RESUMEN
HCoV-OC43 is one of the mildly pathogenic coronaviruses with high infection rates in common population. Here, 43 HCoV-OC43 related cases with pneumonia were reported, corresponding genomes of HCoV-OC43 were obtained. Phylogenetic analyses based on complete genome, orf1ab and spike genes revealed that two novel genotypes of HCoV-OC43 have emerged in China. Obvious recombinant events also can be detected in the analysis of the evolutionary dynamics of novel HCoV-OC43 genotypes. Estimated divergence time analysis indicated that the two novel genotypes had apparently independent evolutionary routes. Efforts should be conducted for further investigation of genomic diversity and evolution analysis of mildly pathogenic coronaviruses.
Asunto(s)
Resfriado Común/epidemiología , Infecciones por Coronavirus/epidemiología , Coronavirus Humano OC43/genética , Genoma Viral , Genotipo , Neumonía Viral/epidemiología , Secuencia de Bases , Teorema de Bayes , Niño , Niño Hospitalizado , Preescolar , China/epidemiología , Resfriado Común/patología , Resfriado Común/transmisión , Resfriado Común/virología , Infecciones por Coronavirus/patología , Infecciones por Coronavirus/transmisión , Infecciones por Coronavirus/virología , Coronavirus Humano OC43/clasificación , Coronavirus Humano OC43/patogenicidad , Monitoreo Epidemiológico , Femenino , Humanos , Lactante , Masculino , Método de Montecarlo , Mutación , Filogenia , Neumonía Viral/patología , Neumonía Viral/transmisión , Neumonía Viral/virología , Recombinación GenéticaRESUMEN
The importance of pre-existing immune responses to seasonal endemic coronaviruses (HCoVs) for the susceptibility to SARS-CoV-2 infection and the course of COVID-19 is the subject of an ongoing scientific debate. Recent studies postulate that immune responses to previous HCoV infections can either have a slightly protective or no effect on SARS-CoV-2 pathogenesis and, consequently, be neglected for COVID-19 risk stratification. Challenging this notion, we provide evidence that pre-existing, anti-nucleocapsid antibodies against endemic α-coronaviruses and S2 domain-specific anti-spike antibodies against ß-coronavirus HCoV-OC43 are elevated in patients with COVID-19 compared to pre-pandemic donors. This finding is particularly pronounced in males and in critically ill patients. Longitudinal evaluation reveals that antibody cross-reactivity or polyclonal stimulation by SARS-CoV-2 infection are unlikely to be confounders. Thus, specific pre-existing immunity to seasonal coronaviruses may increase susceptibility to SARS-CoV-2 and predispose individuals to an adverse COVID-19 outcome, guiding risk management and supporting the development of universal coronavirus vaccines.
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COVID-19/inmunología , Coronavirus/inmunología , SARS-CoV-2/inmunología , Adulto , Anticuerpos/inmunología , Anticuerpos Antivirales/inmunología , COVID-19/etiología , Infecciones por Coronavirus/inmunología , Coronavirus Humano OC43/inmunología , Coronavirus Humano OC43/patogenicidad , Reacciones Cruzadas/inmunología , Femenino , Alemania , Humanos , Inmunidad Humoral/inmunología , Inmunoglobulina G/inmunología , Estudios Longitudinales , Masculino , Persona de Mediana Edad , Pandemias , SARS-CoV-2/patogenicidad , Estaciones del Año , Índice de Severidad de la Enfermedad , Glicoproteína de la Espiga del Coronavirus/inmunologíaRESUMEN
The COVID-19 pandemic caused by SARS-CoV-2 infection poses a serious threat to global public health and the economy. The enzymatic product of cholesterol 25-hydroxylase (CH25H), 25-Hydroxycholesterol (25-HC), was reported to have potent anti-SARS-CoV-2 activity. Here, we found that the combination of 25-HC with EK1 peptide, a pan-coronavirus (CoV) fusion inhibitor, showed a synergistic antiviral activity. We then used the method of 25-HC modification to design and synthesize a series of 25-HC-modified peptides and found that a 25-HC-modified EK1 peptide (EK1P4HC) was highly effective against infections caused by SARS-CoV-2, its variants of concern (VOCs), and other human CoVs, such as HCoV-OC43 and HCoV-229E. EK1P4HC could protect newborn mice from lethal HCoV-OC43 infection, suggesting that conjugation of 25-HC with a peptide-based viral inhibitor was a feasible and universal strategy to improve its antiviral activity.
Asunto(s)
Antivirales/farmacología , Hidroxicolesteroles/química , Lipopéptidos/química , SARS-CoV-2/efectos de los fármacos , Secuencia de Aminoácidos , Animales , Antivirales/química , Antivirales/uso terapéutico , Peso Corporal/efectos de los fármacos , COVID-19/virología , Coronavirus Humano 229E/efectos de los fármacos , Coronavirus Humano 229E/patogenicidad , Infecciones por Coronavirus/tratamiento farmacológico , Infecciones por Coronavirus/mortalidad , Infecciones por Coronavirus/virología , Coronavirus Humano OC43/efectos de los fármacos , Coronavirus Humano OC43/patogenicidad , Modelos Animales de Enfermedad , Sinergismo Farmacológico , Humanos , Hidroxicolesteroles/farmacología , Hidroxicolesteroles/uso terapéutico , Lipopéptidos/farmacología , Lipopéptidos/uso terapéutico , Ratones , Ratones Endogámicos BALB C , Polietilenglicoles/química , SARS-CoV-2/aislamiento & purificación , SARS-CoV-2/fisiología , Tasa de Supervivencia , Internalización del Virus/efectos de los fármacos , Tratamiento Farmacológico de COVID-19RESUMEN
The emergence of a novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and more recently, the independent evolution of multiple SARS-CoV-2 variants has generated renewed interest in virus evolution and cross-species transmission. While all known human coronaviruses (HCoVs) are speculated to have originated in animals, very little is known about their evolutionary history and factors that enable some CoVs to co-exist with humans as low pathogenic and endemic infections (HCoV-229E, HCoV-NL63, HCoV-OC43, HCoV-HKU1), while others, such as SARS-CoV, MERS-CoV and SARS-CoV-2 have evolved to cause severe disease. In this review, we highlight the origins of all known HCoVs and map positively selected for mutations within HCoV proteins to discuss the evolutionary trajectory of SARS-CoV-2. Furthermore, we discuss emerging mutations within SARS-CoV-2 and variants of concern (VOC), along with highlighting the demonstrated or speculated impact of these mutations on virus transmission, pathogenicity, and neutralization by natural or vaccine-mediated immunity.
Asunto(s)
Vacunas contra la COVID-19 , COVID-19/virología , SARS-CoV-2/genética , Animales , COVID-19/transmisión , Coronavirus Humano 229E/genética , Coronavirus Humano 229E/inmunología , Coronavirus Humano 229E/patogenicidad , Coronavirus Humano NL63/genética , Coronavirus Humano NL63/inmunología , Coronavirus Humano NL63/patogenicidad , Coronavirus Humano OC43/genética , Coronavirus Humano OC43/inmunología , Coronavirus Humano OC43/patogenicidad , Humanos , Inmunidad , Mutación , SARS-CoV-2/inmunología , SARS-CoV-2/patogenicidadRESUMEN
Human coronaviruses have become an increasing threat to global health; three highly pathogenic strains have emerged since the early 2000s, including most recently SARS-CoV-2, the cause of COVID-19. A better understanding of the molecular mechanisms of coronavirus pathogenesis is needed, including how these highly virulent strains differ from those that cause milder, common-cold-like disease. While significant progress has been made in understanding how SARS-CoV-2 proteins interact with the host cell, nonstructural protein 3 (nsp3) has largely been omitted from the analyses. Nsp3 is a viral protease with important roles in viral protein biogenesis, replication complex formation, and modulation of host ubiquitinylation and ISGylation. Herein, we use affinity purification-mass spectrometry to study the host-viral protein-protein interactome of nsp3 from five coronavirus strains: pathogenic strains SARS-CoV-2, SARS-CoV, and MERS-CoV; and endemic common-cold strains hCoV-229E and hCoV-OC43. We divide each nsp3 into three fragments and use tandem mass tag technology to directly compare the interactors across the five strains for each fragment. We find that few interactors are common across all variants for a particular fragment, but we identify shared patterns between select variants, such as ribosomal proteins enriched in the N-terminal fragment (nsp3.1) data set for SARS-CoV-2 and SARS-CoV. We also identify unique biological processes enriched for individual homologs, for instance, nuclear protein import for the middle fragment of hCoV-229E, as well as ribosome biogenesis of the MERS nsp3.2 homolog. Lastly, we further investigate the interaction of the SARS-CoV-2 nsp3 N-terminal fragment with ATF6, a regulator of the unfolded protein response. We show that SARS-CoV-2 nsp3.1 directly binds to ATF6 and can suppress the ATF6 stress response. Characterizing the host interactions of nsp3 widens our understanding of how coronaviruses co-opt cellular pathways and presents new avenues for host-targeted antiviral therapeutics.
Asunto(s)
Factor de Transcripción Activador 6/metabolismo , Proteasas Similares a la Papaína de Coronavirus/metabolismo , Interacciones Huésped-Patógeno/fisiología , SARS-CoV-2/patogenicidad , Coronavirus Humano 229E/metabolismo , Coronavirus Humano 229E/patogenicidad , Coronavirus Humano OC43/metabolismo , Coronavirus Humano OC43/patogenicidad , Proteasas Similares a la Papaína de Coronavirus/genética , Degradación Asociada con el Retículo Endoplásmico , Células HEK293 , Humanos , Coronavirus del Síndrome Respiratorio de Oriente Medio/metabolismo , Coronavirus del Síndrome Respiratorio de Oriente Medio/patogenicidad , Mapas de Interacción de Proteínas , SARS-CoV-2/metabolismo , Respuesta de Proteína Desplegada , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/metabolismoRESUMEN
SARS-CoV-2 is an RNA virus whose success as a pathogen relies on its abilities to repurpose host RNA-binding proteins (RBPs) and to evade antiviral RBPs. To uncover the SARS-CoV-2 RNA interactome, we here develop a robust ribonucleoprotein (RNP) capture protocol and identify 109 host factors that directly bind to SARS-CoV-2 RNAs. Applying RNP capture on another coronavirus, HCoV-OC43, revealed evolutionarily conserved interactions between coronaviral RNAs and host proteins. Transcriptome analyses and knockdown experiments delineated 17 antiviral RBPs, including ZC3HAV1, TRIM25, PARP12, and SHFL, and 8 proviral RBPs, such as EIF3D and CSDE1, which are responsible for co-opting multiple steps of the mRNA life cycle. This also led to the identification of LARP1, a downstream target of the mTOR signaling pathway, as an antiviral host factor that interacts with the SARS-CoV-2 RNAs. Overall, this study provides a comprehensive list of RBPs regulating coronaviral replication and opens new avenues for therapeutic interventions.
Asunto(s)
Autoantígenos/genética , COVID-19/genética , ARN Viral/genética , Ribonucleoproteínas/genética , SARS-CoV-2/genética , COVID-19/virología , Coronavirus Humano OC43/genética , Coronavirus Humano OC43/patogenicidad , Células HEK293 , Interacciones Huésped-Patógeno/genética , Humanos , Unión Proteica/genética , Mapas de Interacción de Proteínas/genética , Proteínas de Unión al ARN/genética , SARS-CoV-2/patogenicidad , Serina-Treonina Quinasas TOR/genética , Factores de Transcripción/genética , Transcriptoma/genética , Proteínas de Motivos Tripartitos/genética , Ubiquitina-Proteína Ligasas/genética , Replicación Viral/genética , Antígeno SS-BRESUMEN
Since the emergence of their primitive strains, the complexity surrounding their pathogenesis, constant genetic mutation and translation are contributing factors to the scarcity of a successful vaccine for coronaviruses till moment. Although, the recent announcement of vaccine breakthrough for COVID-19 renews the hope, however, there remains a major challenge of accessibility to urgently match the rapid global therapeutic demand for curtailing the pandemic, thereby creating an impetus for further search. The reassessment of results from a stream of experiments is of enormous importance in identifying bona fide lead-like candidates to fulfil this quest. This review comprehensively highlights the common pathomechanisms and pharmacological targets of HCoV-OC43, SARS-CoV-1, MERS-CoV and SARS-CoV-2, and potent therapeutic potentials from basic and clinical experimental investigations. The implicated targets for the prevention and treatment include the viral proteases (Mpro, PLpro, 3CLpro), viral structural proteins (S- and N-proteins), non-structural proteins (nsp 3, 8, 10, 14, 16), accessory protein (ns12.9), viroporins (3a, E, 8a), enzymes (RdRp, TMPRSS2, ADP-ribosyltransferase, MTase, 2'-O-MTase, TATase, furin, cathepsin, deamidated human triosephosphate isomerase), kinases (MAPK, ERK, PI3K, mTOR, AKT, Abl2), interleukin-6 receptor (IL-6R) and the human host receptor, ACE2. Notably among the 109 overviewed inhibitors include quercetin, eriodictyol, baicalin, luteolin, melatonin, resveratrol and berberine from natural products, GC373, NP164 and HR2P-M2 from peptides, 5F9, m336 and MERS-GD27 from specific human antibodies, imatinib, remdesivir, ivermectin, chloroquine, hydroxychloroquine, nafamostat, interferon-ß and HCQ from repurposing libraries, some iron chelators and traditional medicines. This review represents a model for further translational studies for effective anti-CoV therapeutic designs.
Asunto(s)
Antivirales/farmacología , Infecciones por Coronavirus/etiología , Coronavirus/patogenicidad , Interacciones Huésped-Patógeno , Antivirales/uso terapéutico , Coronavirus/efectos de los fármacos , Coronavirus/metabolismo , Infecciones por Coronavirus/tratamiento farmacológico , Infecciones por Coronavirus/virología , Coronavirus Humano OC43/efectos de los fármacos , Coronavirus Humano OC43/patogenicidad , Humanos , Coronavirus del Síndrome Respiratorio de Oriente Medio/efectos de los fármacos , Coronavirus del Síndrome Respiratorio de Oriente Medio/patogenicidad , Ensayos Clínicos Controlados Aleatorios como Asunto , SARS-CoV-2/efectos de los fármacos , SARS-CoV-2/patogenicidad , Proteínas Virales/química , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
There does not appear to be any studies in the published literature on the stability of SARS-CoV-2 in climbing chalk powder (magnesium carbonate and/or calcium carbonate), which has been hypothesized to pose a potential risk of fomite transmission of coronavirus disease 2019 (COVID-19) within climbing gyms. The aim of this study was to determine the infectivity of a model human coronavirus HCoV-OC43 in the presence of climbing chalk powder on a dry plastic surface. The stability of HCoV-OC43 on a plastic surface dusted with climbing chalk powders (magnesium carbonate, calcium carbonate or a blended chalk) was determined by titration on BHK-21 fibroblast cells. No chalk and no virus controls were included. HCoV-OC43 was stable on the plastic surface for 48 h. The stability of HCoV-OC43 was significantly (P ≤ 0·05) reduced in the presence of magnesium carbonate, calcium carbonate and the chalk blend; the infectivity was reduced by ≥2·29 log10 50% tissue culture infective dose (TCID50 ) immediately upon on contact and by ≥2·46 log10 TCID50 within 1 h of contact. These findings suggest that the infectivity of coronaviruses is reduced by climbing chalk, limiting the risk of potential fomite transmission.
Asunto(s)
Coronavirus Humano OC43/efectos de los fármacos , Animales , Carbonato de Calcio , Línea Celular , Coronavirus Humano OC43/patogenicidad , Cricetinae , Fibroblastos/efectos de los fármacos , Fibroblastos/virología , Fómites , PolvosRESUMEN
Little is known about the role of complement (C') in infections with highly prevalent circulating human coronaviruses such as OC43, a group of viruses of major public health concern. Treatment of OC43-infected human lung cells with human serum resulted in C3 deposition on their surfaces and generation of C5a, indicating robust C' activation. Real-time cell viability assays showed that in vitro C'-mediated lysis of OC43 infected cells requires C3, C5 and C6 but not C7, and was substantially delayed as compared to rapid C'-mediated killing of parainfluenza virus type 5 (PIV5)-infected cells. In cells co-infected with OC43 and PIV5, C'-mediated lysis was delayed, similar to OC43 infected cells alone, suggesting that OC43 infection induced dominant inhibitory signals. When OC43-infected cells were treated with human serum, their cell surfaces contained both Vitronectin (VN) and Clusterin (CLU), two host cell C' inhibitors that can alter membrane attack complex (MAC) formation and C'-mediated killing. VN and CLU were not bound to OC43-infected cells after treatment with antibody-depleted serum. Reconstitution experiments with purified IgG and VN showed that human antibodies are both necessary and sufficient for VN recruitment to OC43-infected lung cells-novel findings with implications for CoV pathogenesis.
Asunto(s)
Anticuerpos/metabolismo , Clusterina/metabolismo , Proteínas Inactivadoras de Complemento/metabolismo , Coronavirus Humano OC43/inmunología , Pulmón/virología , Vitronectina/metabolismo , Línea Celular , Membrana Celular/metabolismo , Supervivencia Celular/inmunología , Activación de Complemento , Complejo de Ataque a Membrana del Sistema Complemento/metabolismo , Proteínas del Sistema Complemento/metabolismo , Coronavirus Humano OC43/patogenicidad , Humanos , Pulmón/metabolismo , Virus de la Parainfluenza 5/inmunologíaRESUMEN
Respiratory viral infection can cause severe disease and hospitalization, especially among children, the elderly, and patients with comorbidities. In Brazil, the official surveillance system of severe acute respiratory infection (SARI) investigates influenza A (IAV) and B (IBV) viruses, respiratory syncytial virus (RSV), adenovirus (HAdV), and parainfluenza viruses (hPIV 1-3). In Rio Grande do Sul (RS), Brazil, many fatalities associated with SARI between 2013 and 2017 occurred among patients without underlying diseases and for whom the causative agent had not been identified using official protocols. This cross-sectional study analyzed the presence of coronaviruses (HCoV), bocavirus (HBoV), metapneumovirus (hMPV), and rhinovirus in patients who died of SARI despite not having comorbidities, and that were negative for IAV, IBV, RSV, HAdV, and hPIV. Nasopharyngeal aspirates/swabs from patients were used for nucleic acid extraction. The presence of HCoVs OC43, HKU1, NL63, and 229E; HBoV; hMPV; and rhinovirus was assessed by quantitative reverse transcription-polymerase chain reaction. Clinical data were also analyzed. Between 2013 and 2017, 16 225 cases of SARI were reported in RS; 9.8% of the patients died; 20% of all fatal cases were patients without comorbidities and for whom no pathogen was detected using standard protocols. Analysis of 271 of these cases identified HCoV in nine cases; HBoV, hMPV, and rhinovirus were detected in 3, 3, and 10 cases, respectively. Of note, patients infected with HCoV were adults. Results reinforce the importance of including coronaviruses in diagnostic panels used by official surveillance systems because besides their pandemic potential, endemic HCoVs are associated to severe disease in healthy adults.
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Infecciones por Coronavirus/mortalidad , Infecciones por Coronavirus/virología , Coronavirus Humano NL63/patogenicidad , Coronavirus Humano OC43/patogenicidad , Adolescente , Adulto , Anciano , Brasil/epidemiología , Niño , Preescolar , Resfriado Común/epidemiología , Resfriado Común/virología , Coronavirus/clasificación , Coronavirus/patogenicidad , Estudios Transversales , Femenino , Voluntarios Sanos , Humanos , Lactante , Masculino , Persona de Mediana Edad , Nasofaringe/virología , Infecciones del Sistema Respiratorio/virología , Rhinovirus/genética , Índice de Severidad de la Enfermedad , Adulto JovenRESUMEN
Human coronaviruses (hCoVs) have become a threat to global health and society, as evident from the SARS outbreak in 2002 caused by SARS-CoV-1 and the most recent COVID-19 pandemic caused by SARS-CoV-2. Despite a high sequence similarity between SARS-CoV-1 and -2, each strain has a distinctive virulence. A better understanding of the basic molecular mechanisms mediating changes in virulence is needed. Here, we profile the virus-host protein-protein interactions of two hCoV nonstructural proteins (nsps) that are critical for virus replication. We use tandem mass tag-multiplexed quantitative proteomics to sensitively compare and contrast the interactomes of nsp2 and nsp4 from three betacoronavirus strains: SARS-CoV-1, SARS-CoV-2, and hCoV-OC43-an endemic strain associated with the common cold. This approach enables the identification of both unique and shared host cell protein binding partners and the ability to further compare the enrichment of common interactions across homologues from related strains. We identify common nsp2 interactors involved in endoplasmic reticulum (ER) Ca2+ signaling and mitochondria biogenesis. We also identify nsp4 interactors unique to each strain, such as E3 ubiquitin ligase complexes for SARS-CoV-1 and ER homeostasis factors for SARS-CoV-2. Common nsp4 interactors include N-linked glycosylation machinery, unfolded protein response associated proteins, and antiviral innate immune signaling factors. Both nsp2 and nsp4 interactors are strongly enriched in proteins localized at mitochondria-associated ER membranes suggesting a new functional role for modulating host processes, such as calcium homeostasis, at these organelle contact sites. Our results shed light on the role these hCoV proteins play in the infection cycle, as well as host factors that may mediate the divergent pathogenesis of OC43 from SARS strains. Our mass spectrometry workflow enables rapid and robust comparisons of multiple bait proteins, which can be applied to additional viral proteins. Furthermore, the identified common interactions may present new targets for exploration by host-directed antiviral therapeutics.
Asunto(s)
COVID-19/metabolismo , Interacciones Huésped-Patógeno/genética , SARS-CoV-2/patogenicidad , Proteínas no Estructurales Virales/metabolismo , COVID-19/virología , Coronavirus Humano OC43/patogenicidad , Retículo Endoplásmico/metabolismo , Células HEK293 , Humanos , Proteínas de la Membrana/metabolismo , Mitocondrias/metabolismo , Unión Proteica , Mapas de Interacción de Proteínas/genética , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/patogenicidad , Síndrome Respiratorio Agudo Grave/metabolismo , Síndrome Respiratorio Agudo Grave/virología , Transfección , Proteínas no Estructurales Virales/genética , Virulencia/genética , Replicación Viral/genéticaRESUMEN
OBJECTIVES: The molecular mechanisms underlying the pathogenesis of human coronavirus OC43 (HCoV-OC43) infection are poorly understood. In this study, we investigated the ability of HCoV-OC43 to antagonize the transcriptional activation of antiviral response elements. METHODS: HCoV-OC43 structural (membrane M and nucleocapsid N) and accessory proteins (ns2a and ns5a) were expressed individually in human embryonic kidney 293 (HEK-293) cells. The transcriptional activation of antiviral response elements was assessed by measuring the levels of firefly luciferase expressed under the control of interferon (IFN)-stimulated response element (ISRE), IFN-ß promoter, or nuclear factor kappa B response element (NF-κB-RE). The antiviral gene expression profile in HEK-293 cells was determined by PCR array. RESULTS: The transcriptional activity of ISRE, IFN-ß promoter, and NF-κB-RE was significantly reduced in the presence of HCoV-OC43 ns2a, ns5a, M, or N protein, following the challenge of cells with Sendai virus, IFN-α or tumor necrosis factor-α. The expression of antiviral genes involved in the type I IFN and NF-κB signaling pathways was also downregulated in the presence of HCoV-OC43 structural or accessory proteins. CONCLUSION: Both structural and accessory HCoV-OC43 proteins are able to inhibit antiviral response elements in HEK-293 cells, and to block the activation of different antiviral signaling pathways.
Asunto(s)
Infecciones por Coronavirus/virología , Coronavirus Humano OC43/patogenicidad , Proteínas de la Nucleocápside/metabolismo , Elementos de Respuesta/genética , Proteínas de la Matriz Viral/metabolismo , Proteínas Reguladoras y Accesorias Virales/metabolismo , Proteínas M de Coronavirus , Proteínas de la Nucleocápside de Coronavirus , Coronavirus Humano OC43/genética , Regulación hacia Abajo , Genes Reporteros , Células HEK293 , Humanos , Interferones/genética , FN-kappa B/genética , FN-kappa B/metabolismo , Proteínas de la Nucleocápside/genética , Regiones Promotoras Genéticas/genética , Transducción de Señal , Activación Transcripcional , Proteínas de la Matriz Viral/genética , Proteínas Reguladoras y Accesorias Virales/genéticaAsunto(s)
Animales Salvajes/virología , Infecciones por Coronavirus/veterinaria , Coronavirus Humano OC43/aislamiento & purificación , Brotes de Enfermedades , Pan troglodytes/virología , Animales , Infecciones por Coronavirus/epidemiología , Infecciones por Coronavirus/transmisión , Coronavirus Humano OC43/genética , Coronavirus Humano OC43/patogenicidad , Heces/virología , HumanosRESUMEN
The Coronaviridae family clusters a number of large RNA viruses, which share several structural and functional features. However, members of this family recognize different cellular receptors and exploit different entry routes, what affects their species specificity and virulence. The aim of this study was to determine how human coronavirus OC43 enters the susceptible cell. Using confocal microscopy and molecular biology tools we visualized early events during infection. We found that the virus employs caveolin-1 dependent endocytosis for the entry and the scission of virus-containing vesicles from the cell surface is dynamin-dependent. Furthermore, the vesicle internalization process requires actin cytoskeleton rearrangements. With our research we strove to broaden the understanding of the infection process, which in future may be beneficial for the development of a potential therapeutics.
Asunto(s)
Infecciones por Coronavirus/genética , Coronavirus Humano OC43/genética , Endocitosis/genética , Internalización del Virus , Citoesqueleto de Actina/genética , Caveolina 1/genética , Línea Celular , Clatrina/genética , Infecciones por Coronavirus/virología , Coronavirus Humano OC43/patogenicidad , Dinaminas/genética , Humanos , Especificidad de la Especie , Virulencia/genéticaRESUMEN
The OC43 strain of human coronavirus (HCoV-OC43) is an ubiquitous respiratory tract pathogen possessing neurotropic capacities. Coronavirus structural envelope (E) protein possesses specific motifs involved in protein-protein interaction or in homo-oligomeric ion channel formation, which are known to play various roles including in virion morphology/assembly and in cell response to infection and/or virulence. Making use of recombinant viruses either devoid of the E protein or harboring mutations either in putative transmembrane domain or PDZ-binding motif, we demonstrated that a fully functional HCoV-OC43 E protein is first needed for optimal production of recombinant infectious viruses. Furthermore, HCoV-OC43 infection of human epithelial and neuronal cell lines, of mixed murine primary cultures from the central nervous system and of mouse central nervous system showed that the E protein is critical for efficient and optimal virus replication and propagation, and thereby for neurovirulence.
Asunto(s)
Sistema Nervioso Central/virología , Infecciones por Coronavirus/virología , Coronavirus Humano OC43/fisiología , Neuronas/virología , Proteínas del Envoltorio Viral/metabolismo , Animales , Línea Celular , Sistema Nervioso Central/patología , Infecciones por Coronavirus/patología , Coronavirus Humano OC43/genética , Coronavirus Humano OC43/patogenicidad , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Neuronas/patología , Proteínas del Envoltorio Viral/genética , VirulenciaRESUMEN
Human coronaviruses (HCoV) are respiratory pathogens with neuroinvasive, neurotropic, and neurovirulent properties, highlighting the importance of studying the potential implication of these viruses in neurological diseases. The OC43 strain (HCoV-OC43) was reported to induce neuronal cell death, which may participate in neuropathogenesis. Here, we show that HCoV-OC43 harboring two point mutations in the spike glycoprotein (rOC/Us183-241) was more neurovirulent than the wild-type HCoV-OC43 (rOC/ATCC) in mice and induced more cell death in murine and human neuronal cells. To evaluate the role of regulated cell death (RCD) in HCoV-OC43-mediated neural pathogenesis, we determined if knockdown of Bax, a key regulator of apoptosis, or RIP1, a key regulator of necroptosis, altered the percentage of neuronal cell death following HCoV-OC43 infection. We found that Bax-dependent apoptosis did not play a significant role in RCD following infection, as inhibition of Bax expression mediated by RNA interference did not confer cellular protection against the cell death process. On the other hand, we demonstrated that RIP1 and MLKL were involved in neuronal cell death, as RIP1 knockdown and chemical inhibition of MLKL significantly increased cell survival after infection. Taken together, these results indicate that RIP1 and MLKL contribute to necroptotic cell death after HCoV-OC43 infection to limit viral replication. However, this RCD could lead to neuronal loss in the mouse CNS and accentuate the neuroinflammation process, reflecting the severity of neuropathogenesis. IMPORTANCE: Because they are naturally neuroinvasive and neurotropic, human coronaviruses are suspected to participate in the development of neurological diseases. Given that the strain OC43 is neurovirulent in mice and induces neuronal cell death, we explored the neuronal response to infection by characterizing the activation of RCD. Our results revealed that classical apoptosis associated with the Bax protein does not play a significant role in HCoV-OC43-induced neuronal cell death and that RIP1 and MLKL, two cellular proteins usually associated with necroptosis (an RCD back-up system when apoptosis is not adequately induced), both play a pivotal role in the process. As necroptosis disrupts cellular membranes and allows the release of damage-associated molecular patterns (DAMP) and possibly induces the production of proinflammatory cytokines, it may represent a proinflammatory cell death mechanism that contributes to excessive neuroinflammation and neurodegeneration and eventually to neurological disorders after a coronavirus infection.
Asunto(s)
Infecciones por Coronavirus/genética , Coronavirus Humano OC43/patogenicidad , Proteínas Activadoras de GTPasa/genética , Interacciones Huésped-Patógeno , Proteínas Quinasas/genética , Glicoproteína de la Espiga del Coronavirus/genética , Animales , Muerte Celular , Línea Celular , Línea Celular Tumoral , Infecciones por Coronavirus/metabolismo , Infecciones por Coronavirus/mortalidad , Infecciones por Coronavirus/virología , Coronavirus Humano OC43/genética , Coronavirus Humano OC43/metabolismo , Embrión de Mamíferos , Femenino , Proteínas Activadoras de GTPasa/metabolismo , Regulación de la Expresión Génica , Humanos , Ratones , Ratones Endogámicos BALB C , Mutación , Neuronas/patología , Neuronas/virología , Cultivo Primario de Células , Proteínas Quinasas/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Glicoproteína de la Espiga del Coronavirus/metabolismo , Análisis de Supervivencia , Virulencia , Proteína X Asociada a bcl-2/antagonistas & inhibidores , Proteína X Asociada a bcl-2/genética , Proteína X Asociada a bcl-2/metabolismoRESUMEN
Human coronaviruses (HCoV) are respiratory pathogens that may be associated with the development of neurological diseases, in view of their neuroinvasive and neurotropic properties. The viral spike (S) glycoprotein is a major virulence factor for several coronavirus species, including the OC43 strain of HCoV (HCoV-OC43). In an attempt to study the role of this protein in virus spread within the central nervous system (CNS) and neurovirulence, as well as to identify amino acid residues important for such functions, we compared the sequence of the S gene found in the laboratory reference strain HCoV-OC43 ATCC VR-759 to S sequences of viruses detected in clinical isolates from the human respiratory tract. We identified one predominant mutation at amino acid 758 (from RRSR↓ G758 to RRSR↓R758), which introduces a putative furin-like cleavage (↓) site. Using a molecular cDNA infectious clone to generate a corresponding recombinant virus, we show for the first time that such point mutation in the HCoV-OC43 S glycoprotein creates a functional cleavage site between the S1 and S2 portions of the S protein. While the corresponding recombinant virus retained its neuroinvasive properties, this mutation led to decreased neurovirulence while potentially modifying the mode of virus spread, likely leading to a limited dissemination within the CNS. Taken together, these results are consistent with the adaptation of HCoV-OC43 to the CNS environment, resulting from the selection of quasi-species harboring mutations that lead to amino acid changes in viral genes, like the S gene in HCoV-OC43, which may contribute to a more efficient establishment of a less pathogenic but persistent CNS infection. This adaptative mechanism could potentially be associated with human encephalitis or other neurological degenerative pathologies.
Asunto(s)
Enfermedades del Sistema Nervioso Central/virología , Infecciones por Coronavirus/virología , Coronavirus Humano OC43/patogenicidad , Proproteína Convertasas/metabolismo , Glicoproteína de la Espiga del Coronavirus/metabolismo , Animales , Coronavirus Humano OC43/aislamiento & purificación , Coronavirus Humano OC43/fisiología , Glicoproteínas/metabolismo , Humanos , Ratones , VirulenciaRESUMEN
IFNs are a family of cytokines that are essential for the antiviral response in vertebrates. Not surprisingly, viruses have adapted to encode virulence factors to cope with the IFN response. Intriguingly, we show here that all three types of interferons, IFN-α, IFN-γ, and IFN-λ, efficiently promote infection by a human coronavirus, HCoV-OC43, one of the major etiological agents of common cold, through the induction of IFN-inducible transmembrane (IFITM) proteins. IFITMs typically exert their antiviral function by inhibiting the entry of a broad spectrum of viruses into their host cells, presumably by trapping and degrading invading virions within the endocytic compartments. In contrast, HCoV-OC43 uses IFN-induced human IFITM2 or IFITM3 as an entry factor to facilitate its infection of host cells. Reverse genetics analyses suggest that the structural motifs critical for the IFITM proteins' enhancement of HCoV-OC43 infection are distinct from those required for inhibiting infection by other viruses. We also present evidence showing that IFITM family members work as homo- and hetero-oligomers to modulate virus entry. The observed enhancement of HCoV-OC43 infection by IFNs may underlie the propensity of the virus to invade the lower respiratory tract under inflammatory conditions.
Asunto(s)
Coronavirus Humano OC43/patogenicidad , Interferones/metabolismo , Proteínas de la Membrana/biosíntesis , Secuencia de Aminoácidos , Animales , Línea Celular , Infecciones por Coronavirus/etiología , Infecciones por Coronavirus/inmunología , Infecciones por Coronavirus/virología , Coronavirus Humano OC43/inmunología , Coronavirus Humano OC43/fisiología , Citocinas/metabolismo , Interacciones Huésped-Patógeno , Humanos , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Ratones , Datos de Secuencia Molecular , Homología de Secuencia de Aminoácido , Virulencia/inmunología , Internalización del VirusRESUMEN
BACKGROUND: It is unknown to what extent the human coronaviruses (HCoVs) OC43, HKU1, 229E and NL63 infect healthy children. Frequencies of infections are only known for hospitalized children. OBJECTIVES: Comparing infection frequencies in children who have mild infections with frequencies in children needing hospital uptake will determine whether infection by one of the four HCoVs leads to more severe disease. In addition, the sequence of seroconversions can reveal whether infection by one HCoV protects from infection by other HCoVs. STUDY DESIGN: Two distinct study groups were monitored: healthy children and children hospitalized due to respiratory infection. HCoV natural infection rates in healthy children were obtained by serology in 25 newborns (followed 0-20months). The frequencies of severe HCoVs infection was determined by real time RT-PCR among 1471 hospitalized infants (<2-years old) with acute respiratory tract disease. RESULTS: The majority of healthy children seroconverted for HCoV-OC43 (n=19) and HCoV-NL63 (n=17), less for HCoV-HKU1 (n=9) and HCoV-229E (n=5). Notably, HCoV-HKU1 seroconversion was absent after HCoV-OC43 infection. Also HCoV-229E infection was rarely observed after HCoV-NL63 infection (1 out of 5). In the hospital 207 (14%) out of 1471 children were HCoV positive. Again we observed most infection by HCoV-OC43 (n=85) and HCoV-NL63 (n=60), followed by HCoV-HKU1 (n=47) and HCoV-229E (n=15). CONCLUSIONS: HCoV-NL63 and HCoV-OC43 infections occur frequently in early childhood, more often than HCoV-HKU1 or HCoV-229E infections. HCoV-OC43 and HCoV-NL63 may elicit immunity that protects from subsequent HCoV-HKU1 and HCoV-229E infection, respectively, which would explain why HCoV-OC43 and HCoV-NL63 are the most frequently infecting HCoVs. There are no indications that infection by one of the HCoVs is more pathogenic than others.
Asunto(s)
Infecciones por Coronavirus/epidemiología , Coronavirus Humano NL63/aislamiento & purificación , Coronavirus Humano OC43/aislamiento & purificación , Infecciones del Sistema Respiratorio/epidemiología , Anticuerpos Antivirales/sangre , Infecciones por Coronavirus/fisiopatología , Infecciones por Coronavirus/virología , Coronavirus Humano NL63/inmunología , Coronavirus Humano NL63/patogenicidad , Coronavirus Humano OC43/inmunología , Coronavirus Humano OC43/patogenicidad , Femenino , Hospitalización/estadística & datos numéricos , Humanos , Lactante , Recién Nacido , Masculino , Infecciones del Sistema Respiratorio/fisiopatología , Infecciones del Sistema Respiratorio/virología , Índice de Severidad de la EnfermedadRESUMEN
Human coronaviruses (HCoV) are recognized respiratory pathogens, and some strains, including HCoV-OC43, can infect human neuronal and glial cells of the central nervous system (CNS) and activate neuroinflammatory mechanisms. Moreover, HCoV-OC43 is neuroinvasive, neurotropic, and neurovirulent in susceptible mice, where it induces chronic encephalitis. Herein, we show that a single point mutation in the viral spike (S) glycoprotein (Y241H), acquired during viral persistence in human neural cells, led to a hind-limb paralytic disease in infected mice. Inhibition of glutamate excitotoxicity using a 2-amino-3-(5-methyl-3-oxo-1,2-oxazol-4-yl)propranoic acid (AMPA) receptor antagonist (GYKI-52466) improved clinical scores related to the paralysis and motor disabilities in S mutant virus-infected mice, as well as protected the CNS from neuronal dysfunctions, as illustrated by restoration of the phosphorylation state of neurofilaments. Expression of the glial glutamate transporter GLT-1, responsible for glutamate homeostasis, was downregulated following infection, and GYKI-52466 also significantly restored its steady-state expression level. Finally, GYKI-52466 treatment of S mutant virus-infected mice led to reduced microglial activation, which may lead to improvement in the regulation of CNS glutamate homeostasis. Taken together, our results strongly suggest an involvement of excitotoxicity in the paralysis-associated neuropathology induced by an HCoV-OC43 mutant which harbors a single point mutation in its spike protein that is acquired upon persistent virus infection.