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1.
Methods Mol Biol ; 2808: 1-7, 2024.
Artículo en Inglés | MEDLINE | ID: mdl-38743358

RESUMEN

We have adopted a real-time assay based on a dual-split reporter to assess cell-cell fusion mediated by the measles virus (MeV) membrane fusion machinery. This reporter system is comprised of two expression vectors, each encoding a segment of Renilla luciferase fused to a segment of GFP. To regain function, the two segments need to associate, which is dependent on cell-cell fusion between effector cells expressing the MeV fusion machinery and target cells expressing the corresponding MeV receptor. By measuring reconstituted luciferase activity, we can follow the kinetics of cell-cell fusion and quantify the extent of fusion. This assay lends itself to the study of the MeV fusion machinery comprised of the attachment and fusion glycoproteins, the matrix protein, and the MeV receptors. Moreover, entry inhibitors targeting attachment or fusion can be readily screened using this assay. Finally, this assay can be easily adopted to study the entry of other members of the Paramyxoviridae, as we have demonstrated for the henipaviruses.


Asunto(s)
Fusión Celular , Virus del Sarampión , Internalización del Virus , Virus del Sarampión/genética , Virus del Sarampión/fisiología , Humanos , Animales , Fusión Celular/métodos , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Chlorocebus aethiops , Línea Celular , Células Vero , Luciferasas de Renilla/genética , Luciferasas de Renilla/metabolismo
2.
Methods Mol Biol ; 2808: 57-70, 2024.
Artículo en Inglés | MEDLINE | ID: mdl-38743362

RESUMEN

RNA viruses generate defective genomes naturally during virus replication. Defective genomes that interfere with the infection dynamics either through resource competition or by interferon stimulation are known as defective interfering (DI) genomes. DI genomes can be successfully packaged into virus-like-particles referred to as defective interfering particles (DIPs). Such DIPs can sustainably coexist with the full-length virus particles and have been shown to negatively impact virus replication in vitro and in vivo. Here, we describe a method to generate a clonal DI genome population by reverse genetics. This method is applicable to other RNA viruses and will enable assessment of DIPs for their antiviral properties.


Asunto(s)
Virus Defectuosos , Genoma Viral , Morbillivirus , Genética Inversa , Replicación Viral , Genética Inversa/métodos , Virus Defectuosos/genética , Animales , Replicación Viral/genética , Morbillivirus/genética , Humanos , Virión/genética , Células Vero , Chlorocebus aethiops , ARN Viral/genética
3.
Methods Mol Biol ; 2808: 209-224, 2024.
Artículo en Inglés | MEDLINE | ID: mdl-38743373

RESUMEN

The plaque reduction neutralization test (PRNT) and the enzyme-linked immunosorbent assay (ELISA) are both widely used to assess immunity to infectious diseases such as measles, but they use two different measurement principles: ELISA measures the ability of antibodies to bind to virus components, while the PRNT detects the aptitude of antibodies to prevent the infection of a susceptible cell. As a result, detection of measles virus (MV) neutralizing antibodies is the gold standard for assessing immunity to measles. However, the assay is laborious and requires experience and excellent technical skills. In addition, the result is only available after several days. Therefore, the classical PRNT is not suitable for high-throughput testing. By using an immunocolorimetric assay (ICA) to detect MV-infected cells, the standard PRNT has been developed into a focus reduction neutralization test (FRNT). This assay is faster and has improved specificity. The FRNT described here is extremely useful when immunity to measles virus needs to be assessed in patients with a specific medical condition, such as immunocompromised individuals in whom presumed residual immunity needs to be assessed. The FRNT is not generally recommended for use with large numbers of specimens, such as in a seroprevalence study.


Asunto(s)
Anticuerpos Neutralizantes , Anticuerpos Antivirales , Virus del Sarampión , Sarampión , Pruebas de Neutralización , Pruebas de Neutralización/métodos , Virus del Sarampión/inmunología , Sarampión/inmunología , Sarampión/diagnóstico , Sarampión/virología , Humanos , Anticuerpos Neutralizantes/inmunología , Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/inmunología , Chlorocebus aethiops , Animales , Células Vero , Ensayo de Placa Viral/métodos , Ensayo de Inmunoadsorción Enzimática/métodos
4.
Molecules ; 29(9)2024 Apr 29.
Artículo en Inglés | MEDLINE | ID: mdl-38731543

RESUMEN

Ribonuclease P (RNase P) complexed with an external guide sequence (EGS) represents a promising nucleic acid-based gene targeting approach for gene expression knock-down and modulation. The RNase P-EGS strategy is unique as an EGS can be designed to basepair any mRNA sequence and recruit intracellular RNase P for hydrolysis of the target mRNA. In this study, we provide the first direct evidence that the RNase P-based approach effectively blocks the gene expression and replication of herpes simplex virus 2 (HSV-2), the causative agent of genital herpes. We constructed EGSs to target the mRNA encoding HSV-2 single-stranded DNA binding protein ICP8, which is essential for viral DNA genome replication and growth. In HSV-2 infected cells expressing a functional EGS, ICP8 levels were reduced by 85%, and viral growth decreased by 3000 folds. On the contrary, ICP8 expression and viral growth exhibited no substantial differences between cells expressing no EGS and those expressing a disabled EGS with mutations precluding RNase P recognition. The anti-ICP8 EGS is specific in targeting ICP8 because it only affects ICP8 expression but does not affect the expression of the other viral immediate-early and early genes examined. This study shows the effective and specific anti-HSV-2 activity of the RNase P-EGS approach and demonstrates the potential of EGS RNAs for anti-HSV-2 applications.


Asunto(s)
Regulación Viral de la Expresión Génica , Herpesvirus Humano 2 , Replicación Viral , Herpesvirus Humano 2/genética , Herpesvirus Humano 2/fisiología , Humanos , Ribonucleasa P/metabolismo , Ribonucleasa P/genética , Animales , Proteínas Virales/genética , Proteínas Virales/metabolismo , Chlorocebus aethiops , ARN Mensajero/genética , ARN Mensajero/metabolismo , Células Vero , Proteínas Inmediatas-Precoces/genética , Proteínas Inmediatas-Precoces/metabolismo , Proteínas de Unión al ADN
5.
BMC Vet Res ; 20(1): 192, 2024 May 11.
Artículo en Inglés | MEDLINE | ID: mdl-38734600

RESUMEN

BACKGROUND: Natural antimicrobial agents such as nisin were used to control the growth of foodborne pathogens in dairy products. The current study aimed to examine the inhibitory effect of pure nisin and nisin nanoparticles (nisin NPs) against methicillin resistant Staphylococcus aureus (MRSA) and E.coli O157:H7 during the manufacturing and storage of yoghurt. Nisin NPs were prepared using new, natural, and safe nano-precipitation method by acetic acid. The prepared NPs were characterized using zeta-sizer and transmission electron microscopy (TEM). In addition, the cytotoxicity of nisin NPs on vero cells was assessed using the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The minimum inhibitory concentrations (MICs) of nisin and its nanoparticles were determined using agar well-diffusion method. Further, fresh buffalo's milk was inoculated with MRSA or E.coli O157:H7 (1 × 106 CFU/ml) with the addition of either nisin or nisin NPs, and then the inoculated milk was used for yoghurt making. The organoleptic properties, pH and bacterial load of the obtained yoghurt were evaluated during storage in comparison to control group. RESULTS: The obtained results showed a strong antibacterial activity of nisin NPs (0.125 mg/mL) against MRSA and E.coli O157:H7 in comparison with control and pure nisin groups. Notably, complete eradication of MRSA and E.coli O157:H7 was observed in yoghurt formulated with nisin NPs after 24 h and 5th day of storage, respectively. The shelf life of yoghurt inoculated with nisin nanoparticles was extended than those manufactured without addition of such nanoparticles. CONCLUSIONS: Overall, the present study indicated that the addition of nisin NPs during processing of yoghurt could be a useful tool for food preservation against MRSA and E.coli O157:H7 in dairy industry.


Asunto(s)
Antibacterianos , Escherichia coli O157 , Staphylococcus aureus Resistente a Meticilina , Pruebas de Sensibilidad Microbiana , Nanopartículas , Nisina , Yogur , Nisina/farmacología , Nisina/química , Yogur/microbiología , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Escherichia coli O157/efectos de los fármacos , Nanopartículas/química , Animales , Antibacterianos/farmacología , Antibacterianos/química , Conservantes de Alimentos/farmacología , Células Vero , Microbiología de Alimentos , Chlorocebus aethiops , Conservación de Alimentos/métodos
6.
Signal Transduct Target Ther ; 9(1): 125, 2024 May 11.
Artículo en Inglés | MEDLINE | ID: mdl-38734691

RESUMEN

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a 'highly transmissible respiratory pathogen, leading to severe multi-organ damage. However, knowledge regarding SARS-CoV-2-induced cellular alterations is limited. In this study, we report that SARS-CoV-2 aberrantly elevates mitochondrial bioenergetics and activates the EGFR-mediated cell survival signal cascade during the early stage of viral infection. SARS-CoV-2 causes an increase in mitochondrial transmembrane potential via the SARS-CoV-2 RNA-nucleocapsid cluster, thereby abnormally promoting mitochondrial elongation and the OXPHOS process, followed by enhancing ATP production. Furthermore, SARS-CoV-2 activates the EGFR signal cascade and subsequently induces mitochondrial EGFR trafficking, contributing to abnormal OXPHOS process and viral propagation. Approved EGFR inhibitors remarkably reduce SARS-CoV-2 propagation, among which vandetanib exhibits the highest antiviral efficacy. Treatment of SARS-CoV-2-infected cells with vandetanib decreases SARS-CoV-2-induced EGFR trafficking to the mitochondria and restores SARS-CoV-2-induced aberrant elevation in OXPHOS process and ATP generation, thereby resulting in the reduction of SARS-CoV-2 propagation. Furthermore, oral administration of vandetanib to SARS-CoV-2-infected hACE2 transgenic mice reduces SARS-CoV-2 propagation in lung tissue and mitigates SARS-CoV-2-induced lung inflammation. Vandetanib also exhibits potent antiviral activity against various SARS-CoV-2 variants of concern, including alpha, beta, delta and omicron, in in vitro cell culture experiments. Taken together, our findings provide novel insight into SARS-CoV-2-induced alterations in mitochondrial dynamics and EGFR trafficking during the early stage of viral infection and their roles in robust SARS-CoV-2 propagation, suggesting that EGFR is an attractive host target for combating COVID-19.


Asunto(s)
COVID-19 , Receptores ErbB , Mitocondrias , SARS-CoV-2 , Replicación Viral , SARS-CoV-2/efectos de los fármacos , Mitocondrias/metabolismo , Mitocondrias/genética , Mitocondrias/efectos de los fármacos , Humanos , Animales , Ratones , COVID-19/virología , COVID-19/metabolismo , COVID-19/genética , Receptores ErbB/metabolismo , Receptores ErbB/genética , Replicación Viral/efectos de los fármacos , Metabolismo Energético/efectos de los fármacos , Metabolismo Energético/genética , Células Vero , Chlorocebus aethiops , Antivirales/farmacología , Tratamiento Farmacológico de COVID-19 , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Fosforilación Oxidativa/efectos de los fármacos , Transducción de Señal/efectos de los fármacos
7.
BMC Mol Cell Biol ; 25(1): 15, 2024 May 13.
Artículo en Inglés | MEDLINE | ID: mdl-38741034

RESUMEN

BACKGROUND: Transfection is an important analytical method for studying gene expression in the cellular environment. There are some barriers to efficient DNA transfection in host cells, including circumventing the plasma membrane, escaping endosomal compartmentalization, autophagy, immune sensing pathways, and translocating the nuclear envelope. Therefore, it would be very useful to introduce an optimum transfection approach to achieve a high transfection efficiency in the Vero cell line. The aim of this study was to compare various transfection techniques and introduce a highly efficient method for gene delivery in Vero cells. METHODS: In the current study, three transfection methods were used, including chemical transfection, electroporation, and lentiviral vector transduction, to obtain the optimum transfection conditions in the Vero cell line. Vero cells were cultured and transfected with chemical transfection reagents, electroporation, or HIV-1-based lentivectors under different experimental conditions. Transfection efficiency was assessed using flow cytometry and fluorescence microscopy to detect GFP-positive cells. RESULTS: Among the tested methods, TurboFect™ chemical transfection exhibited the highest efficiency. Optimal transfection conditions were achieved using 1 µg DNA and 4 µL TurboFect™ in 6 × 104 Vero cells. CONCLUSION: TurboFect™, a cationic polymer transfection reagent, demonstrated superior transfection efficiency in Vero cells compared with electroporation and lentivirus particles, and is the optimal choice for chemical transfection in the Vero cell line.


Asunto(s)
Electroporación , Vectores Genéticos , Transfección , Animales , Chlorocebus aethiops , Células Vero , Electroporación/métodos , Transfección/métodos , Vectores Genéticos/genética , Lentivirus/genética , Transducción Genética/métodos , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos
8.
Org Biomol Chem ; 22(19): 3986-3994, 2024 05 15.
Artículo en Inglés | MEDLINE | ID: mdl-38695061

RESUMEN

Algae-based marine carbohydrate drugs are typically decorated with negative ion groups such as carboxylate and sulfate groups. However, the precise synthesis of highly sulfated alginates is challenging, thus impeding their structure-activity relationship studies. Herein we achieve a microwave-assisted synthesis of a range of highly sulfated mannuronate glycans with up to 17 sulfation sites by overcoming the incomplete sulfation due to the electrostatic repulsion of crowded polyanionic groups. Although the partially sulfated tetrasaccharide had the highest affinity for the receptor binding domain (RBD) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variant, the fully sulfated octasaccharide showed the most potent interference with the binding of the RBD to angiotensin-converting enzyme 2 (ACE2) and Vero E6 cells, indicating that the sulfated oligosaccharides might inhibit the RBD binding to ACE2 in a length-dependent manner.


Asunto(s)
Enzima Convertidora de Angiotensina 2 , Antivirales , Microondas , Polisacáridos , SARS-CoV-2 , SARS-CoV-2/efectos de los fármacos , Antivirales/farmacología , Antivirales/síntesis química , Antivirales/química , Chlorocebus aethiops , Enzima Convertidora de Angiotensina 2/metabolismo , Enzima Convertidora de Angiotensina 2/antagonistas & inhibidores , Enzima Convertidora de Angiotensina 2/química , Células Vero , Polisacáridos/química , Polisacáridos/farmacología , Polisacáridos/síntesis química , Humanos , Animales , Glicoproteína de la Espiga del Coronavirus/antagonistas & inhibidores , Glicoproteína de la Espiga del Coronavirus/metabolismo , Glicoproteína de la Espiga del Coronavirus/química , Ácidos Hexurónicos/química , Ácidos Hexurónicos/farmacología , Ácidos Hexurónicos/síntesis química , Sulfatos/química , Sulfatos/farmacología , Sulfatos/síntesis química , Tratamiento Farmacológico de COVID-19 , Relación Estructura-Actividad
9.
Biochem Biophys Res Commun ; 716: 149954, 2024 Jul 05.
Artículo en Inglés | MEDLINE | ID: mdl-38704887

RESUMEN

Membrane lipids and proteins form dynamic domains crucial for physiological and pathophysiological processes, including viral infection. Many plasma membrane proteins, residing within membrane domains enriched with cholesterol (CHOL) and sphingomyelin (SM), serve as receptors for attachment and entry of viruses into the host cell. Among these, human coronaviruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), use proteins associated with membrane domains for initial binding and internalization. We hypothesized that the interaction of lipid-binding proteins with CHOL in plasma membrane could sequestrate lipids and thus affect the efficiency of virus entry into host cells, preventing the initial steps of viral infection. We have prepared CHOL-binding proteins with high affinities for lipids in the plasma membrane of mammalian cells. Binding of the perfringolysin O domain four (D4) and its variant D4E458L to membrane CHOL impaired the internalization of the receptor-binding domain of the SARS-CoV-2 spike protein and the pseudovirus complemented with the SARS-CoV-2 spike protein. SARS-CoV-2 replication in Vero E6 cells was also decreased. Overall, our results demonstrate that the integrity of CHOL-rich membrane domains and the accessibility of CHOL in the membrane play an essential role in SARS-CoV-2 cell entry.


Asunto(s)
Membrana Celular , Colesterol , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus , Internalización del Virus , Células Vero , Chlorocebus aethiops , Colesterol/metabolismo , Animales , SARS-CoV-2/metabolismo , SARS-CoV-2/fisiología , Membrana Celular/metabolismo , Membrana Celular/virología , Glicoproteína de la Espiga del Coronavirus/metabolismo , Glicoproteína de la Espiga del Coronavirus/química , Humanos , Proteínas Portadoras/metabolismo , COVID-19/virología , COVID-19/metabolismo , Unión Proteica
10.
Arch Microbiol ; 206(6): 269, 2024 May 20.
Artículo en Inglés | MEDLINE | ID: mdl-38767708

RESUMEN

Bacteriocins are ribosomally synthesized bacterial peptides endowed with antibacterial, antiprotozoal, anticancer and antiviral activities. In the present study, we evaluated the antiviral activities of two bacteriocins, enterocin DD14 (EntDD14) and lacticaseicin 30, against herpes simplex virus type 1 (HSV-1), human coronavirus 229E (HCoV-229E) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in Vero, Huh7 and Vero E6 cells, respectively. In addition, the interactions of these bacteriocins with the envelope glycoprotein D of HSV-1 and the receptor binding domains of HCoV-229E and SARS-CoV-2 have been computationally evaluated using protein-protein docking and molecular dynamics simulations. HSV-1 replication in Vero cells was inhibited by EntDD14 and, to a lesser extent, by lacticaseicin 30 added to cells after virus inoculation. EntDD14 and lacticaseicin 30 had no apparent antiviral activity against HCoV-229E; however, EntDD14 was able to inhibit SARS-CoV-2 in Vero E6 cells. Further studies are needed to elucidate the antiviral mechanism of these bacteriocins.


Asunto(s)
Antivirales , Bacteriocinas , SARS-CoV-2 , Bacteriocinas/farmacología , Chlorocebus aethiops , Animales , Antivirales/farmacología , Células Vero , Humanos , SARS-CoV-2/efectos de los fármacos , Replicación Viral/efectos de los fármacos , Herpesvirus Humano 1/efectos de los fármacos , Herpesvirus Humano 1/fisiología , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Hidrocarburos Aromáticos con Puentes
11.
Int J Mol Sci ; 25(9)2024 May 02.
Artículo en Inglés | MEDLINE | ID: mdl-38732185

RESUMEN

Herpes simplex virus (HSV) infections are highly widespread among humans, producing symptoms ranging from ulcerative lesions to severe diseases such as blindness and life-threatening encephalitis. At present, there are no vaccines available, and some existing antiviral treatments can be ineffective or lead to adverse effects. As a result, there is a need for new anti-HSV drugs. In this report, the in vitro anti-HSV effect of 9,9'-norharmane dimer (nHo-dimer), which belongs to the ß-carboline (ßC) alkaloid family, was evaluated. The dimer exhibited no virucidal properties and did not impede either the attachment or penetration steps of viral particles. The antiviral effect was only exerted under the constant presence of the dimer in the incubation media, and the mechanism of action was found to involve later events of virus infection. Analysis of fluorescence lifetime imaging data showed that the nHo-dimer internalized well into the cells when present in the extracellular incubation medium, with a preferential accumulation into perinuclear organelles including mitochondria. After washing the host cells with fresh medium free of nHo-dimer, the signal decreased, suggesting the partial release of the compound from the cells. This agrees with the observation that the antiviral effect is solely manifested when the alkaloid is consistently present in the incubation media.


Asunto(s)
Antivirales , Antivirales/farmacología , Antivirales/química , Chlorocebus aethiops , Humanos , Células Vero , Animales , Simplexvirus/efectos de los fármacos , Simplexvirus/fisiología , Herpes Simple/tratamiento farmacológico , Herpes Simple/virología , Carbolinas/farmacología , Carbolinas/química , Herpesvirus Humano 1/efectos de los fármacos , Herpesvirus Humano 1/fisiología , Harmina/farmacología , Harmina/química , Harmina/análogos & derivados
12.
Int J Mol Sci ; 25(9)2024 May 03.
Artículo en Inglés | MEDLINE | ID: mdl-38732202

RESUMEN

Acquiring resistance against antiviral drugs is a significant problem in antimicrobial therapy. In order to identify novel antiviral compounds, the antiviral activity of eight plants indigenous to the southern region of Hungary against herpes simplex virus-2 (HSV-2) was investigated. The plant extracts and the plant compound carnosic acid were tested for their effectiveness on both the extracellular and intracellular forms of HSV-2 on Vero and HeLa cells. HSV-2 replication was measured by a direct quantitative PCR (qPCR). Among the tested plant extracts, Salvia rosmarinus (S. rosmarinus) exhibited a 90.46% reduction in HSV-2 replication at the 0.47 µg/mL concentration. Carnosic acid, a major antimicrobial compound found in rosemary, also demonstrated a significant dose-dependent inhibition of both extracellular and intracellular forms of HSV-2. The 90% inhibitory concentration (IC90) of carnosic acid was between 25 and 6.25 µg/mL. Proteomics and high-resolution respirometry showed that carnosic acid suppressed key ATP synthesis pathways such as glycolysis, citrate cycle, and oxidative phosphorylation. Inhibition of oxidative phosphorylation also suppressed HSV-2 replication up to 39.94-fold. These results indicate that the antiviral action of carnosic acid includes the inhibition of ATP generation by suppressing key energy production pathways. Carnosic acid holds promise as a potential novel antiviral agent against HSV-2.


Asunto(s)
Abietanos , Adenosina Trifosfato , Antivirales , Herpesvirus Humano 2 , Extractos Vegetales , Replicación Viral , Abietanos/farmacología , Replicación Viral/efectos de los fármacos , Chlorocebus aethiops , Células Vero , Adenosina Trifosfato/metabolismo , Adenosina Trifosfato/biosíntesis , Humanos , Animales , Herpesvirus Humano 2/efectos de los fármacos , Herpesvirus Humano 2/fisiología , Antivirales/farmacología , Extractos Vegetales/farmacología , Extractos Vegetales/química , Células HeLa
13.
BMC Vet Res ; 20(1): 187, 2024 May 10.
Artículo en Inglés | MEDLINE | ID: mdl-38730463

RESUMEN

BACKGROUND: Porcine epidemic diarrhea virus (PEDV), a type of coronavirus, is one of the main pathogens that can infect pigs of all ages. It causes diarrhea and acute death of newborn piglets, resulting in massive economic losses to the worldwide swine industry. While vaccination remains the primary approach in combating PEDV, it often fails to address all the challenges posed by the infection, particularly in light of the emergence of evolving mutant strains. Therefore, there is a critical need to identify potent antiviral drugs that can effectively safeguard pigs against PEDV infection. RESULTS: In this study, the antiviral efficacy of SP2509, a specific antagonist of Lysine-specific demethylase 1(LSD1), was evaluated in vitro. The RT-qPCR, Western blot, TCID50, and IFA showed that at a concentration of 1µmol/L, SP2509 significantly inhibited PEDV infection. Additionally, viral life cycle assays showed that SP2509 operates by impeding PEDV internalization and replication rather than attachment and release. Regarding mechanism, in Huh-7 cells, knockdowns LSD1 can suppress PEDV replication. This indicated that the inhibition effect of SP2509 on PEDV largely depends on the activity of its target protein, LSD1. CONCLUSION: Our results in vitro show that SP2509 can inhibit PEDV infection during the internalization and replication stage and revealed a role of LSD1 as a restriction factor for PEDV. These imply that LSD1 might be a target for interfering with the viral infection, and SP2509 could be developed as an effective anti-PEDV agent.


Asunto(s)
Antivirales , Histona Demetilasas , Virus de la Diarrea Epidémica Porcina , Replicación Viral , Virus de la Diarrea Epidémica Porcina/efectos de los fármacos , Animales , Antivirales/farmacología , Replicación Viral/efectos de los fármacos , Histona Demetilasas/antagonistas & inhibidores , Porcinos , Chlorocebus aethiops , Enfermedades de los Porcinos/virología , Enfermedades de los Porcinos/tratamiento farmacológico , Infecciones por Coronavirus/veterinaria , Infecciones por Coronavirus/tratamiento farmacológico , Infecciones por Coronavirus/virología , Células Vero
14.
Biochem Biophys Res Commun ; 715: 149994, 2024 Jun 30.
Artículo en Inglés | MEDLINE | ID: mdl-38692139

RESUMEN

Many virus lysis/transport buffers used in molecular diagnostics, including the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA, contain guanidine-based chaotropic salts, primarily guanidine hydrochloride (GuHCl) or guanidine isothiocyanate (GITC). Although the virucidal effects of GuHCl and GITC alone against some enveloped viruses have been established, standardized data on their optimum virucidal concentrations against SARS-CoV-2 and effects on viral RNA stability are scarce. Thus, we aimed to determine the optimum virucidal concentrations of GuHCl and GITC against SARS-CoV-2 compared to influenza A virus (IAV), another enveloped respiratory virus. We also evaluated the effectiveness of viral RNA stabilization at the determined optimum virucidal concentrations under high-temperature conditions (35°C) using virus-specific real-time reverse transcription polymerase chain reaction. Both viruses were potently inactivated by 1.0 M GITC and 2.5 M GuHCl, but the GuHCl concentration for efficient SARS-CoV-2 inactivation was slightly higher than that for IAV inactivation. GITC showed better viral RNA stability than GuHCl at the optimum virucidal concentrations. An increased concentration of GuHCl or GITC increased viral RNA degradation at 35°C. Our findings highlight the need to standardize GuHCl and GITC concentrations in virus lysis/transport buffers and the potential application of these guanidine-based salts alone as virus inactivation solutions in SARS-CoV-2 and IAV molecular diagnostics.


Asunto(s)
Guanidina , Virus de la Influenza A , ARN Viral , SARS-CoV-2 , Manejo de Especímenes , SARS-CoV-2/efectos de los fármacos , SARS-CoV-2/genética , Virus de la Influenza A/efectos de los fármacos , Virus de la Influenza A/genética , Guanidina/farmacología , Guanidina/química , ARN Viral/genética , Humanos , Manejo de Especímenes/métodos , Genoma Viral , COVID-19/virología , COVID-19/diagnóstico , Chlorocebus aethiops , Células Vero , Inactivación de Virus/efectos de los fármacos , Animales , Estabilidad del ARN/efectos de los fármacos , Contención de Riesgos Biológicos , Guanidinas/farmacología , Guanidinas/química , Sales (Química)/farmacología , Sales (Química)/química
15.
Proc Natl Acad Sci U S A ; 121(22): e2314166121, 2024 May 28.
Artículo en Inglés | MEDLINE | ID: mdl-38768348

RESUMEN

The nonstructural protein 1 (Nsp1) of SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2) is a virulence factor that targets multiple cellular pathways to inhibit host gene expression and antiviral response. However, the underlying mechanisms of the various Nsp1-mediated functions and their contributions to SARS-CoV-2 virulence remain unclear. Among the targets of Nsp1 is the mRNA (messenger ribonucleic acid) export receptor NXF1-NXT1, which mediates nuclear export of mRNAs from the nucleus to the cytoplasm. Based on Nsp1 crystal structure, we generated mutants on Nsp1 surfaces and identified an acidic N-terminal patch that is critical for interaction with NXF1-NXT1. Photoactivatable Nsp1 probe reveals the RNA Recognition Motif (RRM) domain of NXF1 as an Nsp1 N-terminal binding site. By mutating the Nsp1 N-terminal acidic patch, we identified a separation-of-function mutant of Nsp1 that retains its translation inhibitory function but substantially loses its interaction with NXF1 and reverts Nsp1-mediated mRNA export inhibition. We then generated a recombinant (r)SARS-CoV-2 mutant on the Nsp1 N-terminal acidic patch and found that this surface is key to promote NXF1 binding and inhibition of host mRNA nuclear export, viral replication, and pathogenicity in vivo. Thus, these findings provide a mechanistic understanding of Nsp1-mediated mRNA export inhibition and establish the importance of this pathway in the virulence of SARS-CoV-2.


Asunto(s)
Transporte Activo de Núcleo Celular , COVID-19 , Proteínas de Transporte Nucleocitoplasmático , ARN Mensajero , Proteínas de Unión al ARN , SARS-CoV-2 , Proteínas no Estructurales Virales , Humanos , SARS-CoV-2/metabolismo , SARS-CoV-2/patogenicidad , SARS-CoV-2/genética , Proteínas no Estructurales Virales/metabolismo , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/química , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Proteínas de Transporte Nucleocitoplasmático/genética , Animales , COVID-19/virología , COVID-19/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Replicación Viral , Núcleo Celular/metabolismo , Células Vero , Virulencia , Chlorocebus aethiops , Células HEK293
16.
Proc Natl Acad Sci U S A ; 121(19): e2401341121, 2024 May 07.
Artículo en Inglés | MEDLINE | ID: mdl-38696466

RESUMEN

Neurotropic alphaherpesviruses, including herpes simplex virus type 1 (HSV-1), recruit microtubule motor proteins to invade cells. The incoming viral particle traffics to nuclei in a two-step process. First, the particle uses the dynein-dynactin motor to sustain transport to the centrosome. In neurons, this step is responsible for long-distance retrograde axonal transport and is an important component of the neuroinvasive property shared by these viruses. Second, a kinesin-dependent mechanism redirects the particle from the centrosome to the nucleus. We have reported that the kinesin motor used during the second step of invasion is assimilated into nascent virions during the previous round of infection. Here, we report that the HSV-1 pUL37 tegument protein suppresses the assimilated kinesin-1 motor during retrograde axonal transport. Region 2 (R2) of pUL37 was required for suppression and functioned independently of the autoinhibitory mechanism native to kinesin-1. Furthermore, the motor domain and proximal coiled coil of kinesin-1 were sufficient for HSV-1 assimilation, pUL37 suppression, and nuclear trafficking. pUL37 localized to the centrosome, the site of assimilated kinesin-1 activation during infection, when expressed in cells in the absence of other viral proteins; however, pUL37 did not suppress kinesin-1 in this context. These results indicate that the pUL37 tegument protein spatially and temporally regulates kinesin-1 via the amino-terminal motor region in the context of the incoming viral particle.


Asunto(s)
Herpesvirus Humano 1 , Cinesinas , Proteínas Estructurales Virales , Cinesinas/metabolismo , Herpesvirus Humano 1/fisiología , Herpesvirus Humano 1/metabolismo , Humanos , Animales , Transporte Axonal/fisiología , Chlorocebus aethiops , Centrosoma/metabolismo , Neuronas/metabolismo , Neuronas/virología , Células Vero , Núcleo Celular/metabolismo , Núcleo Celular/virología
17.
ACS Appl Bio Mater ; 7(5): 2862-2871, 2024 May 20.
Artículo en Inglés | MEDLINE | ID: mdl-38699864

RESUMEN

Mosquito-borne viruses are a major worldwide health problem associated with high morbidity and mortality rates and significant impacts on national healthcare budgets. The development of antiviral drugs for both the treatment and prophylaxis of these diseases is thus of considerable importance. To address the need for therapeutics with antiviral activity, a library of heparan sulfate mimetic polymers was screened against dengue virus (DENV), Yellow fever virus (YFV), Zika virus (ZIKV), and Ross River virus (RRV). The polymers were prepared by RAFT polymerization of various acidic monomers with a target MW of 20 kDa (average Mn ∼ 27 kDa by GPC). Among the polymers, poly(SS), a homopolymer of sodium styrenesulfonate, was identified as a broad spectrum antiviral with activity against all the tested viruses and particularly potent inhibition of YFV (IC50 = 310 pM). Our results further uncovered that poly(SS) exhibited a robust inhibition of ZIKV infection in both mosquito and human cell lines, which points out the potential functions of poly(SS) in preventing mosquito-borne viruses associated diseases by blocking viral transmission in their mosquito vectors and mitigating viral infection in patients.


Asunto(s)
Antivirales , Heparitina Sulfato , Polímeros , Antivirales/farmacología , Antivirales/química , Antivirales/síntesis química , Heparitina Sulfato/química , Heparitina Sulfato/farmacología , Animales , Humanos , Polímeros/química , Polímeros/farmacología , Materiales Biocompatibles/química , Materiales Biocompatibles/farmacología , Materiales Biocompatibles/síntesis química , Culicidae/efectos de los fármacos , Culicidae/virología , Pruebas de Sensibilidad Microbiana , Ensayo de Materiales , Tamaño de la Partícula , Línea Celular , Estructura Molecular , Chlorocebus aethiops , Materiales Biomiméticos/química , Materiales Biomiméticos/farmacología , Virus Zika/efectos de los fármacos
18.
Nat Commun ; 15(1): 4056, 2024 May 14.
Artículo en Inglés | MEDLINE | ID: mdl-38744813

RESUMEN

The fusion peptide of SARS-CoV-2 spike protein is functionally important for membrane fusion during virus entry and is part of a broadly neutralizing epitope. However, sequence determinants at the fusion peptide and its adjacent regions for pathogenicity and antigenicity remain elusive. In this study, we perform a series of deep mutational scanning (DMS) experiments on an S2 region spanning the fusion peptide of authentic SARS-CoV-2 in different cell lines and in the presence of broadly neutralizing antibodies. We identify mutations at residue 813 of the spike protein that reduced TMPRSS2-mediated entry with decreased virulence. In addition, we show that an F823Y mutation, present in bat betacoronavirus HKU9 spike protein, confers resistance to broadly neutralizing antibodies. Our findings provide mechanistic insights into SARS-CoV-2 pathogenicity and also highlight a potential challenge in developing broadly protective S2-based coronavirus vaccines.


Asunto(s)
Anticuerpos Neutralizantes , COVID-19 , Mutación , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus , Internalización del Virus , Glicoproteína de la Espiga del Coronavirus/inmunología , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/metabolismo , Humanos , SARS-CoV-2/inmunología , SARS-CoV-2/genética , Anticuerpos Neutralizantes/inmunología , COVID-19/virología , COVID-19/inmunología , Animales , Anticuerpos Antivirales/inmunología , Serina Endopeptidasas/genética , Serina Endopeptidasas/inmunología , Serina Endopeptidasas/metabolismo , Chlorocebus aethiops , Células HEK293 , Células Vero , Epítopos/inmunología , Epítopos/genética , Línea Celular , Ratones
19.
Methods Cell Biol ; 187: 99-116, 2024.
Artículo en Inglés | MEDLINE | ID: mdl-38705632

RESUMEN

Correlative Light Electron Microscopy (CLEM) is a powerful technique to investigate the ultrastructure of specific cells and organelles at sub-cellular resolution. Transmission Electron Microscopy (TEM) is particularly useful to the field of virology, given the small size of the virion, which is below the limit of detection by light microscopy. Furthermore, viral infection results in the rearrangement of host organelles to form spatially defined compartments that facilitate the replication of viruses. With the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), there has been great interest to study the viral replication complex using CLEM. In this chapter we provide an exemplary workflow describing the safe preparation and processing of cells grown on coverslips and infected with SARS-CoV-2.


Asunto(s)
COVID-19 , SARS-CoV-2 , SARS-CoV-2/ultraestructura , Humanos , COVID-19/virología , Células Vero , Chlorocebus aethiops , Animales , Microscopía Electrónica de Transmisión/métodos , Replicación Viral , Microscopía Electrónica/métodos
20.
Biol Pharm Bull ; 47(5): 930-940, 2024.
Artículo en Inglés | MEDLINE | ID: mdl-38692871

RESUMEN

The coronavirus disease 2019 (COVID-19) is caused by the etiological agent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). COVID-19, with the recurrent epidemics of new variants of SARS-CoV-2, remains a global public health problem, and new antivirals are still required. Some cholesterol derivatives, such as 25-hydroxycholesterol, are known to have antiviral activity against a wide range of enveloped and non-enveloped viruses, including SARS-CoV-2. At the entry step of SARS-CoV-2 infection, the viral envelope fuses with the host membrane dependent of viral spike (S) glycoproteins. From the screening of cholesterol derivatives, we found a new compound 26,27-dinorcholest-5-en-24-yne-3ß,20-diol (Nat-20(S)-yne) that inhibited the SARS-CoV-2 S protein-dependent membrane fusion in a syncytium formation assay. Nat-20(S)-yne exhibited the inhibitory activities of SARS-CoV-2 pseudovirus entry and intact SARS-CoV-2 infection in a dose-dependent manner. Among the variants of SARS-CoV-2, inhibition of infection by Nat-20(S)-yne was stronger in delta and Wuhan strains, which predominantly invade into cells via fusion at the plasma membrane, than in omicron strains. The interaction between receptor-binding domain of S proteins and host receptor ACE2 was not affected by Nat-20(S)-yne. Unlike 25-hydroxycholesterol, which regulates various steps of cholesterol metabolism, Nat-20(S)-yne inhibited only de novo cholesterol biosynthesis. As a result, plasma membrane cholesterol content was substantially decreased in Nat-20(S)-yne-treated cells, leading to inhibition of SARS-CoV-2 infection. Nat-20(S)-yne having a new mechanism of action may be a potential therapeutic candidate for COVID-19.


Asunto(s)
Antivirales , COVID-19 , Colesterol , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus , SARS-CoV-2/efectos de los fármacos , Antivirales/farmacología , Humanos , COVID-19/virología , Colesterol/metabolismo , Células Vero , Chlorocebus aethiops , Glicoproteína de la Espiga del Coronavirus/metabolismo , Animales , Internalización del Virus/efectos de los fármacos , Betacoronavirus/efectos de los fármacos , Pandemias , Tratamiento Farmacológico de COVID-19 , Infecciones por Coronavirus/tratamiento farmacológico , Infecciones por Coronavirus/virología , Enzima Convertidora de Angiotensina 2/metabolismo , Neumonía Viral/tratamiento farmacológico , Neumonía Viral/virología
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