RESUMO
RNA viruses are among the most prevalent pathogens and are a major burden on society. Although RNA viruses have been studied extensively, little is known about the processes that occur during the first several hours of infection because of a lack of sensitive assays. Here we develop a single-molecule imaging assay, virus infection real-time imaging (VIRIM), to study translation and replication of individual RNA viruses in live cells. VIRIM uncovered a striking heterogeneity in replication dynamics between cells and revealed extensive coordination between translation and replication of single viral RNAs. Furthermore, using VIRIM, we identify the replication step of the incoming viral RNA as a major bottleneck of successful infection and identify host genes that are responsible for inhibition of early virus replication. Single-molecule imaging of virus infection is a powerful tool to study virus replication and virus-host interactions that may be broadly applicable to RNA viruses.
Assuntos
Biossíntese de Proteínas , Vírus de RNA/fisiologia , Replicação Viral/fisiologia , Linhagem Celular Tumoral , Sobrevivência Celular , Células HEK293 , Interações Hospedeiro-Patógeno , Humanos , Interferons/metabolismo , Transporte de RNA , RNA Viral/genética , Reprodutibilidade dos Testes , Imagem Individual de Molécula , Fatores de TempoRESUMO
Coronavirus spike proteins mediate receptor binding and membrane fusion, making them prime targets for neutralizing antibodies. In the cases of severe acute respiratory syndrome coronavirus, severe acute respiratory syndrome coronavirus 2 and Middle East respiratory syndrome coronavirus, spike proteins transition freely between open and closed conformations to balance host cell attachment and immune evasion1-5. Spike opening exposes domain S1B, allowing it to bind to proteinaceous receptors6,7, and is also thought to enable protein refolding during membrane fusion4,5. However, with a single exception, the pre-fusion spike proteins of all other coronaviruses studied so far have been observed exclusively in the closed state. This raises the possibility of regulation, with spike proteins more commonly transitioning to open states in response to specific cues, rather than spontaneously. Here, using cryogenic electron microscopy and molecular dynamics simulations, we show that the spike protein of the common cold human coronavirus HKU1 undergoes local and long-range conformational changes after binding a sialoglycan-based primary receptor to domain S1A. This binding triggers the transition of S1B domains to the open state through allosteric interdomain crosstalk. Our findings provide detailed insight into coronavirus attachment, with possibilities of dual receptor usage and priming of entry as a means of immune escape.
Assuntos
Betacoronavirus , Polissacarídeos , Ácidos Siálicos , Glicoproteína da Espícula de Coronavírus , Humanos , Regulação Alostérica , Betacoronavirus/química , Betacoronavirus/ultraestrutura , Resfriado Comum/virologia , Microscopia Crioeletrônica , Simulação de Dinâmica Molecular , Polissacarídeos/química , Polissacarídeos/metabolismo , Ligação Proteica , Conformação Proteica , Ácidos Siálicos/química , Ácidos Siálicos/metabolismo , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/metabolismo , Glicoproteína da Espícula de Coronavírus/ultraestrutura , Evasão da Resposta ImuneRESUMO
The discovery that extracellular vesicles (EVs) serve as carriers of virus particles calls for a reevaluation of the release strategies of non-enveloped viruses. Little is currently known about the molecular mechanisms that determine the release and composition of EVs produced by virus-infected cells, as well as conservation of these mechanisms among viruses. We previously described an important role for the Leader protein of the picornavirus encephalomyocarditis virus (EMCV) in the induction of virus-carrying EV subsets with distinct molecular and physical properties. EMCV L acts as a 'viral security protein' by suppressing host antiviral stress and type-I interferon (IFN) responses. Here, we tested the ability of functionally related picornavirus proteins of Theilers murine encephalitis virus (TMEV L), Saffold virus (SAFV L), and coxsackievirus B3 (CVB3 2Apro), to rescue EV and EV-enclosed virus release when introduced in Leader-deficient EMCV. We show that all viral security proteins tested were able to promote virus packaging in EVs, but that only the expression of EMCV L and CVB3 2Apro increased overall EV production. We provide evidence that one of the main antiviral pathways counteracted by this class of picornaviral proteins, i.e. the inhibition of PKR-mediated stress responses, affected EV and EV-enclosed virus release during infection. Moreover, we show that the enhanced capacity of the viral proteins EMCV L and CVB3 2Apro to promote EV-enclosed virus release is linked to their ability to simultaneously promote the activation of the stress kinase P38 MAPK. Taken together, we demonstrate that cellular stress pathways involving the kinases PKR and P38 are modulated by the activity of non-structural viral proteins to increase the release EV-enclosed viruses during picornavirus infections. These data shed new light on the molecular regulation of EV production in response to virus infection.
Assuntos
Vesículas Extracelulares , Picornaviridae , Proteínas Virais , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/virologia , Humanos , Picornaviridae/metabolismo , Picornaviridae/fisiologia , Proteínas Virais/metabolismo , Proteínas Virais/genética , Animais , eIF-2 Quinase/metabolismo , Liberação de Vírus/fisiologia , Camundongos , Theilovirus/metabolismo , Infecções por Cardiovirus/virologia , Infecções por Cardiovirus/metabolismo , Vírus da Encefalomiocardite/metabolismo , Vírus da Encefalomiocardite/fisiologiaRESUMO
Viruses actively reprogram the metabolism of the host to ensure the availability of sufficient building blocks for virus replication and spreading. However, relatively little is known about how picornaviruses-a large family of small, non-enveloped positive-strand RNA viruses-modulate cellular metabolism for their own benefit. Here, we studied the modulation of host metabolism by coxsackievirus B3 (CVB3), a member of the enterovirus genus, and encephalomyocarditis virus (EMCV), a member of the cardiovirus genus, using steady-state as well as 13C-glucose tracing metabolomics. We demonstrate that both CVB3 and EMCV increase the levels of pyrimidine and purine metabolites and provide evidence that this increase is mediated through degradation of nucleic acids and nucleotide recycling, rather than upregulation of de novo synthesis. Finally, by integrating our metabolomics data with a previously acquired phosphoproteomics dataset of CVB3-infected cells, we identify alterations in phosphorylation status of key enzymes involved in nucleotide metabolism, providing insight into the regulation of nucleotide metabolism during infection.
Assuntos
Cardiovirus , Infecções por Enterovirus , Enterovirus , Picornaviridae , Humanos , Enterovirus/fisiologia , Vírus da Encefalomiocardite/fisiologia , Replicação Viral , Enterovirus Humano B/fisiologia , Células HeLaRESUMO
Picornaviridae represent a large family of single-stranded positive RNA viruses of which different members can infect both humans and animals. These include the enteroviruses (e.g., poliovirus, coxsackievirus, and rhinoviruses) as well as the cardioviruses (e.g., encephalomyocarditis virus). Picornaviruses have evolved to interact with, use, and/or evade cellular host systems to create the optimal environment for replication and spreading. It is known that viruses modify kinase activity during infection, but a proteome-wide overview of the (de)regulation of cellular kinases during picornavirus infection is lacking. To study the kinase activity landscape during picornavirus infection, we here applied dedicated targeted mass spectrometry-based assays covering â¼40% of the human kinome. Our data show that upon infection, kinases of the MAPK pathways become activated (e.g., ERK1/2, RSK1/2, JNK1/2/3, and p38), while kinases involved in regulating the cell cycle (e.g., CDK1/2, GWL, and DYRK3) become inactivated. Additionally, we observed the activation of CHK2, an important kinase involved in the DNA damage response. Using pharmacological kinase inhibitors, we demonstrate that several of these activated kinases are essential for the replication of encephalomyocarditis virus. Altogether, the data provide a quantitative understanding of the regulation of kinome activity induced by picornavirus infection, providing a resource important for developing novel antiviral therapeutic interventions.
Assuntos
Infecções por Picornaviridae , Picornaviridae , Humanos , Picornaviridae/fisiologia , Picornaviridae/enzimologia , Infecções por Picornaviridae/virologia , Infecções por Picornaviridae/metabolismo , Células HeLa , Proteoma/metabolismo , Proteínas Quinases/metabolismo , Replicação Viral , FosforilaçãoRESUMO
To become established upon zoonotic transfer, influenza A viruses (IAV) need to switch binding from "avian-type" α2-3-linked sialic acid receptors (2-3Sia) to "human-type" Siaα2-6-linked sialic acid receptors (2-6Sia). For the 1968 H3N2 pandemic virus, this was accomplished by two canonical amino acid substitutions in its hemagglutinin (HA) although a full specificity shift had not occurred. The receptor repertoire on epithelial cells is highly diverse and simultaneous interaction of a virus particle with a range of low- to very low-affinity receptors results in tight heteromultivalent binding. How this range of affinities determines binding selectivity and virus motility remains largely unknown as the analysis of low-affinity monovalent HA-receptor interactions is technically challenging. Here, a biolayer interferometry assay enabled a comprehensive analysis of receptor-binding kinetics evolution upon host-switching. Virus-binding kinetics of H3N2 virus isolates slowly evolved from 1968 to 1979 from mixed 2-3/2-6Sia specificity to high 2-6Sia specificity, surprisingly followed by a decline in selectivity after 1992. By using genetically tuned HEK293 cells, presenting either a simplified 2-3Sia- or 2-6Sia-specific receptor repertoire, receptor-specific binding was shown to correlate strongly with receptor-specific entry. In conclusion, the slow and continuous evolution of entry and receptor-binding specificity of seasonal H3N2 viruses contrasts with the paradigm that human IAVs need to rapidly acquire and maintain a high specificity for 2-6Sia. Analysis of the kinetic parameters of receptor binding provides a basis for understanding virus-binding specificity, motility, and HA/neuraminidase balance at the molecular level.
Assuntos
Vírus da Influenza A , Influenza Humana , Humanos , Vírus da Influenza A/metabolismo , Vírus da Influenza A Subtipo H3N2/genética , Sítios de Ligação , Células HEK293 , Pandemias , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Receptores Virais/metabolismoRESUMO
The ongoing COVID-19 pandemic has had great societal and health consequences. Despite the availability of vaccines, infection rates remain high due to immune evasive Omicron sublineages. Broad-spectrum antivirals are needed to safeguard against emerging variants and future pandemics. We used messenger RNA (mRNA) display under a reprogrammed genetic code to find a spike-targeting macrocyclic peptide that inhibits SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) Wuhan strain infection and pseudoviruses containing spike proteins of SARS-CoV-2 variants or related sarbecoviruses. Structural and bioinformatic analyses reveal a conserved binding pocket between the receptor-binding domain, N-terminal domain, and S2 region, distal to the angiotensin-converting enzyme 2 receptor-interaction site. Our data reveal a hitherto unexplored site of vulnerability in sarbecoviruses that peptides and potentially other drug-like molecules can target.
Assuntos
COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Pandemias/prevenção & controle , Peptídeos/farmacologiaRESUMO
Many viruses initiate infection by binding to sialoglycan receptors at the cell surface. Binding to such receptors comes at a cost, however, as the sheer abundance of sialoglycans e.g. in mucus, may immobilize virions to non-functional decoy receptors. As a solution, sialoglycan-binding as well as sialoglycan-cleavage activities are often present in these viruses, which for paramyxoviruses are combined in the hemagglutinin-neuraminidase (HN) protein. The dynamic interactions of sialoglycan-binding paramyxoviruses with their receptors are thought to be key determinants of species tropism, replication and pathogenesis. Here we used biolayer interferometry to perform kinetic analyses of receptor interactions of animal and human paramyxoviruses (Newcastle disease virus, Sendai virus, and human parainfluenza virus 3). We show that these viruses display strikingly different receptor interaction dynamics, which correlated with their receptor-binding and -cleavage activities and the presence of a second sialic acid binding site. Virion binding was followed by sialidase-driven release, during which virions cleaved sialoglycans until a virus-specific density was reached, which was largely independent of virion concentration. Sialidase-driven virion release was furthermore shown to be a cooperative process and to be affected by pH. We propose that paramyxoviruses display sialidase-driven virion motility on a receptor-coated surface, until a threshold receptor density is reached at which virions start to dissociate. Similar motility has previously been observed for influenza viruses and is likely to also apply to sialoglycan-interacting embecoviruses. Analysis of the balance between receptor-binding and -cleavage increases our understanding of host species tropism determinants and zoonotic potential of viruses.
Assuntos
Neuraminidase , Proteínas Virais , Animais , Humanos , Neuraminidase/metabolismo , Cinética , Ligação Proteica , Proteínas Virais/metabolismo , Vírion/metabolismo , Proteína HN/genética , Proteína HN/metabolismoRESUMO
The nucleocapsid protein N of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enwraps and condenses the viral genome for packaging but is also an antagonist of the innate antiviral defense. It suppresses the integrated stress response (ISR), purportedly by interacting with stress granule (SG) assembly factors G3BP1 and 2, and inhibits type I interferon responses. To elucidate its mode of action, we systematically deleted and over-expressed distinct regions and domains. We show that N via domain N2b blocks PKR-mediated ISR activation, as measured by suppression of ISR-induced translational arrest and SG formation. N2b mutations that prevent dsRNA binding abrogate these activities also when introduced in the intact N protein. Substitutions reported to block post-translation modifications of N or its interaction with G3BP1/2 did not have a detectable additive effect. In an encephalomyocarditis virus-based infection model, N2b - but not a derivative defective in RNA binding-prevented PKR activation, inhibited ß-interferon expression and promoted virus replication. Apparently, SARS-CoV-2 N inhibits innate immunity by sequestering dsRNA to prevent activation of PKR and RIG-I-like receptors. Similar observations were made for the N protein of human coronavirus 229E, suggesting that this may be a general trait conserved among members of other orthocoronavirus (sub)genera.
Assuntos
COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/genética , DNA Helicases , Proteínas de Ligação a Poli-ADP-Ribose , RNA Helicases , Proteínas com Motivo de Reconhecimento de RNA/genética , Motivos de Ligação ao RNA , Vírus da EncefalomiocarditeRESUMO
Many RNA viruses remodel intracellular membranes to generate specialized sites for RNA replication. How membranes are remodeled and what properties make them conducive for replication are unknown. Here we show how RNA viruses can manipulate multiple components of the cellular secretory pathway to generate organelles specialized for replication that are distinct in protein and lipid composition from the host cell. Specific viral proteins modulate effector recruitment by Arf1 GTPase and its guanine nucleotide exchange factor GBF1, promoting preferential recruitment of phosphatidylinositol-4-kinase IIIbeta (PI4KIIIbeta) to membranes over coat proteins, yielding uncoated phosphatidylinositol-4-phosphate (PI4P) lipid-enriched organelles. The PI4P-rich lipid microenvironment is essential for both enteroviral and flaviviral RNA replication; PI4KIIIbeta inhibition interferes with this process; and enteroviral RNA polymerases specifically bind PI4P. These findings reveal how RNA viruses can selectively exploit specific elements of the host to form specialized organelles where cellular phosphoinositide lipids are key to regulating viral RNA replication.
Assuntos
Enterovirus/metabolismo , Flavivirus/metabolismo , RNA Viral/metabolismo , Via Secretória , Replicação Viral , Retículo Endoplasmático/metabolismo , Células HeLa , Humanos , Fosfatos de Fosfatidilinositol/metabolismoRESUMO
Self-amplifying mRNA (SAM) vaccines can be rapidly deployed in the event of disease outbreaks. A legitimate safety concern is the potential for recombination between alphavirus-based SAM vaccines and circulating viruses. This theoretical risk needs to be assessed in the regulatory process for SAM vaccine approval. Herein, we undertake extensive in vitro and in vivo assessments to explore recombination between SAM vaccine and a wide selection of alphaviruses and a coronavirus. SAM vaccines were found to effectively limit alphavirus co-infection through superinfection exclusion, although some co-replication was still possible. Using sensitive cell-based assays, replication-competent alphavirus chimeras were generated in vitro as a result of rare, but reproducible, RNA recombination events. The chimeras displayed no increased fitness in cell culture. Viable alphavirus chimeras were not detected in vivo in C57BL/6J, Rag1-/- and Ifnar-/- mice, in which high levels of SAM vaccine and alphavirus co-replicated in the same tissue. Furthermore, recombination between a SAM-spike vaccine and a swine coronavirus was not observed. In conclusion we state that although the ability of SAM vaccines to recombine with alphaviruses might be viewed as an environmental safety concern, several key factors substantially mitigate against in vivo emergence of chimeric viruses from SAM vaccine recipients.
Assuntos
Alphavirus , Recombinação Genética , Vacinas de mRNA , Animais , Camundongos , Alphavirus/genética , Alphavirus/imunologia , Camundongos Endogâmicos C57BL , Humanos , Receptor de Interferon alfa e beta/genética , Replicação Viral , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/imunologia , Vacinas Sintéticas/imunologia , Vacinas Sintéticas/efeitos adversos , Camundongos Knockout , SARS-CoV-2/genética , SARS-CoV-2/imunologia , Vacinas Virais/imunologia , Vacinas Virais/genética , Vacinas Virais/efeitos adversosRESUMO
IMPORTANCE: Influenza A viruses (IAVs) contain hemagglutinin (HA) proteins involved in sialoglycan receptor binding and neuraminidase (NA) proteins that cleave sialic acids. While the importance of the NA protein in virion egress is well established, its role in virus entry remains to be fully elucidated. NA activity is needed for the release of virions from mucus decoy receptors, but conflicting results have been reported on the importance of NA activity in virus entry in the absence of decoy receptors. We now show that inhibition of NA activity affects virus entry depending on the receptor-binding properties of HA and the receptor repertoire present on cells. Inhibition of entry by the presence of mucus correlated with the importance of NA activity for virus entry, with the strongest inhibition being observed when mucus and OsC were combined. These results shed light on the importance in virus entry of the NA protein, an important antiviral drug target.
Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza , Vírus da Influenza A , Neuraminidase , Receptores Virais , Proteínas Virais , Internalização do Vírus , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Vírus da Influenza A/enzimologia , Vírus da Influenza A/metabolismo , Influenza Humana/enzimologia , Influenza Humana/metabolismo , Neuraminidase/antagonistas & inibidores , Neuraminidase/metabolismo , Ligação Proteica , Receptores Virais/metabolismo , Especificidade por Substrato , Proteínas Virais/antagonistas & inibidores , Proteínas Virais/metabolismo , Linhagem Celular , MucoRESUMO
Plus-strand RNA viruses are the largest group of viruses. Many are human pathogens that inflict a socio-economic burden. Interestingly, plus-strand RNA viruses share remarkable similarities in their replication. A hallmark of plus-strand RNA viruses is the remodeling of intracellular membranes to establish replication organelles (so-called "replication factories"), which provide a protected environment for the replicase complex, consisting of the viral genome and proteins necessary for viral RNA synthesis. In the current study, we investigate pan-viral similarities and virus-specific differences in the life cycle of this highly relevant group of viruses. We first measured the kinetics of viral RNA, viral protein, and infectious virus particle production of hepatitis C virus (HCV), dengue virus (DENV), and coxsackievirus B3 (CVB3) in the immuno-compromised Huh7 cell line and thus without perturbations by an intrinsic immune response. Based on these measurements, we developed a detailed mathematical model of the replication of HCV, DENV, and CVB3 and showed that only small virus-specific changes in the model were necessary to describe the in vitro dynamics of the different viruses. Our model correctly predicted virus-specific mechanisms such as host cell translation shut off and different kinetics of replication organelles. Further, our model suggests that the ability to suppress or shut down host cell mRNA translation may be a key factor for in vitro replication efficiency, which may determine acute self-limited or chronic infection. We further analyzed potential broad-spectrum antiviral treatment options in silico and found that targeting viral RNA translation, such as polyprotein cleavage and viral RNA synthesis, may be the most promising drug targets for all plus-strand RNA viruses. Moreover, we found that targeting only the formation of replicase complexes did not stop the in vitro viral replication early in infection, while inhibiting intracellular trafficking processes may even lead to amplified viral growth.
Assuntos
Hepatite C , Vírus de RNA , Humanos , Antivirais/farmacologia , Replicação Viral/fisiologia , RNA Viral/genética , Modelos TeóricosRESUMO
There is a great need for antiviral drugs to treat enterovirus (EV) and rhinovirus (RV) infections, which can be severe and occasionally life-threatening. The conserved nonstructural protein 2C, which is an AAA+ ATPase, is a promising target for drug development. Here, we present a structure-activity relationship study of a previously identified compound that targets the 2C protein of EV-A71 and several EV-B species members, but not poliovirus (PV) (EV-C species). This compound is structurally related to the Food and Drug Administration (FDA)-approved drug fluoxetine-which also targets 2C-but has favorable chemical properties. We identified several compounds with increased antiviral potency and broadened activity. Four compounds showed broad-spectrum EV and RV activity and inhibited contemporary strains of emerging EVs of public health concern, including EV-A71, coxsackievirus (CV)-A24v, and EV-D68. Importantly, unlike (S)-fluoxetine, these compounds are no longer neuroactive. By raising resistant EV-A71, CV-B3, and EV-D68 variants against one of these inhibitors, we identified novel 2C resistance mutations. Reverse engineering of these mutations revealed a conserved mechanism of resistance development. Resistant viruses first acquired a mutation in, or adjacent to, the α2 helix of 2C. This mutation disrupted compound binding and provided drug resistance, but this was at the cost of viral fitness. Additional mutations at distantly localized 2C residues were then acquired to increase resistance and/or to compensate for the loss of fitness. Using computational methods to identify solvent accessible tunnels near the α2 helix in the EV-A71 and PV 2C crystal structures, a conserved binding pocket of the inhibitors is proposed.
Assuntos
Antivirais/farmacologia , Proteínas de Transporte/efeitos dos fármacos , Enterovirus/efeitos dos fármacos , Proteínas não Estruturais Virais/efeitos dos fármacos , Antígenos Virais , Proteínas de Transporte/metabolismo , Descoberta de Drogas/métodos , Enterovirus/patogenicidade , Infecções por Enterovirus/virologia , Fluoxetina/farmacologia , Células HeLa , Humanos , Relação Estrutura-Atividade , Proteínas não Estruturais Virais/metabolismo , Replicação ViralRESUMO
Picornaviruses are a leading cause of human and veterinary infections that result in various diseases, including polio and the common cold. As archetypical non-enveloped viruses, their biology has been extensively studied. Although a range of different cell-surface receptors are bound by different picornaviruses, it is unclear whether common host factors are needed for them to reach the cytoplasm. Using genome-wide haploid genetic screens, here we identify the lipid-modifying enzyme PLA2G16 (refs 8, 9, 10, 11) as a picornavirus host factor that is required for a previously unknown event in the viral life cycle. We find that PLA2G16 functions early during infection, enabling virion-mediated genome delivery into the cytoplasm, but not in any virion-assigned step, such as cell binding, endosomal trafficking or pore formation. To resolve this paradox, we screened for suppressors of the ΔPLA2G16 phenotype and identified a mechanism previously implicated in the clearance of intracellular bacteria. The sensor of this mechanism, galectin-8 (encoded by LGALS8), detects permeated endosomes and marks them for autophagic degradation, whereas PLA2G16 facilitates viral genome translocation and prevents clearance. This study uncovers two competing processes triggered by virus entry: activation of a pore-activated clearance pathway and recruitment of a phospholipase to enable genome release.
Assuntos
Citoplasma/virologia , Genoma Viral , Fatores Celulares Derivados do Hospedeiro/metabolismo , Fosfolipases A2 Independentes de Cálcio/metabolismo , Picornaviridae/genética , Picornaviridae/fisiologia , Proteínas Supressoras de Tumor/metabolismo , Internalização do Vírus , Animais , Autofagia , Transporte Biológico , Linhagem Celular , Citoplasma/genética , Endossomos/metabolismo , Feminino , Galectinas/genética , Galectinas/metabolismo , Fatores Celulares Derivados do Hospedeiro/deficiência , Fatores Celulares Derivados do Hospedeiro/genética , Humanos , Masculino , Camundongos , Mutação , Fenótipo , Fosfolipases A2 Independentes de Cálcio/deficiência , Fosfolipases A2 Independentes de Cálcio/genética , Supressão Genética , Proteínas Supressoras de Tumor/deficiência , Proteínas Supressoras de Tumor/genética , Vírion/genética , Vírion/metabolismo , Replicação ViralRESUMO
Human coronaviruses OC43 and HKU1 are respiratory pathogens of zoonotic origin that have gained worldwide distribution. OC43 apparently emerged from a bovine coronavirus (BCoV) spillover. All three viruses attach to 9-O-acetylated sialoglycans via spike protein S with hemagglutinin-esterase (HE) acting as a receptor-destroying enzyme. In BCoV, an HE lectin domain promotes esterase activity toward clustered substrates. OC43 and HKU1, however, lost HE lectin function as an adaptation to humans. Replaying OC43 evolution, we knocked out BCoV HE lectin function and performed forced evolution-population dynamics analysis. Loss of HE receptor binding selected for second-site mutations in S, decreasing S binding affinity by orders of magnitude. Irreversible HE mutations led to cooperativity in virus swarms with low-affinity S minority variants sustaining propagation of high-affinity majority phenotypes. Salvageable HE mutations induced successive second-site substitutions in both S and HE. Apparently, S and HE are functionally interdependent and coevolve to optimize the balance between attachment and release. This mechanism of glycan-based receptor usage, entailing a concerted, fine-tuned activity of two envelope protein species, is unique among CoVs, but reminiscent of that of influenza A viruses. Apparently, general principles fundamental to virion-sialoglycan interactions prompted convergent evolution of two important groups of human and animal pathogens.
Assuntos
Coronavirus/fisiologia , Hemaglutininas Virais/genética , Glicoproteína da Espícula de Coronavírus/genética , Proteínas Virais de Fusão/genética , Vírion/metabolismo , Animais , Evolução Biológica , Linhagem Celular , Coronavirus/genética , Coronavirus/metabolismo , Infecções por Coronavirus/virologia , Coronavirus Humano OC43/genética , Coronavirus Humano OC43/metabolismo , Coronavirus Humano OC43/fisiologia , Coronavirus Bovino/genética , Coronavirus Bovino/metabolismo , Coronavirus Bovino/fisiologia , Hemaglutininas Virais/química , Hemaglutininas Virais/metabolismo , Humanos , Lectinas/genética , Lectinas/metabolismo , Camundongos , Mutação , Ligação Proteica , Domínios Proteicos , Receptores Virais/metabolismo , Seleção Genética , Ácidos Siálicos/metabolismo , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/metabolismo , Proteínas Virais de Fusão/química , Proteínas Virais de Fusão/metabolismo , Vírion/genética , Ligação Viral , Liberação de VírusRESUMO
Influenza A viruses (IAVs) cause seasonal epidemics and occasional pandemics. Most pandemics occurred upon adaptation of avian IAVs to humans. This adaptation includes a hallmark receptor-binding specificity switch of hemagglutinin (HA) from avian-type α2,3- to human-type α2,6-linked sialic acids. Complementary changes of the receptor-destroying neuraminidase (NA) are considered to restore the precarious, but poorly described, HA-NA-receptor balance required for virus fitness. In comparison to the detailed functional description of adaptive mutations in HA, little is known about the functional consequences of mutations in NA in relation to their effect on the HA-NA balance and host tropism. An understudied feature of NA is the presence of a second sialic acid-binding site (2SBS) in avian IAVs and absence of a 2SBS in human IAVs, which affects NA catalytic activity. Here we demonstrate that mutation of the 2SBS of avian IAV H5N1 disturbs the HA-NA balance. Passaging of a 2SBS-negative H5N1 virus on MDCK cells selected for progeny with a restored HA-NA balance. These viruses obtained mutations in NA that restored a functional 2SBS and/or in HA that reduced binding of avian-type receptors. Importantly, a particular HA mutation also resulted in increased binding of human-type receptors. Phylogenetic analyses of avian IAVs show that also in the field, mutations in the 2SBS precede mutations in HA that reduce binding of avian-type receptors and increase binding of human-type receptors. Thus, 2SBS mutations in NA can drive acquisition of mutations in HA that not only restore the HA-NA balance, but may also confer increased zoonotic potential.
Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Vírus da Influenza A/genética , Mutação , Neuraminidase/genética , Infecções por Orthomyxoviridae/virologia , Ácidos Siálicos/metabolismo , Replicação Viral , Substituição de Aminoácidos , Animais , Sítios de Ligação , Cães , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Vírus da Influenza A/isolamento & purificação , Células Madin Darby de Rim Canino , Neuraminidase/química , Neuraminidase/metabolismo , Infecções por Orthomyxoviridae/genética , Infecções por Orthomyxoviridae/patologia , Ligação ProteicaRESUMO
The type I interferon response is an important innate antiviral pathway. Recognition of viral RNA by RIG-I-like receptors (RLRs) activates a signaling cascade that leads to type I interferon (IFN-α/ß) gene transcription. Multiple proteins in this signaling pathway (e.g. RIG-I, MDA5, MAVS, TBK1, IRF3) are regulated by (de)ubiquitination events. Most viruses have evolved mechanisms to counter this antiviral response. The leader protease (Lpro) of foot-and-mouth-disease virus (FMDV) has been recognized to reduce IFN-α/ß gene transcription; however, the exact mechanism is unknown. The proteolytic activity of Lpro is vital for releasing itself from the viral polyprotein and for cleaving and degrading specific host cell proteins, such as eIF4G and NF-κB. In addition, Lpro has been demonstrated to have deubiquitination/deISGylation activity. Lpro's deubiquitination/deISGylation activity and the cleavage/degradation of signaling proteins have both been postulated to be important for reduced IFN-α/ß gene transcription. Here, we demonstrate that TBK1, the kinase that phosphorylates and activates the transcription factor IRF3, is cleaved by Lpro in FMDV-infected cells as well as in cells infected with a recombinant EMCV expressing Lpro. In vitro cleavage experiments revealed that Lpro cleaves TBK1 at residues 692-694. We also observed cleavage of MAVS in HeLa cells infected with EMCV-Lpro, but only observed decreasing levels of MAVS in FMDV-infected porcine LFPK αVß6 cells. We set out to dissect Lpro's ability to cleave RLR signaling proteins from its deubiquitination/deISGylation activity to determine their relative contributions to the reduction of IFN-α/ß gene transcription. The introduction of specific mutations, of which several were based on the recently published structure of Lpro in complex with ISG15, allowed us to identify specific amino acid substitutions that separate the different proteolytic activities of Lpro. Characterization of the effects of these mutations revealed that Lpro's ability to cleave RLR signaling proteins but not its deubiquitination/deISGylation activity correlates with the reduced IFN-ß gene transcription.
Assuntos
Proteína DEAD-box 58/metabolismo , Endopeptidases/metabolismo , Vírus da Febre Aftosa/metabolismo , Interferon Tipo I/biossíntese , Animais , Linhagem Celular , Endopeptidases/genética , Febre Aftosa/imunologia , Febre Aftosa/metabolismo , Vírus da Febre Aftosa/imunologia , Humanos , ProteóliseRESUMO
The lipid kinase PI4KB, which generates phosphatidylinositol 4-phosphate (PI4P), is a key enzyme in regulating membrane transport and is also hijacked by multiple picornaviruses to mediate viral replication. PI4KB can interact with multiple protein binding partners, which are differentially manipulated by picornaviruses to facilitate replication. The protein c10orf76 is a PI4KB-associated protein that increases PI4P levels at the Golgi and is essential for the viral replication of specific enteroviruses. We used hydrogen-deuterium exchange mass spectrometry to characterize the c10orf76-PI4KB complex and reveal that binding is mediated by the kinase linker of PI4KB, with formation of the heterodimeric complex modulated by PKA-dependent phosphorylation. Complex-disrupting mutations demonstrate that PI4KB is required for membrane recruitment of c10orf76 to the Golgi, and that an intact c10orf76-PI4KB complex is required for the replication of c10orf76-dependent enteroviruses. Intriguingly, c10orf76 also contributed to proper Arf1 activation at the Golgi, providing a putative mechanism for the c10orf76-dependent increase in PI4P levels at the Golgi.
Assuntos
Enterovirus , Animais , Enterovirus/genética , Enterovirus/metabolismo , Complexo de Golgi/metabolismo , Fosfatos de Fosfatidilinositol , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Ligação Proteica , Células Sf9 , Replicação ViralRESUMO
Activation of the integrated stress response (ISR) by a variety of stresses triggers phosphorylation of the α-subunit of translation initiation factor eIF2. P-eIF2α inhibits eIF2B, the guanine nucleotide exchange factor that recycles inactive eIF2â¢GDP to active eIF2â¢GTP. eIF2 phosphorylation thereby represses translation. Persistent activation of the ISR has been linked to the development of several neurological disorders, and modulation of the ISR promises new therapeutic strategies. Recently, a small-molecule ISR inhibitor (ISRIB) was identified that rescues translation in the presence of P-eIF2α by facilitating the assembly of more active eIF2B. ISRIB enhances cognitive memory processes and has therapeutic effects in brain-injured mice without displaying overt side effects. While using ISRIB to investigate the ISR in picornavirus-infected cells, we observed that ISRIB rescued translation early in infection when P-eIF2α levels were low, but not late in infection when P-eIF2α levels were high. By treating cells with varying concentrations of poly(I:C) or arsenite to induce the ISR, we provide additional proof that ISRIB is unable to inhibit the ISR when intracellular P-eIF2α concentrations exceed a critical threshold level. Together, our data demonstrate that the effects of pharmacological activation of eIF2B are tuned by P-eIF2α concentration. Thus, ISRIB can mitigate undesirable outcomes of low-level ISR activation that may manifest neurological disease but leaves the cytoprotective effects of acute ISR activation intact. The insensitivity of cells to ISRIB during acute ISR may explain why ISRIB does not cause overt toxic side effects in vivo.