RESUMO
The kinetics of the immune changes in COVID-19 across severity groups have not been rigorously assessed. Using immunophenotyping, RNA sequencing, and serum cytokine analysis, we analyzed serial samples from 207 SARS-CoV2-infected individuals with a range of disease severities over 12 weeks from symptom onset. An early robust bystander CD8+ T cell immune response, without systemic inflammation, characterized asymptomatic or mild disease. Hospitalized individuals had delayed bystander responses and systemic inflammation that was already evident near symptom onset, indicating that immunopathology may be inevitable in some individuals. Viral load did not correlate with this early pathological response but did correlate with subsequent disease severity. Immune recovery is complex, with profound persistent cellular abnormalities in severe disease correlating with altered inflammatory responses, with signatures associated with increased oxidative phosphorylation replacing those driven by cytokines tumor necrosis factor (TNF) and interleukin (IL)-6. These late immunometabolic and immune defects may have clinical implications.
Assuntos
Linfócitos T CD8-Positivos/imunologia , COVID-19/imunologia , COVID-19/virologia , Interações Hospedeiro-Patógeno/imunologia , Ativação Linfocitária/imunologia , SARS-CoV-2/imunologia , Biomarcadores , Linfócitos T CD8-Positivos/metabolismo , COVID-19/diagnóstico , COVID-19/genética , Citocinas/metabolismo , Suscetibilidade a Doenças , Perfilação da Expressão Gênica , Humanos , Mediadores da Inflamação/metabolismo , Estudos Longitudinais , Ativação Linfocitária/genética , Fosforilação Oxidativa , Fenótipo , Prognóstico , Espécies Reativas de Oxigênio/metabolismo , Índice de Gravidade de Doença , TranscriptomaRESUMO
The emergence of SARS-CoV-2 variants of concern suggests viral adaptation to enhance human-to-human transmission1,2. Although much effort has focused on the characterization of changes in the spike protein in variants of concern, mutations outside of spike are likely to contribute to adaptation. Here, using unbiased abundance proteomics, phosphoproteomics, RNA sequencing and viral replication assays, we show that isolates of the Alpha (B.1.1.7) variant3 suppress innate immune responses in airway epithelial cells more effectively than first-wave isolates. We found that the Alpha variant has markedly increased subgenomic RNA and protein levels of the nucleocapsid protein (N), Orf9b and Orf6-all known innate immune antagonists. Expression of Orf9b alone suppressed the innate immune response through interaction with TOM70, a mitochondrial protein that is required for activation of the RNA-sensing adaptor MAVS. Moreover, the activity of Orf9b and its association with TOM70 was regulated by phosphorylation. We propose that more effective innate immune suppression, through enhanced expression of specific viral antagonist proteins, increases the likelihood of successful transmission of the Alpha variant, and may increase in vivo replication and duration of infection4. The importance of mutations outside the spike coding region in the adaptation of SARS-CoV-2 to humans is underscored by the observation that similar mutations exist in the N and Orf9b regulatory regions of the Delta and Omicron variants.
Assuntos
COVID-19/imunologia , COVID-19/virologia , Evolução Molecular , Evasão da Resposta Imune , Imunidade Inata/imunologia , SARS-CoV-2/genética , SARS-CoV-2/imunologia , COVID-19/transmissão , Proteínas do Nucleocapsídeo de Coronavírus/química , Proteínas do Nucleocapsídeo de Coronavírus/metabolismo , Humanos , Imunidade Inata/genética , Interferons/imunologia , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial/metabolismo , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Fosforilação , Proteômica , RNA Viral/genética , RNA-Seq , SARS-CoV-2/classificação , SARS-CoV-2/crescimento & desenvolvimentoRESUMO
The spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is critical for virus infection through the engagement of the human ACE2 protein1 and is a major antibody target. Here we show that chronic infection with SARS-CoV-2 leads to viral evolution and reduced sensitivity to neutralizing antibodies in an immunosuppressed individual treated with convalescent plasma, by generating whole-genome ultra-deep sequences for 23 time points that span 101 days and using in vitro techniques to characterize the mutations revealed by sequencing. There was little change in the overall structure of the viral population after two courses of remdesivir during the first 57 days. However, after convalescent plasma therapy, we observed large, dynamic shifts in the viral population, with the emergence of a dominant viral strain that contained a substitution (D796H) in the S2 subunit and a deletion (ΔH69/ΔV70) in the S1 N-terminal domain of the spike protein. As passively transferred serum antibodies diminished, viruses with the escape genotype were reduced in frequency, before returning during a final, unsuccessful course of convalescent plasma treatment. In vitro, the spike double mutant bearing both ΔH69/ΔV70 and D796H conferred modestly decreased sensitivity to convalescent plasma, while maintaining infectivity levels that were similar to the wild-type virus.The spike substitution mutant D796H appeared to be the main contributor to the decreased susceptibility to neutralizing antibodies, but this mutation resulted in an infectivity defect. The spike deletion mutant ΔH69/ΔV70 had a twofold higher level of infectivity than wild-type SARS-CoV-2, possibly compensating for the reduced infectivity of the D796H mutation. These data reveal strong selection on SARS-CoV-2 during convalescent plasma therapy, which is associated with the emergence of viral variants that show evidence of reduced susceptibility to neutralizing antibodies in immunosuppressed individuals.
Assuntos
Tratamento Farmacológico da COVID-19 , COVID-19/terapia , COVID-19/virologia , Evolução Molecular , Mutagênese/efeitos dos fármacos , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/genética , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/farmacologia , Monofosfato de Adenosina/uso terapêutico , Idoso , Alanina/análogos & derivados , Alanina/farmacologia , Alanina/uso terapêutico , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , COVID-19/imunologia , Doença Crônica , Genoma Viral/efeitos dos fármacos , Genoma Viral/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Evasão da Resposta Imune/efeitos dos fármacos , Evasão da Resposta Imune/genética , Evasão da Resposta Imune/imunologia , Tolerância Imunológica/efeitos dos fármacos , Tolerância Imunológica/imunologia , Imunização Passiva , Terapia de Imunossupressão , Masculino , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/imunologia , Mutação , Filogenia , SARS-CoV-2/imunologia , SARS-CoV-2/metabolismo , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia , Fatores de Tempo , Carga Viral/efeitos dos fármacos , Eliminação de Partículas Virais , Soroterapia para COVID-19RESUMO
To initiate infection, many viruses enter their host cells by triggering endocytosis following receptor engagement. However, the mechanisms by which non-enveloped viruses escape the endosome are poorly understood. Here we present near-atomic-resolution cryo-electron microscopy structures for feline calicivirus both undecorated and labelled with a soluble fragment of its cellular receptor, feline junctional adhesion molecule A. We show that VP2, a minor capsid protein encoded by all caliciviruses1,2, forms a large portal-like assembly at a unique three-fold axis of symmetry, following receptor engagement. This assembly-which was not detected in undecorated virions-is formed of twelve copies of VP2, arranged with their hydrophobic N termini pointing away from the virion surface. Local rearrangement at the portal site leads to the opening of a pore in the capsid shell. We hypothesize that the portal-like assembly functions as a channel for the delivery of the calicivirus genome, through the endosomal membrane, into the cytoplasm of a host cell, thereby initiating infection. VP2 was previously known to be critical for the production of infectious virus3; our findings provide insights into its structure and function that advance our understanding of the Caliciviridae.
Assuntos
Calicivirus Felino/metabolismo , Calicivirus Felino/ultraestrutura , Proteínas do Capsídeo/metabolismo , Proteínas do Capsídeo/ultraestrutura , Microscopia Crioeletrônica , Molécula A de Adesão Juncional/ultraestrutura , Receptores Virais/ultraestrutura , Montagem de Vírus , Animais , Calicivirus Felino/química , Calicivirus Felino/crescimento & desenvolvimento , Proteínas do Capsídeo/química , Gatos , Linhagem Celular , Endossomos/metabolismo , Endossomos/virologia , Genoma Viral , Interações Hidrofóbicas e Hidrofílicas , Molécula A de Adesão Juncional/química , Molécula A de Adesão Juncional/metabolismo , Modelos Moleculares , Receptores Virais/química , Receptores Virais/metabolismo , Eletricidade Estática , Vírion/química , Vírion/genética , Vírion/metabolismo , Vírion/ultraestruturaRESUMO
Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) infects cells by binding to the host cell receptor ACE2 and undergoing virus-host membrane fusion. Fusion is triggered by the protease TMPRSS2, which processes the viral Spike (S) protein to reveal the fusion peptide. SARS-CoV-2 has evolved a multibasic site at the S1-S2 boundary, which is thought to be cleaved by furin in order to prime S protein for TMPRSS2 processing. Here we show that CRISPR-Cas9 knockout of furin reduces, but does not prevent, the production of infectious SARS-CoV-2 virus. Comparing S processing in furin knockout cells to multibasic site mutants reveals that while loss of furin substantially reduces S1-S2 cleavage it does not prevent it. SARS-CoV-2 S protein also mediates cell-cell fusion, potentially allowing virus to spread virion-independently. We show that loss of furin in either donor or acceptor cells reduces, but does not prevent, TMPRSS2-dependent cell-cell fusion, unlike mutation of the multibasic site that completely prevents syncytia formation. Our results show that while furin promotes both SARS-CoV-2 infectivity and cell-cell spread it is not essential, suggesting furin inhibitors may reduce but not abolish viral spread.
Assuntos
Fusão Celular , Furina/genética , Glicoproteína da Espícula de Coronavírus/química , Internalização do Vírus , Animais , COVID-19 , Sistemas CRISPR-Cas , Chlorocebus aethiops , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Estrutura Terciária de Proteína , SARS-CoV-2 , Serina Endopeptidases , Células VeroRESUMO
Vesivirus 2117 is an adventitious agent that has been responsible for lost productivity in biopharmaceutical production following contamination of Chinese hamster ovary cell cultures in commercial bioreactors. A member of the Caliciviridae, 2117 is classified within the Vesivirus genus in a clade that includes canine and mink caliciviruses but is distinct from the vesicular exanthema of swine virus (VESV) clade, which includes the extensively studied feline calicivirus (FCV). We have used cryogenic electron microscopy (cryo-EM) to determine the structure of the capsid of this small, icosahedral, positive-sense-RNA-containing virus. We show that the outer face of the dimeric capsomeres, which contains the receptor binding site and major immunodominant epitopes in all caliciviruses studied thus far, is quite different from that of FCV. This is a consequence of a 22-amino-acid insertion in the sequence of the FCV major capsid protein that forms a "cantilevered arm" that both plays an important role in receptor engagement and undergoes structural rearrangements thought to be important for genome delivery to the cytosol. Our data highlight a potentially important difference in the attachment and entry pathways employed by the different clades of the Vesivirus genus. IMPORTANCE Vesivirus 2117 has caused significant losses in manufacturing of biopharmaceutical products following contamination of cell cultures used in their production. We report the structure of the vesivirus 2117 capsid, the shell that encloses the virus's genome. Comparison of this structure with that of a related vesivirus, feline calicivirus (FCV), highlighted potentially important differences related to virus attachment and entry. Our findings suggest that these two viruses may bind differently to receptors at the host cell surface. We also show that a region of the capsid protein of FCV that rearranges following receptor engagement is not present in vesivirus 2117. These structural changes in the FCV capsid have been shown to allow the assembly of a portal-like structure that is hypothesized to deliver the viral genome to the cell's interior. Our data suggest that the 2117 portal assembly may employ a different means of anchoring to the outer face of the capsid.
Assuntos
Capsídeo/metabolismo , Vesivirus/metabolismo , Animais , Células CHO , Proteínas do Capsídeo/genética , Linhagem Celular , Cricetinae , Cricetulus , Microscopia Crioeletrônica , Estrutura Quaternária de Proteína/fisiologia , Vírion/metabolismo , Ligação ViralRESUMO
Murine norovirus (MNV) infection results in a late translation shutoff that is proposed to contribute to the attenuated and delayed innate immune response observed both in vitro and in vivo. Recently, we further demonstrated the activation of the α subunit of eukaryotic initiation factor 2 (eIF2α) kinase GCN2 during MNV infection, which has been previously linked to immunomodulation and resistance to inflammatory signaling during metabolic stress. While viral infection is usually associated with activation of double-stranded RNA (dsRNA) binding pattern recognition receptor PKR, we hypothesized that the establishment of a metabolic stress in infected cells is a proviral event, exploited by MNV to promote replication through weakening the activation of the innate immune response. In this study, we used multi-omics approaches to characterize cellular responses during MNV replication. We demonstrate the activation of pathways related to the integrated stress response, a known driver of anti-inflammatory phenotypes in macrophages. In particular, MNV infection causes an amino acid imbalance that is associated with GCN2 and ATF2 signaling. Importantly, this reprogramming lacks the features of a typical innate immune response, with the ATF/CHOP target GDF15 contributing to the lack of antiviral responses. We propose that MNV-induced metabolic stress supports the establishment of host tolerance to viral replication and propagation. IMPORTANCE During viral infection, host defenses are typically characterized by the secretion of proinflammatory autocrine and paracrine cytokines, potentiation of the interferon (IFN) response, and induction of the antiviral response via activation of JAK and Stat signaling. To avoid these and propagate, viruses have evolved strategies to evade or counteract host sensing. In this study, we demonstrate that murine norovirus controls the antiviral response by activating a metabolic stress response that activates the amino acid response and impairs inflammatory signaling. This highlights novel tools in the viral countermeasures arsenal and demonstrates the importance of the currently poorly understood metabolic reprogramming occurring during viral infections.
Assuntos
Infecções por Caliciviridae/imunologia , Macrófagos/virologia , Fator 2 Ativador da Transcrição/metabolismo , Animais , Antivirais , Infecções por Caliciviridae/metabolismo , Linhagem Celular , Fator de Iniciação 2 em Eucariotos/metabolismo , Imunidade Inata/imunologia , Inflamação/imunologia , Interferons , Macrófagos/imunologia , Camundongos , Norovirus/patogenicidade , Proteínas Serina-Treonina Quinases/metabolismo , Células RAW 264.7 , RNA de Cadeia Dupla/genética , Transdução de Sinais/imunologia , Proteínas não Estruturais Virais/metabolismo , Replicação Viral/genéticaRESUMO
Viral infections impose major stress on the host cell. In response, stress pathways can rapidly deploy defence mechanisms by shutting off the protein synthesis machinery and triggering the accumulation of mRNAs into stress granules to limit the use of energy and nutrients. Because this threatens viral gene expression, viruses need to evade these pathways to propagate. Human norovirus is responsible for gastroenteritis outbreaks worldwide. Here we examined how norovirus interacts with the eIF2α signaling axis controlling translation and stress granules. While norovirus infection represses host cell translation, our mechanistic analyses revealed that eIF2α signaling mediated by the stress kinase GCN2 is uncoupled from translational stalling. Moreover, infection results in a redistribution of the RNA-binding protein G3BP1 to replication complexes and remodelling of its interacting partners, allowing the avoidance from canonical stress granules. These results define novel strategies by which norovirus undergo efficient replication whilst avoiding the host stress response and manipulating the G3BP1 interactome.
Assuntos
Infecções por Caliciviridae/virologia , DNA Helicases/metabolismo , Fator de Iniciação 2 em Eucariotos/metabolismo , Norovirus/fisiologia , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Biossíntese de Proteínas , RNA Helicases/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Animais , Infecções por Caliciviridae/genética , Linhagem Celular , Grânulos Citoplasmáticos/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Células RAW 264.7 , RNA/metabolismo , Transdução de Sinais , Replicação ViralRESUMO
Human noroviruses (HuNoVs) are increasingly becoming the main cause of transmissible gastroenteritis worldwide, with hundreds of thousands of deaths recorded annually. Yet, decades after their discovery, there is still no effective treatment or vaccine. Efforts aimed at developing vaccines or treatment will benefit from a greater understanding of norovirus-host interactions, including the host response to infection. In this review, we provide a concise overview of the evidence establishing the significance of type I and type III interferon (IFN) responses in the restriction of noroviruses. We also critically examine our current understanding of the molecular mechanisms of IFN induction in norovirus-infected cells, and outline the diverse strategies deployed by noroviruses to supress and/or avoid host IFN responses. It is our hope that this review will facilitate further discussion and increase interest in this area.
Assuntos
Infecções por Caliciviridae/imunologia , Infecções por Caliciviridae/virologia , Interferons/fisiologia , Norovirus/imunologia , Norovirus/patogenicidade , Animais , Linhagem Celular , Humanos , Evasão da Resposta Imune , Imunidade Inata , Interferons/biossíntese , Proteínas Virais/metabolismo , Replicação ViralRESUMO
Murine norovirus (MNV) is widely used as a model for studying norovirus biology. While MNV isolates vary in their pathogenesis, infection of immunocompetent mice mostly results in persistent infection. The ability of a virus to establish a persistent infection is dependent on its ability to subvert or avoid the host immune response. Previously, we described the identification and characterization of virulence factor 1 (VF1) in MNV, and demonstrated its role as an innate immune antagonist. Here, we explore the role of VF1 during persistent MNV infection in an immunocompetent host. Using reverse genetics, we generated MNV-3 viruses carrying a single or a triple termination codon inserted in the VF1 ORF. VF1-deleted MNV-3 replicated to comparable levels to the wildtype virus in tissue culture. Comparative studies between MNV-3 and an acute MNV-1 strain show that MNV-3 VF1 exerts the same functions as MNV-1 VF1, but with reduced potency. C57BL/6 mice infected with VF1-deleted MNV-3 showed significantly reduced replication kinetics during the acute phase of the infection, but viral loads rapidly reached the levels seen in mice infected with wildtype virus after phenotypic restoration of VF1 expression. Infection with an MNV-3 mutant that had three termination codons inserted into VF1, in which reversion was suppressed, resulted in consistently lower replication throughout a 3 month persistent infection in mice, suggesting a role for VF1 in viral fitness in vivo. Our results indicate that VF1 expressed by a persistent strain of MNV also functions to antagonize the innate response to infection. We found that VF1 is not essential for viral persistence, but instead contributes to viral fitness in mice. These data fit with the hypothesis that noroviruses utilize multiple mechanisms to avoid and/or control the host response to infection and that VF1 is just one component of this.
Assuntos
Infecções por Caliciviridae/virologia , Norovirus/patogenicidade , Proteínas Virais/metabolismo , Fatores de Virulência/metabolismo , Animais , Apoptose , Infecções por Caliciviridae/imunologia , Linhagem Celular , Imunidade Inata , Interferon beta/biossíntese , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Norovirus/genética , Norovirus/fisiologia , Proteínas Virais/genética , Virulência , Fatores de Virulência/genética , Replicação Viral , Eliminação de Partículas ViraisRESUMO
RNA-remodeling proteins, including RNA helicases and chaperones, act to remodel RNA structures and/or protein-RNA interactions and are required for all processes involving RNAs. Although many viruses encode RNA helicases and chaperones, their in vitro activities and their roles in infected cells largely remain elusive. Noroviruses are a diverse group of positive-strand RNA viruses in the family Caliciviridae and constitute a significant and potentially fatal threat to human health. Here, we report that the protein NS3 encoded by human norovirus has both ATP-dependent RNA helicase activity that unwinds RNA helices and ATP-independent RNA-chaperoning activity that can remodel structured RNAs and facilitate strand annealing. Moreover, NS3 can facilitate viral RNA synthesis in vitro by norovirus polymerase. NS3 may therefore play an important role in norovirus RNA replication. Lastly, we demonstrate that the RNA-remodeling activity of NS3 is inhibited by guanidine hydrochloride, an FDA-approved compound, and, more importantly, that it reduces the replication of the norovirus replicon in cultured human cells. Altogether, these findings are the first to demonstrate the presence of RNA-remodeling activities encoded by Caliciviridae and highlight the functional significance of NS3 in the noroviral life cycle.IMPORTANCE Noroviruses are a diverse group of positive-strand RNA viruses, which annually cause hundreds of millions of human infections and over 200,000 deaths worldwide. For RNA viruses, cellular or virus-encoded RNA helicases and/or chaperones have long been considered to play pivotal roles in viral life cycles. However, neither RNA helicase nor chaperoning activity has been demonstrated to be associated with any norovirus-encoded proteins, and it is also unknown whether norovirus replication requires the participation of any viral or cellular RNA helicases/chaperones. We found that a norovirus protein, NS3, not only has ATP-dependent helicase activity, but also acts as an ATP-independent RNA chaperone. Also, NS3 can facilitate in vitro viral RNA synthesis, suggesting the important role of NS3 in norovirus replication. Moreover, NS3 activities can be inhibited by an FDA-approved compound, which also suppresses norovirus replicon replication in human cells, raising the possibility that NS3 could be a target for antinoroviral drug development.
Assuntos
Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Norovirus/enzimologia , Norovirus/genética , RNA Helicases/genética , RNA Helicases/metabolismo , Proteínas não Estruturais Virais/metabolismo , Sequência de Aminoácidos , Linhagem Celular , Guanidina/antagonistas & inibidores , Humanos , Estágios do Ciclo de Vida , Chaperonas Moleculares/efeitos dos fármacos , Norovirus/efeitos dos fármacos , Norovirus/crescimento & desenvolvimento , Nucleosídeo-Trifosfatase/genética , Nucleosídeo-Trifosfatase/metabolismo , Ligação Proteica , Dobramento de Proteína , RNA Helicases/efeitos dos fármacos , RNA Viral/química , RNA Viral/efeitos dos fármacos , RNA Viral/genética , RNA Viral/metabolismo , Replicon/efeitos dos fármacos , Alinhamento de Sequência , Análise de Sequência , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/efeitos dos fármacos , Proteínas não Estruturais Virais/genética , Replicação Viral/efeitos dos fármacos , Replicação Viral/fisiologiaRESUMO
Translation initiation is a critical early step in the replication cycle of the positive-sense, single-stranded RNA genome of noroviruses, a major cause of gastroenteritis in humans. Norovirus RNA, which has neither a 5´ m7G cap nor an internal ribosome entry site (IRES), adopts an unusual mechanism to initiate protein synthesis that relies on interactions between the VPg protein covalently attached to the 5´-end of the viral RNA and eukaryotic initiation factors (eIFs) in the host cell. For murine norovirus (MNV) we previously showed that VPg binds to the middle fragment of eIF4G (4GM; residues 652-1132). Here we have used pull-down assays, fluorescence anisotropy, and isothermal titration calorimetry (ITC) to demonstrate that a stretch of ~20 amino acids at the C terminus of MNV VPg mediates direct and specific binding to the HEAT-1 domain within the 4GM fragment of eIF4G. Our analysis further reveals that the MNV C terminus binds to eIF4G HEAT-1 via a motif that is conserved in all known noroviruses. Fine mutagenic mapping suggests that the MNV VPg C terminus may interact with eIF4G in a helical conformation. NMR spectroscopy was used to define the VPg binding site on eIF4G HEAT-1, which was confirmed by mutagenesis and binding assays. We have found that this site is non-overlapping with the binding site for eIF4A on eIF4G HEAT-1 by demonstrating that norovirus VPg can form ternary VPg-eIF4G-eIF4A complexes. The functional significance of the VPg-eIF4G interaction was shown by the ability of fusion proteins containing the C-terminal peptide of MNV VPg to inhibit in vitro translation of norovirus RNA but not cap- or IRES-dependent translation. These observations define important structural details of a functional interaction between norovirus VPg and eIF4G and reveal a binding interface that might be exploited as a target for antiviral therapy.
Assuntos
Fator de Iniciação Eucariótico 4G/metabolismo , Norovirus/fisiologia , Iniciação Traducional da Cadeia Peptídica/fisiologia , Proteínas Virais/metabolismo , Motivos de Aminoácidos , Animais , Calorimetria , Linhagem Celular , Cromatografia em Gel , Imunoprecipitação , Espectroscopia de Ressonância Magnética , Camundongos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Estrutura Secundária de ProteínaRESUMO
Vesivirus 2117 is an adventitious agent that, in 2009, was identified as a contaminant of Chinese hamster ovary cells propagated in bioreactors at a pharmaceutical manufacturing plant belonging to Genzyme. The consequent interruption in supply of Fabrazyme and Cerezyme (drugs used to treat Fabry and Gaucher diseases, respectively) caused significant economic losses. Vesivirus 2117 is a member of the Caliciviridae, a family of small icosahedral viruses encoding a positive-sense RNA genome. We have used cryo-electron microscopy and three-dimensional image reconstruction to calculate a structure of vesivirus 2117 virus-like particles as well as feline calicivirus and a chimeric sapovirus. We present a structural comparison of several members of the Caliciviridae, showing that the distal P domain of vesivirus 2117 is morphologically distinct from that seen in other known vesivirus structures. Furthermore, at intermediate resolutions, we found a high level of structural similarity between vesivirus 2117 and Caliciviridae from other genera: sapovirus and rabbit hemorrhagic disease virus. Phylogenetic analysis confirms vesivirus 2117 as a vesivirus closely related to canine vesiviruses. We postulate that morphological differences in virion structure seen between vesivirus clades may reflect differences in receptor usage.
Assuntos
Capsídeo/ultraestrutura , Lagovirus/ultraestrutura , Sapovirus/ultraestrutura , Vesivirus/ultraestrutura , Animais , Cricetulus , Microscopia Crioeletrônica , Imageamento Tridimensional , Filogenia , RNA Viral/genética , Análise de Sequência de DNA , Vesivirus/genéticaRESUMO
Until recently, molecular studies on human norovirus (HuNoV), a major causative agent of gastroenteritis, have been hampered by the lack of an efficient cell culture system. Murine norovirus-1 (MNV-1) has served as a surrogate model system for norovirus research, due to the availability of robust cell culture systems and reverse genetics. To identify amino acids involved in RNA synthesis by the viral RNA-dependent RNA polymerase (NS7), we constructed NS7 mutants in which basic amino acids surrounding the catalytic site were substituted with alanine. Electrophoretic mobility shift assay revealed that these residues are important for RNA binding, particularly R396. Furthermore, in vitro RNA synthesis and reverse genetics were used to identify conserved amino acids essential for RNA synthesis and viral replication. These results provide additional functional insights into highly conserved amino acids in NS7 and provide potential methods of rational attenuation of norovirus replication.
Assuntos
Aminoácidos/genética , Aminoácidos/metabolismo , Norovirus/enzimologia , RNA Viral/metabolismo , RNA Polimerase Dependente de RNA/genética , RNA Polimerase Dependente de RNA/metabolismo , Replicação Viral , Substituição de Aminoácidos , Animais , Sítios de Ligação , Linhagem Celular , Cricetinae , Análise Mutacional de DNA , Ensaio de Desvio de Mobilidade Eletroforética , Mutagênese Sítio-Dirigida , Ligação Proteica , Genética ReversaRESUMO
UNLABELLED: In response to stress such as virus infection, cells can stall translation by storing mRNAs away in cellular compartments called stress granules (SGs). This defense mechanism favors cell survival by limiting the use of energy and nutrients until the stress is resolved. In some cases it may also block viral propagation as viruses are dependent on the host cell resources to produce viral proteins. Human norovirus is a member of the Caliciviridae family responsible for gastroenteritis outbreaks worldwide. Previous studies on caliciviruses have identified mechanisms by which they can usurp the host translational machinery, using the viral protein genome-linked VPg, or regulate host protein synthesis through the mitogen-activated protein kinase (MAPK) pathway. Here, we examined the effect of feline calicivirus (FCV) infection on SG accumulation. We show that FCV infection impairs the assembly of SGs despite an increased phosphorylation of eukaryotic initiation factor eIF2α, a hallmark of stress pathway activation. Furthermore, SGs did not accumulate in FCV-infected cells that were stressed with arsenite or hydrogen peroxide. FCV infection resulted in the cleavage of the SG-nucleating protein Ras-GTPase activating SH3 domain-binding protein (G3BP1), which is mediated by the viral 3C-like proteinase NS6(Pro) Using mutational analysis, we identified the FCV-induced cleavage site within G3BP1, which differs from the poliovirus 3C proteinase cleavage site previously identified. Finally, we showed that NS6(Pro)-mediated G3BP1 cleavage impairs SG assembly. In contrast, murine norovirus (MNV) infection did not impact arsenite-induced SG assembly or G3BP1 integrity, suggesting that related caliciviruses have distinct effects on the stress response pathway. IMPORTANCE: Human noroviruses are a major cause of viral gastroenteritis, and it is important to understand how they interact with the infected host cell. Feline calicivirus (FCV) and murine norovirus (MNV) are used as models to understand norovirus biology. Recent studies have suggested that the assembly of stress granules is central in orchestrating stress and antiviral responses to restrict viral replication. Overall, our study provides the first insight on how caliciviruses impair stress granule assembly by targeting the nucleating factor G3BP1 via the viral proteinase NS6(Pro) This work provides new insights into host-pathogen interactions that regulate stress pathways during FCV infection.
Assuntos
Infecções por Caliciviridae/virologia , Calicivirus Felino/patogenicidade , Proteínas de Transporte/metabolismo , Grânulos Citoplasmáticos/metabolismo , Interações Hospedeiro-Patógeno , Replicação Viral , Proteases Virais 3C , Animais , Infecções por Caliciviridae/metabolismo , Infecções por Caliciviridae/patologia , Proteínas de Transporte/genética , Gatos , Cisteína Endopeptidases/metabolismo , Grânulos Citoplasmáticos/virologia , DNA Helicases , Fator de Iniciação 2 em Eucariotos/metabolismo , Células HeLa , Humanos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosforilação , Proteínas de Ligação a Poli-ADP-Ribose , RNA Helicases , Proteínas com Motivo de Reconhecimento de RNA , Proteínas Virais/metabolismoRESUMO
The replication enzyme of RNA viruses must preferentially recognize their RNAs in an environment that contains an abundance of cellular RNAs. The factors responsible for specific RNA recognition are not well understood, in part because viral RNA synthesis takes place within enzyme complexes associated with modified cellular membrane compartments. Recombinant RNA-dependent RNA polymerases (RdRps) from the human norovirus and the murine norovirus (MNV) were found to preferentially recognize RNA segments that contain the promoter and a short template sequence for subgenomic RNA synthesis. Both the promoter and template sequence contribute to stable RdRp binding, accurate initiation of the subgenomic RNAs and efficient RNA synthesis. Using a method that combines RNA crosslinking and mass spectrometry, residues near the template channel of the MNV RdRp were found to contact the hairpin RNA motif. Mutations in the hairpin contact site in the MNV RdRp reduced MNV replication and virus production in cells. This work demonstrates that the specific recognition of the norovirus subgenomic promoter is through binding by the viral RdRp.
Assuntos
Norovirus/enzimologia , Norovirus/genética , Regiões Promotoras Genéticas , RNA Viral/biossíntese , RNA Polimerase Dependente de RNA/química , RNA Polimerase Dependente de RNA/metabolismo , Animais , Sítios de Ligação , Linhagem Celular , Genoma Viral , Camundongos , Mutação , Ligação Proteica , Elementos Reguladores de Transcrição , Iniciação da Transcrição GenéticaRESUMO
UNLABELLED: Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most economically important viruses affecting the swine industry worldwide. Our previous research showed that PRRSV downregulates the expression of heme oxygenase-1 (HO-1), a pivotal cytoprotective enzyme, postinfection and that overexpression of HO-1 inhibits PRRSV replication. MicroRNAs regulate gene expression at the posttranscriptional level and have recently been demonstrated to play vital roles in pathogen-host interactions. The present study sought to determine whether microRNAs modulate HO-1 expression and, by doing so, regulate PRRSV replication. Using bioinformatic prediction and experimental verification, we demonstrate that HO-1 expression is regulated by miR-24-3p. A direct interaction between miR-24-3p and HO-1 mRNA was confirmed using a number of approaches. Overexpression of miR-24-3p significantly decreased HO-1 mRNA and protein levels. PRRSV infection induced miR-24-3p expression to facilitate viral replication. The suppressive effect of HO-1 induction by protoporphyrin IX cobalt chloride (CoPP; a classical inducer of HO-1 expression) on PRRSV replication in MARC-145 cells and primary porcine alveolar macrophages could also be reversed by overexpression of miR-24-3p. Collectively, these results suggested that miR-24-3p promotes PRRSV replication through suppression of HO-1 expression, which not only provides new insights into virus-host interactions during PRRSV infection but also suggests potential new antiviral strategies against PRRSV infection. IMPORTANCE: MicroRNAs (miRNAs) play vital roles in viral infections by regulating the expression of viral or host genes at the posttranscriptional level. Heme oxygenase-1 (HO-1), a pivotal cytoprotective enzyme, has antiviral activity for a number of viruses, such as Ebola virus, hepatitis C virus, human immunodeficiency virus, and our focus, PRRSV, which causes great economic losses each year in the swine industry worldwide. Here, we show that PRRSV infection induces host miRNA miR-24-3p expression and that miR-24-3p regulates HO-1 expression through both mRNA degradation and translation repression. Suppression of HO-1 expression by miR-24-3p facilitates PRRSV replication. This work lends credibility to the hypothesis that an arterivirus can manipulate cellular miRNAs to enhance virus replication by regulating antiviral responses following viral infection. Therefore, our findings provide new insights into the pathogenesis of PRRSV.
Assuntos
Regulação da Expressão Gênica/genética , Heme Oxigenase-1/metabolismo , Interações Hospedeiro-Patógeno/genética , Vírus da Síndrome Respiratória e Reprodutiva Suína/fisiologia , Replicação Viral/fisiologia , Análise de Variância , Animais , Western Blotting , Linhagem Celular , Chlorocebus aethiops , Biologia Computacional , Primers do DNA/genética , Citometria de Fluxo , Imunoprecipitação , Luciferases , Macaca mulatta , Vírus da Síndrome Respiratória e Reprodutiva Suína/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Sus scrofa , Replicação Viral/genéticaRESUMO
Norovirus infection in humans typically results in acute gastroenteritis but may also occur in many animal species. Noroviruses are recognized as one of the most common causes of acute gastroenteritis in the world, being responsible for almost 20% of all cases. Despite their prevalence and impact, our knowledge of the norovirus life cycle and the pathological processes associated with norovirus-induced disease is limited. Whilst infection of the intestine is the norm, extraintestinal spread and associated pathologies have also been described. In addition, long-term chronic infections are now recognized as a significant cause of morbidity and mortality in the immunocompromised. This review aims to summarize the current state of knowledge with respect to norovirus pathology and the underlying mechanisms that have been characterized to date.