolismo , Grânulos de Estresse/genética , Grânulos de Estresse/imunologia , Proteínas da Matriz Viral/genética , Proteínas da Matriz Viral/imunologia , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
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.
Assuntos
Proteases 3C de Coronavírus/genética , Proteínas do Nucleocapsídeo de Coronavírus/genética , Proteína DEAD-box 58/genética , DNA Helicases/genética , Proteínas de Ligação a Poli-ADP-Ribose/genética , RNA Helicases/genética , Proteínas com Motivo de Reconhecimento de RNA/genética , Proteínas de Ligação a RNA/genética , Receptores Imunológicos/genética , SARS-CoV-2/genética , Grânulos de Estresse/genética , Animais , Chlorocebus aethiops , Proteases 3C de Coronavírus/imunologia , Proteínas do Nucleocapsídeo de Coronavírus/imunologia , Proteína DEAD-box 58/imunologia , DNA Helicases/imunologia , Regulação da Expressão Gênica , Células HEK293 , Células HeLa , Humanos , Evasão da Resposta Imune , Fosfoproteínas/genética , Fosfoproteínas/imunologia , Poli I-C/farmacologia , Proteínas de Ligação a Poli-ADP-Ribose/imunologia , Ligação Proteica , RNA Helicases/imunologia , Proteínas com Motivo de Reconhecimento de RNA/imunologia , RNA de Cadeia Dupla/genética , RNA de Cadeia Dupla/imunologia , Proteínas de Ligação a RNA/imunologia , Receptores Imunológicos/imunologia , SARS-CoV-2/imunologia , SARS-CoV-2/patogenicidade , Vírus Sendai/genética , Vírus Sendai/imunologia , Transdução de Sinais , Grânulos de Estresse/efeitos dos fármacos , Grânulos de Estresse/imunologia , Grânulos de Estresse/virologia , Células Vero , Vesiculovirus/genética , Vesiculovirus/imunologiaRESUMO
Stress granules (SGs) are highly dynamic cytoplasmic foci that form in response to activation of the integrated stress response (ISR) that results in eIF2α phosphorylation and global translation shutdown. Stress granules, which are largely nucleated by G3BP1, serve as hubs for mRNA triage, but there is mounting evidence that they also perform cell signaling functions that are vital to cell survival, particularly during viral infection. We previously showed that SG formation leads to NFκB activation and JNK signaling and that this association may be due in part to G3BP1-dependent recruitment of PKR to SGs. Others have reported close associations between G3BP1 and various innate immune PRRs of the type 1 interferon signaling system, including RIG-I. We also reported SG assembly dynamics is dependent on the arginine-methylation status of G3BP1. Another protein that rapidly localizes to SGs, TDRD3, is a methyl reader protein that performs transcriptional activation and adaptor functions within the nucleus, but neither the mechanism nor its function in SGs is clear. Here, we present evidence that TDRD3 localizes to SGs partly based upon methylation potential of G3BP1. We also characterize granules that TDRD3 forms during overexpression and show that these granules can form in the absence of G3BP but also contain translation components found in canonical SGs. We also show for the first time that SGs recruit additional interferon effectors IRF3, IRF7, TBK1, and Sting, and provide evidence that TDRD3 may play a role in recruitment of these factors. We also present evidence that TDRD3 is a novel antiviral protein that is cleaved by enteroviral 2A proteinase. G3BP1 and TDRD3 knockdown in cells results in altered transcriptional regulation of numerous IFN effectors in complex modulatory patterns that are distinctive for G3BP1 and TDRD3. Overall, we describe a novel role of TDRD3 in innate immunity in which G3BP1 and TDRD3 may coordinate to play important roles in regulation of innate antiviral defenses.
Assuntos
DNA Helicases/imunologia , Imunidade Inata/imunologia , Proteínas de Ligação a Poli-ADP-Ribose/imunologia , Proteínas/imunologia , RNA Helicases/imunologia , Proteínas com Motivo de Reconhecimento de RNA/imunologia , Viroses/imunologia , Linhagem Celular , Humanos , Interferons/imunologia , Transdução de Sinais/imunologia , Grânulos de Estresse/imunologiaRESUMO
To rapidly adapt to stresses such as infections, cells have evolved several mechanisms, which include the activation of stress response pathways and the innate immune response. These stress responses result in the rapid inhibition of translation and condensation of stalled mRNAs with RNA-binding proteins and signalling components into cytoplasmic biocondensates called stress granules (SGs). Increasing evidence suggests that SGs contribute to antiviral defence, and thus viruses need to evade these responses to propagate. We previously showed that feline calicivirus (FCV) impairs SG assembly by cleaving the scaffolding protein G3BP1. We also observed that uninfected bystander cells assembled G3BP1-positive granules, suggesting a paracrine response triggered by infection. We now present evidence that virus-free supernatant generated from infected cells can induce the formation of SG-like foci, which we name paracrine granules. They are linked to antiviral activity and exhibit specific kinetics of assembly-disassembly, and protein and RNA composition that are different from canonical SGs. We propose that this paracrine induction reflects a novel cellular defence mechanism to limit viral propagation and promote stress responses in bystander cells.
Assuntos
Infecções por Caliciviridae , Grânulos de Estresse , Animais , Infecções por Caliciviridae/imunologia , Calicivirus Felino/imunologia , Gatos , Proteínas de Ligação a Poli-ADP-Ribose/imunologia , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Grânulos de Estresse/imunologia , Replicação Viral/fisiologiaRESUMO
Cells have to deal with conditions that can cause damage to biomolecules and eventually cell death. To protect against these adverse conditions and promote recovery, cells undergo dramatic changes upon exposure to stress. This involves activation of signaling pathways, cell cycle arrest, translational reprogramming, and reorganization of the cytoplasm. Notably, many stress conditions cause a global inhibition of mRNA translation accompanied by the formation of cytoplasmic condensates called stress granules (SGs), which sequester mRNA together with RNA-binding proteins, translation initiation factors, and other components. SGs are highly conserved in eukaryotes, suggesting that they perform an important function during the stress response. Over the years, many different roles have been assigned to SGs, including translational control, mRNA storage, regulation of mRNA decay, antiviral innate immune response, and modulation of signaling pathways. Most of our understanding, however, has been deduced from correlative data based upon the composition of SGs and only recently have technological innovations allowed hypotheses for SG function to be directly tested. Here, we discuss these challenges and explore the evidence related to the function of SGs.
Assuntos
Grânulos Citoplasmáticos/genética , Imunidade Inata/genética , RNA Mensageiro/genética , Grânulos de Estresse/genética , Grânulos Citoplasmáticos/imunologia , Resposta ao Choque Térmico/genética , Humanos , Estresse Oxidativo/genética , Estabilidade de RNA/genética , Estabilidade de RNA/imunologia , Grânulos de Estresse/imunologiaRESUMO
Cellular stress can induce cytoplasmic ribonucleoprotein complexes called stress granules that allow the cells to survive. Stress granules are also central to cellular responses to infections, in which they can act as platforms for viral sensing or modulate innate immune signaling through pattern recognition receptors. However, the effect of innate immune signaling on stress granules is poorly understood. In this study, we report that prior induction of innate immune signaling through TLRs inhibited stress granule assembly in a TLR ligand dose-dependent manner in murine bone marrow-derived macrophages. Time course analysis suggests that TLR stimulation can reverse stress granule assembly even after it has begun. Additionally, both MYD88- and TRIF-mediated TLR signaling inhibited stress granule assembly in response to endoplasmic reticulum stress in bone marrow-derived macrophages and the chemotherapeutic drug oxaliplatin in murine B16 melanoma cells. This inhibition was not due to a decrease in expression of the critical stress granule proteins G3BP1 and DDX3X and was independent of IRAK1/4, JNK, ERK and P38 kinase activity but dependent on IKK complex kinase activity. Overall, we have identified the TLR-IKK complex signaling axis as a regulator of stress granule assembly-disassembly dynamics, highlighting cross-talk between processes that are critical in health and disease.