RESUMEN
Angiotensin converting enzyme 2 (ACE2), the host receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, is differentially expressed in a wide variety of tissues and cell types. The expression of ACE2 is under tight regulation, but the mechanisms regulating ACE2 expression have not yet been well defined. Through a genome-wide CRISPR knockout screen, we discovered that host factors TRAF3, DYRK1A, and RAD54L2 (TDR) form a complex to regulate the expression of ACE2. Knockout of TRAF3, DYRK1A, or RAD54L2 reduces the mRNA levels of ACE2 and inhibits the cellular entry of SARS-CoV-2. On the other hand, SARS-CoV-2 continuously evolves by genetic mutations for the adaption to the host. We have identified mutations in spike (S) (P1079T) and nucleocapsid (N) (S194L) that enhance the replication of SARS-CoV-2 in cells that express ACE2 at a low level. Our results have revealed the mechanisms for the transcriptional regulation of ACE2 and the adaption of SARS-CoV-2. IMPORTANCE: The expression of ACE2 is essential for the entry of SARS-CoV-2 into host cells. We identify a new complex-the TDR complex-that acts to maintain the abundance of ACE2 in host cells. The identification and characterization of the TDR complex provide new targets for the development of therapeutics against SARS-CoV-2 infection. By analysis of SARS-CoV-2 virus replicating in cells expressing low levels of ACE2, we identified mutations in spike (P1079T) and nucleocapsid (S194L) that overcome the restriction of limited ACE2. Functional analysis of these key amino acids in S and N extends our knowledge of the impact of SARS-CoV-2 variants on virus infection and transmission.
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Enzima Convertidora de Angiotensina 2 , COVID-19 , SARS-CoV-2 , Humanos , Enzima Convertidora de Angiotensina 2/metabolismo , Enzima Convertidora de Angiotensina 2/genética , COVID-19/virología , COVID-19/metabolismo , COVID-19/genética , Mutación , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Tirosina Quinasas/metabolismo , Proteínas Tirosina Quinasas/genética , SARS-CoV-2/genética , SARS-CoV-2/fisiología , SARS-CoV-2/metabolismo , Glicoproteína de la Espiga del Coronavirus/metabolismo , Glicoproteína de la Espiga del Coronavirus/genética , Internalización del Virus , Células Vero , Chlorocebus aethiops , Animales , Línea CelularRESUMEN
Using multiple cell types and isolates of Delta and Omicron variants of SARS-CoV-2, we report differences in virus production, replication, and infectivity in vitro. Ancestral and Delta SARS-CoV-2 variant exhibit reduced virus production and replication at 34°C compared to 37°C while Omicron replication is balanced between temperatures.
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COVID-19 , Humanos , SARS-CoV-2/genética , TemperaturaRESUMEN
The development of antivirals against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been hampered by the lack of efficient cell-based replication systems that are amenable to high-throughput screens in biosafety level 2 laboratories. Here we report that stable cell clones harboring autonomously replicating SARS-CoV-2 RNAs without spike (S), membrane (M), and envelope (E) genes can be efficiently derived from the baby hamster kidney (BHK-21) cell line when a pair of mutations were introduced into the non-structural protein 1 (Nsp1) of SARS-CoV-2 to ameliorate cellular toxicity associated with virus replication. In a proof-of-concept experiment we screened a 273-compound library using replicon cells and identified three compounds as novel inhibitors of SARS-CoV-2 replication. Altogether, this work establishes a robust, cell-based system for genetic and functional analyses of SARS-CoV-2 replication and for the development of antiviral drugs. IMPORTANCE SARS-CoV-2 replicon systems that have been reported up to date were unsuccessful in deriving stable cell lines harboring non-cytopathic replicons. The transient expression of viral sgmRNA or a reporter gene makes it impractical for industry-scale screening of large compound libraries using these systems. Here, for the first time, we derived stable cell clones harboring the SARS-CoV-2 replicon. These clones may now be conveniently cultured in a standard BSL-2 laboratory for high throughput screen of compound libraries. Additionally, our stable replicon cells represent a new model system to study SARS-CoV-2 replication.
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Antivirales , Tratamiento Farmacológico de COVID-19 , Evaluación Preclínica de Medicamentos , SARS-CoV-2 , Animales , Antivirales/farmacología , Línea Celular , Células Clonales , Cricetinae , Evaluación Preclínica de Medicamentos/métodos , ARN Viral , Replicón , SARS-CoV-2/efectos de los fármacos , Replicación ViralRESUMEN
Biochemical and structural analyses suggest that SARS-CoV-2 is well-adapted to infecting humans and the presence of four residues (PRRA) at the S1/S2 site within the spike (S) protein, which may lead to unexpected tissue or host tropism. Here we report that SARS-CoV-2 efficiently utilized ACE2 of 9 species to infect 293T cells. Similarly, pseudoviruses bearing S protein derived from either the bat RaTG13 or pangolin GX, two closely related animal coronaviruses, utilized ACE2 of a diverse range of animal species to gain entry. Removal of PRRA from SARS-CoV-2 S protein displayed distinct effects on pseudoviral entry into different cell types. Unexpectedly, insertion of PRRA into the RaTG13 S protein selectively abrogated the usage of horseshoe bat and pangolin ACE2 but enhanced the usage of mouse ACE2 by the relevant pseudovirus to enter cells. Together, our findings identified a previously unrecognized effect of the PRRA insert on SARS-CoV-2 and RaTG13 S proteins.ImportanceThe four-residue insert (PRRA) at the boundary between the S1and S2 subunits of SARS-CoV-2 has been widely recognized since day 1 for its role in SARS-CoV-2 S protein processing and activation. As this PRRA insert is unique to SARS-CoV-2 among group b betacoronaviruses, it is thought to affect the tissue and species tropism of SARS-CoV-2. We compared the usage of 10 ACE2 orthologs and found that the presence of PRRA not only affects the cellular tropism of SARS-CoV-2 but also modulates the usage of ACE2 orthologs by the closely related bat RaTG13 S protein. The binding of pseudovirions carrying RaTG13 S with a PRRA insert to mouse ACE2 was nearly 2-fold higher than that of pseudovirions carrying RaTG13 S.
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COVID-19 vaccines provide high levels of protection against severe disease and hospitalization due to severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) infection. Vaccination may be less effective in preventing shedding of infectious viruses from otherwise immune patients. In this study, we describe breakthrough infections and shedding of infectious viruses in convalescent hamsters without significant replication in the lower respiratory tract following reinfection by Alpha and Delta variants despite high levels of circulating antibodies in sera. Using convalescent hamsters with long-term immunity (up to 1 year) following infection by ancestral SARS-CoV-2, we can model aspects of recurring COVID-19 in the context of preexisting immunity.
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COVID-19 , Animales , COVID-19/prevención & control , Vacunas contra la COVID-19 , Cricetinae , Humanos , Mesocricetus , SARS-CoV-2 , TráqueaRESUMEN
The mechanistic interplay between SARS-CoV-2 infection, inflammation, and oxygen homeostasis is not well defined. Here, we show that the hypoxia-inducible factor (HIF-1α) transcriptional pathway is activated, perhaps due to a lack of oxygen or an accumulation of mitochondrial reactive oxygen species (ROS) in the lungs of adult Syrian hamsters infected with SARS-CoV-2. Prominent nuclear localization of HIF-1α and increased expression of HIF-1α target proteins, including glucose transporter 1 (Glut1), lactate dehydrogenase (LDH), and pyruvate dehydrogenase kinase-1 (PDK1), were observed in areas of lung consolidation filled with infiltrating monocytes/macrophages. Upregulation of these HIF-1α target proteins was accompanied by a rise in glycolysis as measured by extracellular acidification rate (ECAR) in lung homogenates. A concomitant reduction in mitochondrial respiration was also observed as indicated by a partial loss of oxygen consumption rates (OCR) in isolated mitochondrial fractions of SARS-CoV-2-infected hamster lungs. Proteomic analysis further revealed specific deficits in the mitochondrial ATP synthase (Atp5a1) within complex V and in the ATP/ADP translocase (Slc25a4). The activation of HIF-1α in inflammatory macrophages may also drive proinflammatory cytokine production and complement activation and oxidative stress in infected lungs. Together, these findings support a role for HIF-1α as a central mediator of the metabolic reprogramming, inflammation, and bioenergetic dysfunction associated with SARS-CoV-2 infection.
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COVID-19 , Subunidad alfa del Factor 1 Inducible por Hipoxia , Estrés Oxidativo , Cricetinae , COVID-19/metabolismo , Metabolismo Energético , Glucólisis , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Inflamación , Oxígeno , Proteómica , SARS-CoV-2RESUMEN
The newly emerging Kappa, Delta, and Lambda SARS-CoV-2 variants are worrisome, characterized with the double mutations E484Q/L452R, T478K/L452R, and F490S/L452Q, respectively, in their receptor binding domains (RBDs) of the spike proteins. As revealed in crystal structures, most of these residues (e.g., 452 and 484 in RBDs) are not in direct contact with interfacial residues in the angiotensin-converting enzyme 2 (ACE2). This suggests that albeit there are some possibly nonlocal effects, these mutations might not significantly affect RBD's binding with ACE2, which is an important step for viral entry into host cells. Thus, without knowing the molecular mechanism, these successful mutations (from the point of view of SARS-CoV-2) may be hypothesized to evade human antibodies. Using all-atom molecular dynamics (MD) simulation, here, we show that the E484Q/L452R mutations significantly reduce the binding affinity between the RBD of the Kappa variant and the antibody LY-CoV555 (also named as Bamlanivimab), which was efficacious for neutralizing the wild-type SARS-CoV-2. To verify simulation results, we further carried out experiments with both pseudovirions- and live virus-based neutralization assays and demonstrated that LY-CoV555 completely lost neutralizing activity against the L452R/E484Q mutant. Similarly, we show that mutations in the Delta and Lambda variants can also destabilize the RBD's binding with LY-CoV555. With the revealed molecular mechanism on how these variants evade LY-CoV555, we expect that more specific therapeutic antibodies can be accordingly designed and/or a precise mixing of antibodies can be achieved as a cocktail treatment for patients infected with these variants.
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COVID-19 , SARS-CoV-2 , Humanos , Mutación , Unión Proteica , Glicoproteína de la Espiga del Coronavirus/metabolismoRESUMEN
Detection and degradation of foreign nucleic acids is an ancient form of host defense. However, the underlying mechanisms are not completely clear. MCPIP1 is an endoribonuclease and an important regulator in both innate and adaptive immunity by targeting inflammatory mRNA degradation. Here we report that MCPIP1 RNase can also selectively detect and degrade the mRNAs encoded by transfected plasmids. In transient transfection, MCPIP1 expression potently degraded the mRNA from exogenously transfected vectors, which is independent on the vector, genes and cell types used. Conversely, the expression of transfected plasmids in MCPIP1-null cells is significantly higher than that in wild-type cells. Interestingly, overexpression of MCPIP1 or MCPIP1 deficiency does not affect the expression of the exogenous genes incorporated into the host genome in a stable cell line or the global gene expression of host genome. This ability is not associated with PKR/RNase L system, as PKR inhibitors does not block MCPIP1-mediated mRNA degradation of exogenously transfected genes. Lastly, expression of MCPIP1 suppressed replication of Zika virus in infected cells. The study may provide a model for understanding the antiviral mechanisms of MCPIP1, and a putative tool to increase the expression of transfected exogenous genes.
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Estabilidad del ARN , ARN Mensajero/química , ARN Viral/química , Ribonucleasas/fisiología , Factores de Transcripción/fisiología , Replicación Viral/fisiología , Infección por el Virus Zika/genética , Virus Zika/genética , Vectores Genéticos , Células HEK293 , Células HeLa , Humanos , TransfecciónRESUMEN
As a unique rocaglate (flavagline) natural product, aglaroxin C displays intriguing biological activity by inhibiting hepatitis C viral entry. To further elucidate structure-activity relationships and diversify the pyrimidinone scaffold, we report a concise synthesis of aglaroxin C utilizing a highly regioselective pyrimidinone condensation. We have prepared more than 40 aglaroxin C analogues utilizing various amidine condensation partners. Through biological evaluation of analogues, we have discovered two lead compounds, CMLD012043 and CMLD012044, which show preferential bias for the inhibition of hepatitis C viral entry vs translation inhibition. Overall, the study demonstrates the power of chemical synthesis to produce natural product variants with both target inhibition bias and improved therapeutic indexes.
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Antivirales/farmacología , Benzofuranos/farmacología , Hepacivirus/efectos de los fármacos , Pirimidinonas/farmacología , Antivirales/síntesis química , Antivirales/toxicidad , Benzofuranos/síntesis química , Benzofuranos/toxicidad , Línea Celular , Humanos , Modelos Químicos , Estructura Molecular , Pirimidinonas/síntesis química , Pirimidinonas/toxicidad , Estereoisomerismo , Relación Estructura-Actividad , Internalización del Virus/efectos de los fármacosRESUMEN
Rocaglates are a family of natural products isolated from the genus Aglaia which possess a highly substituted cyclopenta[b]benzofuran skeleton and inhibit cap-dependent protein synthesis. Rocaglates are attractive compounds due to their potential for inhibiting tumor cell maintenance in vivo by specifically targeting eukaryotic initiation factor 4A (eIF4A) and interfering with recruitment of ribosomes to mRNA. In this paper, we describe an intercepted retro-Nazarov reaction utilizing intramolecular tosyl migration to generate a reactive oxyallyl cation on the rocaglate skeleton. Trapping of the oxyallyl cation with a diverse range of nucleophiles has been used to generate over 50 novel amidino-rocaglate (ADR) and amino-rocaglate derivatives. Subsequently, these derivatives were evaluated for their ability to inhibit cap-dependent protein synthesis where they were found to outperform previous lead compounds including the rocaglate hydroxamate CR-1-31-B.
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Amidinas/farmacología , Antineoplásicos/farmacología , Factor 4A Eucariótico de Iniciación/antagonistas & inhibidores , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Amidinas/síntesis química , Animales , Antineoplásicos/síntesis química , Línea Celular Tumoral , Ensayos de Selección de Medicamentos Antitumorales , Compuestos Heterocíclicos de 4 o más Anillos/síntesis química , Humanos , Ratones , Modelos Químicos , Estructura Molecular , Células 3T3 NIH , Relación Estructura-ActividadRESUMEN
BACKGROUND: Asthmatic and allergic inflammation is mediated by TH2 cytokines (IL-4, IL-5, and IL-13). Although we have learned much about how TH2 cells are differentiated, the TH2 checkpoint mechanisms remain elusive. OBJECTIVES: In this study we investigate how monocyte chemotactic protein-induced protein 1 (MCPIP1; encoded by the Zc3h12a gene) regulates IL-5-producing TH2 cell differentiation and TH2-mediated inflammation. METHODS: The functions of Zc3h12a-/- CD4 T cells were evaluated by checking the expression of TH2 cytokines and transcription factors in vivo and in vitro. Allergic airway inflammation of Zc3h12a-/- mice was examined with murine asthma models. In addition, antigen-specific CD4 T cells deficient in MCPIP1 were transferred to wild-type recipient mice, challenged with ovalbumin (OVA) or house dust mite (HDM), and accessed for TH2 inflammation. RESULTS: Zc3h12a-/- mice have spontaneous severe lung inflammation, with an increase in mainly IL-5- and IL-13-producing but not IL-4-producing TH2 cells in the lung. Mechanistically, differentiation of IL-5-producing Zc3h12a-/- TH2 cells is mediated through Notch signaling and Gata3 independent of IL-4. Gata3 mRNA is stabilized in Zc3h12a-/- TH2 cells. MCPIP1 promotes Gata3 mRNA decay through the RNase domain. Furthermore, deletion of MCPIP1 in OVA- or HDM-specific T cells leads to significantly increased TH2-mediated airway inflammation in OVA or HDM murine models of asthma. CONCLUSIONS: Our study reveals that MCPIP1 regulates the development and function of IL-5-producing TH2 cells through the Notch/Gata3 pathway. MCPIP1 represents a new and promising target for the treatment of asthma and other TH2-mediated diseases.
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Asma/inmunología , Inflamación/inmunología , Hipersensibilidad Respiratoria/inmunología , Ribonucleasas/metabolismo , Células Th2/inmunología , Traslado Adoptivo , Animales , Diferenciación Celular , Células Cultivadas , Modelos Animales de Enfermedad , Factor de Transcripción GATA3/metabolismo , Humanos , Terapia de Inmunosupresión , Interleucina-13/metabolismo , Interleucina-5/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores Notch/metabolismo , Ribonucleasas/genética , Transducción de Señal , Células Th2/trasplanteRESUMEN
RATIONALE: The mosquito-borne Zika virus (ZIKV) is now recognized as a blood-borne pathogen, raising an important question about how the virus gets into human bloodstream. The imminent threat of the ZIKV epidemic to the global blood supply also demands novel therapeutics to stop virus transmission though transfusion. OBJECTIVE: We intend to characterize ZIKV tropism for human endothelial cells (ECs) and provide potential targets for intervention. METHODS AND RESULTS: We conducted immunostaining, plaque assay, and quantitative reverse transcription-polymerase chain reaction of ZIKV RNA to evaluate the possible infection of ECs by ZIKV. Both the African and the South American ZIKV strains readily infect human umbilical vein endothelial cells and human ECs derived from aortic and coronary artery, as well as the saphenous vein. Infected ECs released infectious progeny virus. Compared with the African strains, South American ZIKV isolates replicate faster in ECs and are partially cytopathic, suggesting enhanced virulence of these isolates. Flow cytometric analyses showed that the susceptibility of ECs positively correlated with the cell surface levels of tyrosine-protein kinase receptor UFO (AXL) receptor tyrosine kinase. Gain- and loss-of-function studies further revealed that AXL is required for ZIKV entry at a postbinding step. Finally, small-molecule inhibitors of the AXL kinase significantly reduced ZIKA infection of ECs. CONCLUSIONS: We identified EC as a key cell type for ZIKV infection. These data support the view of hematogenous dissemination of ZIKV and implicate AXL as a new target for antiviral therapy.
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Células Endoteliales de la Vena Umbilical Humana/enzimología , Células Endoteliales de la Vena Umbilical Humana/virología , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Infección por el Virus Zika/enzimología , Virus Zika/enzimología , Anilidas/farmacología , Animales , Línea Celular Transformada , Chlorocebus aethiops , Cricetinae , Relación Dosis-Respuesta a Droga , Células Endoteliales/efectos de los fármacos , Células Endoteliales/enzimología , Células Endoteliales/virología , Células HEK293 , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Humanos , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Piridinas/farmacología , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Células Vero , Virus Zika/efectos de los fármacos , Tirosina Quinasa del Receptor AxlRESUMEN
Toll-like receptor-3 (TLR3) senses double-stranded RNA intermediates produced during hepatitis C virus (HCV) replication, leading to activation of interferon regulatory factor-3 (IRF3) and NF-κB and subsequent antiviral and proinflammatory responses. Yet, how this TLR3-dependent pathway operates in hepatocytes is unclear. Upon fractionating cultured hepatocytes into various cellular organelles, we observed that TLR3 predominantly resides in endolysosomes of hepatocytes. To determine the critical regulators of TLR3 signaling in response to HCV infection in human hepatocytes, we isolated endolysosome fractions from mock-infected and HCV-infected hepatoma Huh7.5 cells that had been reconstituted for TLR3 expression, separated these fractions on two-dimensional gels, and identified up-regulated/down-regulated proteins by mass spectrometry. Approximately a dozen of cellular proteins were found to be differentially expressed in endolysosome fractions following HCV infection. Of these, expression of several molecular chaperone proteins was elevated. Knockdown of one of these chaperones, glucose-regulated protein 78 kDa (GRP78), compromised TLR3-dependent induction of interferon-stimulated genes and chemokines following HCV infection or poly(I:C) stimulation in cultured hepatocytes. Consistent with this finding, GRP78 depletion impaired TLR3-mediated establishment of an antiviral state. Mechanistically, although TLR3 trafficking to endolysosomes was not affected, phosphorylated IRF3 diminished faster following GRP78 knockdown. Remarkably, GRP78 transcript was significantly up-regulated in liver biopsies of chronic hepatitis C patients as compared with normal liver tissues. Moreover, the GRP78 expression level correlated with that of RANTES (regulated upon activation, normal T-cell expressed and secreted) and CXCL10, two inflammatory chemokines most frequently elevated in HCV-infected liver. Altogether, our data suggest that GRP78 contributes to TLR3-mediated, IRF3-dependent innate immune response to HCV in hepatocytes.
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Proteínas de Choque Térmico/metabolismo , Hepacivirus/inmunología , Hepatocitos/metabolismo , Inmunidad Innata , Receptor Toll-Like 3/metabolismo , Adulto , Línea Celular Tumoral , Células Cultivadas , Quimiocina CCL5/genética , Quimiocina CCL5/metabolismo , Quimiocina CXCL10/genética , Quimiocina CXCL10/metabolismo , Electroforesis en Gel Bidimensional , Chaperón BiP del Retículo Endoplásmico , Endosomas/metabolismo , Endosomas/virología , Femenino , Expresión Génica/efectos de los fármacos , Proteínas de Choque Térmico/genética , Hepacivirus/fisiología , Hepatitis C Crónica/genética , Hepatitis C Crónica/metabolismo , Hepatitis C Crónica/virología , Hepatocitos/efectos de los fármacos , Hepatocitos/virología , Interacciones Huésped-Patógeno , Humanos , Factor 3 Regulador del Interferón/genética , Factor 3 Regulador del Interferón/metabolismo , Lisosomas/metabolismo , Lisosomas/virología , Masculino , Microscopía Confocal , Persona de Mediana Edad , Poli I-C/farmacología , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Receptor Toll-Like 3/genéticaRESUMEN
The innate immune system plays a critical role in pathogen recognition and initiation of protective immune response through the recognition of pathogen associated molecular patterns (PAMPs) by its pattern recognition receptors (PRRs). Nucleic acids including RNA and DNA have been recognized as very important PAMPs of pathogens especially for viruses. RNA are the major PAMPs of RNA viruses, to which most severe disease causing viruses belong thus posing a tougher challenge to human and animal health. Therefore, the understanding of the immune biology of RNA PRRs is critical for control of pathogen infections especially for RNA virus infections. RNA PRRs are comprised of TLR3, TLR7, TLR8, RIG-I, MDA5, NLRP3, NOD2, and some other minorities. This review introduces these RNA PRRs by describing the cellular localizations, ligand recognitions, activation mechanisms, cell signaling pathways, and recognition of pathogens; the cross-talks between various RNA PRRs are also reviewed. The deep insights of these RNA PRRs can be utilized to improve anti-viral immune response. © 2017 IUBMB Life, 69(5):297-304, 2017.
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Inmunidad Innata/fisiología , Moléculas de Patrón Molecular Asociado a Patógenos , Virus ARN/patogenicidad , ARN/inmunología , Receptores de Reconocimiento de Patrones/fisiología , Animales , Interacciones Huésped-Patógeno/inmunología , Humanos , ARN/metabolismo , Infecciones por Virus ARN/inmunología , ARN Viral/inmunología , ARN Viral/metabolismo , Receptores Toll-Like/inmunología , Receptores Toll-Like/metabolismoRESUMEN
BACKGROUND: Middle East Respiratory Syndrome coronavirus (MERS-CoV) is an emerging viral pathogen that causes severe morbidity and mortality. Up to date, there is no approved or licensed vaccine or antiviral medicines can be used to treat MERS-CoV-infected patients. Here, we analyzed the antiviral activities of resveratrol, a natural compound found in grape seeds and skin and in red wine, against MERS-CoV infection. METHODS: We performed MTT and neutral red uptake assays to assess the survival rates of MERS-infected Vero E6 cells. In addition, quantitative PCR, western blotting, and immunofluorescent assays determined the intracellular viral RNA and protein expression. For viral productivity, we utilized plaque assays to confirm the antiviral properties of resveratrol against MERS-CoV. RESULTS: Resveratrol significantly inhibited MERS-CoV infection and prolonged cellular survival after virus infection. We also found that the expression of nucleocapsid (N) protein essential for MERS-CoV replication was decreased after resveratrol treatment. Furthermore, resveratrol down-regulated the apoptosis induced by MERS-CoV in vitro. By consecutive administration of resveratrol, we were able to reduce the concentration of resveratrol while achieving inhibitory effectiveness against MERS-CoV. CONCLUSION: In this study, we first demonstrated that resveratrol is a potent anti-MERS agent in vitro. We perceive that resveratrol can be a potential antiviral agent against MERS-CoV infection in the near future.
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Antivirales/farmacología , Infecciones por Coronavirus/tratamiento farmacológico , Infecciones por Coronavirus/virología , Coronavirus del Síndrome Respiratorio de Oriente Medio/efectos de los fármacos , Coronavirus del Síndrome Respiratorio de Oriente Medio/patogenicidad , Estilbenos/farmacología , Animales , Chlorocebus aethiops , Unión Proteica , ARN Viral/metabolismo , Proteínas de Unión al ARN/metabolismo , Resveratrol , Células Vero , Proteínas Virales/metabolismoRESUMEN
We have previously shown that the cellular RNase MCPIP1/regnase-1 potently blocks HIV-1 infection in resting CD4+ T-cells. As simian immunodeficiency virus (SIV) encodes an accessory protein named Vpx, which enhances viral replication in resting CD4+ T-cells by degrading the cellular restriction factor SAMHD1, we investigated whether MCPIP1 restricts SIV infection and whether Vpx protein antagonizes MCPIP1-mediated restriction. In co-transfection studies, human MCPIP1 markedly reduced the production of infectious SIV, whereas MCPIP2 and MCPIP3 had little effect. MCPIP1 derived from cynomolgus monkey also inhibited human immunodeficiency virus (HIV-1) and SIV production, albeit to a lesser degree. Lastly, expression of SIV Vpx protein did not reduce MCPIP1 at the protein level, nor did it ablate the MCPIP1-mediated restriction. In conclusion, both human and cynomolgus monkey MCPIP1 restrict SIV replication. Unlike SAMHD1, MCPIP1-mediated HIV-1 restriction cannot be overcome by SIV Vpx.
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VIH-1/metabolismo , Interacciones Huésped-Patógeno/fisiología , Ribonucleasas/metabolismo , Virus de la Inmunodeficiencia de los Simios/metabolismo , Factores de Transcripción/metabolismo , Proteínas Reguladoras y Accesorias Virales/metabolismo , Replicación Viral/fisiología , Desaminasa APOBEC-3G/metabolismo , Animales , Linfocitos T CD4-Positivos/virología , Línea Celular , Células HEK293 , Células HeLa , Humanos , Macaca fascicularis , Proteínas de Unión al GTP Monoméricas/metabolismo , ARN Mensajero/metabolismo , ARN Viral/metabolismo , Proteína 1 que Contiene Dominios SAM y HDRESUMEN
Antigenic assessments of SARS-CoV-2 variants inform decisions to update COVID-19 vaccines. Primary infection sera are often used for assessments, but such sera are rare due to population immunity from SARS-CoV-2 infections and COVID-19 vaccinations. Here, we show that neutralization titers and breadth of matched human and hamster pre-Omicron variant primary infection sera correlate well and generate similar antigenic maps. The hamster antigenic map shows modest antigenic drift among XBB sub-lineage variants, with JN.1 and BA.4/BA.5 variants within the XBB cluster, but with five to six-fold antigenic differences between these variants and XBB.1.5. Compared to sera following only ancestral or bivalent COVID-19 vaccinations, or with post-vaccination infections, XBB.1.5 booster sera had the broadest neutralization against XBB sub-lineage variants, although a five-fold titer difference was still observed between JN.1 and XBB.1.5 variants. These findings suggest that antibody coverage of antigenically divergent JN.1 could be improved with a matched vaccine antigen.
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Background: Neutralising antibodies against SARS-CoV-2 are a vital component in the fight against COVID-19 pandemic, having the potential of both therapeutic and prophylactic applications. Bispecific antibodies (BsAbs) against SARS-CoV-2 are particularly promising, given their ability to bind simultaneously to two distinct sites of the receptor-binding domain (RBD) of the viral spike protein. Such antibodies are complex molecules associated with multi-faceted mechanisms of action that require appropriate bioassays to ensure product quality and manufacturing consistency. Methods: We developed procedures for biolayer interferometry (BLI) and a cell-based virus neutralisation assay, the focus reduction neutralisation test (FRNT). Using both assays, we tested a panel of five BsAbs against different spike variants (Ancestral, Delta and Omicron) to evaluate the use of these analytical methods in assessing binding and neutralisation activities of anti-SARS-CoV-2 therapeutics. Results: We found comparable trends between BLI-derived binding affinity and FRNT-based virus neutralisation activity. Antibodies that displayed high binding affinity against a variant were often followed by potent neutralisation at lower concentrations, whereas those with low binding affinity also demonstrated reduced neutralisation activity. Conclusion: The results support the utility of BLI and FRNT assays in measuring variant-specific binding and virus neutralisation activity of anti-SARS-CoV-2 antibodies.
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Detection of secretory antibodies in the airway is highly desirable when evaluating mucosal protection by vaccines against a respiratory virus, such as the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We show that intranasal delivery of an attenuated SARS-CoV-2 (Nsp1-K164A/H165A) induces both mucosal and systemic IgA and IgG in male Syrian hamsters. Interestingly, either direct intranasal immunization or airborne transmission-mediated delivery of Nsp1-K164A/H165A in Syrian hamsters offers protection against heterologous challenge with variants of concern (VOCs) including Delta, Omicron BA.1, BA.2.12.1 and BA.5. Vaccinated animals show significant reduction in both tissue viral loads and lung inflammation. Similarly attenuated viruses bearing BA.1 and BA.5 spike boost variant-specific neutralizing antibodies in male mice that were first vaccinated with modified vaccinia virus Ankara vectors (MVA) expressing full-length WA1/2020 Spike protein. Together, these results demonstrate that our attenuated virus may be a promising nasal vaccine candidate for boosting mucosal immunity against future SARS-CoV-2 VOCs.
Asunto(s)
COVID-19 , Masculino , Cricetinae , Animales , Ratones , COVID-19/prevención & control , Mesocricetus , Aerosoles y Gotitas Respiratorias , SARS-CoV-2 , Anticuerpos Neutralizantes , Glicoproteína de la Espiga del Coronavirus/genética , Anticuerpos AntiviralesRESUMEN
Despite being more transmissible, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variant only causes milder diseases in laboratory animals, often accompanied by a lower viral load compared with previous variants of concern. In this study, we report the structural basis for a robust interaction between the receptor-binding domain of the Omicron spike protein and mouse ACE2. We show that pseudovirus bearing the Omicron spike protein efficiently utilizes mouse ACE2 for entry. By comparing viral load and disease severity among laboratory mice infected by a natural Omicron variant or recombinant ancestral viruses bearing either the entire Omicron spike or only the N501Y/Q493R mutations in its spike, we find that mutations outside the spike protein in the Omicron variant may be responsible for the observed lower viral load. Together, our results imply that a post-entry block to the Omicron variant exists in laboratory mice.