RESUMEN
Resistance-associated substitutions (RASs) of hepatitis C virus (HCV) affect the efficacy of direct-acting antivirals (DAAs). In this study, we aimed to clarify the susceptibility of the coexistence of nonstructural (NS) 5A Q24K/L28M/R30Q (or R30E)/A92K RASs, which were observed in patients with DAAs re-treatment failure and to consider new therapeutic agents. We used a subgenomic replicon system in which HCV genotype 1B strain 1B-4 was electroporated into OR6c cells derived from HuH-7 cells (Wild-type [WT]). We converted WT genes to NS5A Q24K/L28M/R30Q/A92K or Q24/L28K/R30E/A92K. Compared with the WT, the Q24K/L28M/R30Q/A92K RASs was 36,000-fold resistant to daclatasvir, 440,000-fold resistant to ledipasvir, 6300-fold resistant to velpatasvir, 3100-fold resistant to elbasvir, and 1.8-fold resistant to pibrentasvir. Compared with the WT, the Q24K/L28M/R30E/A92K RASs was 640,000-fold resistant to daclatasvir and ledipasvir, 150,000-fold resistant to velpatasvir, 44,000-fold resistant to elbasvir, and 1500-fold resistant to pibrentasvir. The Q24K/L28M/R30E/A92K RASs was 816.3 times more resistant to pibrentasvir than the Q24K/L28M/R30Q/A92K RASs. Furthermore, a combination of pibrentasvir and sofosbuvir showed therapeutic efficacy against these RASs. Combination regimens may eradicate HCV with NS5A Q24K/L28M/R30E/A92K RASs.
Asunto(s)
Antivirales , Bencimidazoles , Farmacorresistencia Viral , Hepacivirus , Imidazoles , Proteínas no Estructurales Virales , Hepacivirus/efectos de los fármacos , Hepacivirus/genética , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/antagonistas & inhibidores , Humanos , Antivirales/farmacología , Antivirales/uso terapéutico , Farmacorresistencia Viral/genética , Farmacorresistencia Viral/efectos de los fármacos , Bencimidazoles/farmacología , Imidazoles/farmacología , Carbamatos/farmacología , Fluorenos/farmacología , Sofosbuvir/farmacología , Pirrolidinas/farmacología , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Valina/análogos & derivados , Valina/farmacología , Genotipo , Replicón/efectos de los fármacos , Replicón/genética , Sulfonamidas/farmacología , Benzofuranos/farmacología , Pirazinas/farmacología , Benzopiranos , ARN Polimerasa Dependiente del ARNRESUMEN
The COVID-19 pandemic continues to threaten healthcare systems worldwide due to the limited access to vaccines, suboptimal treatment options, and the continuous emergence of new and more transmissible SARS-CoV-2 variants. Reverse-genetics studies of viral genes and mutations have proven highly valuable in advancing basic virus research, leading to the development of therapeutics. We developed a functional and highly versatile full-length SARS-CoV-2 infectious system by cloning the sequence of a COVID-19 associated virus isolate (DK-AHH1) into a bacterial artificial chromosome (BAC). Viruses recovered after RNA-transfection of in vitro transcripts into Vero E6 cells showed growth kinetics and remdesivir susceptibility similar to the DK-AHH1 virus isolate. Insertion of reporter genes, green fluorescent protein, and nanoluciferase into the ORF7 genomic region led to high levels of reporter activity, which facilitated high throughput treatment experiments. We found that putative coronavirus remdesivir resistance-associated substitutions F480L and V570L-and naturally found polymorphisms A97V, P323L, and N491S, all in nsp12-did not decrease SARS-CoV-2 susceptibility to remdesivir. A nanoluciferase reporter clone with deletion of spike (S), envelope (E), and membrane (M) proteins exhibited high levels of transient replication, was inhibited by remdesivir, and therefore could function as an efficient non-infectious subgenomic replicon system. The developed SARS-CoV-2 reverse-genetics systems, including recombinants to modify infectious viruses and non-infectious subgenomic replicons with autonomous genomic RNA replication, will permit high-throughput cell culture studies-providing fundamental understanding of basic biology of this coronavirus. We have proven the utility of the systems in rapidly introducing mutations in nsp12 and studying their effect on the efficacy of remdesivir, which is used worldwide for the treatment of COVID-19. Our system provides a platform to effectively test the antiviral activity of drugs and the phenotype of SARS-CoV-2 mutants.
Asunto(s)
Antivirales/farmacología , Farmacorresistencia Viral/genética , Genética Inversa/métodos , SARS-CoV-2/efectos de los fármacos , SARS-CoV-2/genética , Replicación Viral/genética , Sustitución de Aminoácidos , Animales , Chlorocebus aethiops , Cromosomas Artificiales Bacterianos/genética , Humanos , Polimorfismo Genético , Replicón/efectos de los fármacos , Replicón/genética , Células VeroRESUMEN
Despite the excellent antiviral potency of direct-acting antivirals (DAAs) against hepatitis C virus (HCV), emergence of drug-resistant viral mutations remains a potential challenge. Sofobuvir (SOF), a nucleotide analog targeting HCV NS5B - RNA-dependent RNA polymerase (RdRp), constitutes a key component of many anti-HCV cocktail regimens and confers a high barrier for developing drug resistance. The serine to threonine mutation at the amino acid position 282 of NS5B (S282T) is the mostly documented SOF resistance-associated substitution (RAS), but severely hampers the virus fitness. In this study, we first developed new genotype 1b (GT1b) subgenomic replicon cells, denoted PR52D4 and PR52D9, directly from a GT1b clinical isolate. Next, we obtained SOF-resistant and replication-competent PR52D4 replicon by culturing the replicon cells in the presence of SOF. Sequencing analysis showed that the selected replicon harbored two mutations K74R and S282T in NS5B. Reverse genetics analysis showed that while PR52D4 consisting of either single mutation K74R or S282T could not replicate efficiently, the engineering of the both mutations led to a replication-competent and SOF-resistant PR52D4 replicon. Furthermore, we showed that the K74R mutation could also rescue the replication deficiency of the S282T mutation in Con1, another GT1b replicon as well as in JFH1, a GT2a replicon. Structural modeling analysis suggested that K74R might help maintain an active catalytic conformation of S282T by engaging with Y296. In conclusion, we identified the combination of two NS5B mutations S282T and K74R as a novel RAS that confers a substantial resistance to SOF while retains the HCV replication capacity.
Asunto(s)
Antivirales/farmacología , Farmacorresistencia Viral/genética , Variación Genética , Hepacivirus/efectos de los fármacos , Hepacivirus/genética , Replicón/genética , Sofosbuvir/farmacología , Genotipo , Hepacivirus/aislamiento & purificación , Hepatitis C/virología , Humanos , Replicón/efectos de los fármacosRESUMEN
Hepatitis C virus (HCV) genotype 3 is widely distributed, and genotype 3-infected patients achieve a lower cure rate in direct-acting antiviral (DAA) therapy and are associated with a higher risk of hepatic steatosis than patients with other genotypes. Thus, the study of the virology and pathogenesis of genotype 3 HCV is increasingly relevant. Here, we developed a full-length infectious clone and a subgenomic replicon for the genotype 3a isolate, CH3a. From an infected serum, we constructed a full-length CH3a clone, however, it was nonviable in Huh7.5.1 cells. Next, we systematically adapted several intergenotypic recombinants containing Core-NS2 and 5'UTR-NS5A from CH3a, and other sequences from a replication-competent genotype 2 a clone JFH1. Adaptive mutations were identified, of which several combinations facilitated the replication of CH3a-JFH1 recombinants; however, they failed to adapt to the full-length CH3a and the recombinants containing CH3a NS5B. Thus, we attempted to separately adapt CH3a NS5B-3'UTR by constructing an intragenotypic recombinant using 5'UTR-NS5A from an infectious genotype 3a clone, DBN3acc, from which L3004P/M in NS5B and a deletion of 11 nucleotides (Δ11nt) downstream of the polyU/UC tract of the 3'UTR were identified and demonstrated to efficiently improve virus production. Finally, we combined functional 5'UTR-NS5A and NS5B-3'UTR sequences that carried the selected mutations to generate full-length CH3a with 26 or 27 substitutions (CH3acc), and both revealed efficient replication and virus spread in transfected and infected cells, releasing HCV of 104.2 f.f.u. ml-1. CH3acc was inhibited by DAAs targeting NS3/4A, NS5A and NS5B in a dose-dependent manner. The selected mutations permitted the development of subgenomic replicon CH3a-SGRep, by which L3004P, L3004M and Δ11nt were proven, together with a single-cycle virus production assay, to facilitate virus assembly, release, and RNA replication. CH3acc clones and CH3a-SGRep replicon provide new tools for the study of HCV genotype 3.
Asunto(s)
Antivirales/farmacología , Genotipo , Hepacivirus/genética , Hepatitis C/tratamiento farmacológico , Proteínas no Estructurales Virales/genética , Regiones no Traducidas 5' , Carcinoma Hepatocelular/prevención & control , Línea Celular Tumoral , Células Clonales , Hepacivirus/efectos de los fármacos , Hepatitis C/virología , Humanos , Mutación , Replicón/efectos de los fármacos , Replicación Viral/efectos de los fármacosRESUMEN
The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) virus, which is highly pathogenic and classified as a biosafety level 3 (BSL-3) agent, has greatly threatened global health and efficacious antivirals are urgently needed. The high requirement of facilities to manipulate the live virus has limited the development of antiviral study. Here, we constructed a reporter replicon of SARS-CoV-2, which can be handled in a BSL-2 laboratory. The Renilla luciferase activity effectively reflected the transcription and replication levels of the replicon genome. We identified the suitability of the replicon in antiviral screening using the known inhibitors, and thus established the replicon-based high-throughput screening (HTS) assay for SARS-CoV-2. The application of the HTS assay was further validated using a few hit natural compounds, which were screened out in a SARS-CoV-2 induced cytopathic-effect-based HTS assay in our previous study. This replicon-based HTS assay will be a safe platform for SARS-CoV-2 antiviral screening in a BSL-2 laboratory without the live virus.
Asunto(s)
Antivirales/farmacología , Evaluación Preclínica de Medicamentos/métodos , Replicón/efectos de los fármacos , SARS-CoV-2/efectos de los fármacos , Animales , Chlorocebus aethiops , Descubrimiento de Drogas , Ensayos Analíticos de Alto Rendimiento/métodos , Humanos , Replicón/genética , SARS-CoV-2/genética , Células Vero , Replicación Viral/efectos de los fármacos , Tratamiento Farmacológico de COVID-19RESUMEN
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) research and antiviral discovery are hampered by the lack of a cell-based virus replication system that can be readily adopted without biosafety level 3 (BSL-3) restrictions. Here, the construction of a noninfectious SARS-CoV-2 reporter replicon and its application in deciphering viral replication mechanisms and evaluating SARS-CoV-2 inhibitors are presented. The replicon genome is replication competent but does not produce progeny virions. Its replication can be inhibited by RdRp mutations or by known SARS-CoV-2 antiviral compounds. Using this system, a high-throughput antiviral assay has also been developed. Significant differences in potencies of several SARS-CoV-2 inhibitors in different cell lines were observed, which highlight the challenges of discovering antivirals capable of inhibiting viral replication in vivo and the importance of testing compounds in multiple cell culture models. The generation of a SARS-CoV-2 replicon provides a powerful platform to expand the global research effort to combat COVID-19.
Asunto(s)
Antivirales/farmacología , COVID-19/virología , Ensayos Analíticos de Alto Rendimiento/métodos , Replicón/efectos de los fármacos , SARS-CoV-2/efectos de los fármacos , Células A549 , Animales , Chlorocebus aethiops , ARN Polimerasa Dependiente de ARN de Coronavirus/genética , Células HEK293 , Humanos , Replicón/genética , SARS-CoV-2/genética , Células Vero , Replicación Viral/efectos de los fármacosRESUMEN
The etiologic agent of COVID-19 is highly contagious and has caused a severe global pandemic. Until now, there has been no simple and reliable system available in a lower-biosafety-grade laboratory for SARS-CoV-2 virologic research and inhibitor screening. In this study, we reported a replicon system which consists of four plasmids expressing the required segments of SARS-CoV-2. Our study revealed that the features for viral RNA synthesis and responses to antivirus drugs of the replicon are similar to those of wild-type viruses. Further analysis indicated that ORF6 provided potent in trans stimulation of the viral replication. Some viral variations, such as 5'UTR-C241T and ORF8-(T28144C) L84S mutation, also exhibit their different impact upon viral replication. Besides, the screening of clinically used drugs identified that several tyrosine kinase inhibitors and DNA-Top II inhibitors potently inhibit the replicon, as well as authentic SARS-CoV-2 viruses. Collectively, this replicon system provides a biosafety-worry-free platform for studying SARS-CoV-2 virology, monitoring the functional impact of viral mutations, and developing viral inhibitors.IMPORTANCE COVID-19 has caused a severe global pandemic. Until now, there has been no simple and reliable system available in a lower-biosafety-grade laboratory for SARS-CoV-2 virologic research and inhibitor screening. We reported a replicon system which consists of four ordinary plasmids expressing the required segments of SARS-CoV-2. Using the replicon system, we developed three application scenarios: (i) to identify the effects of viral proteins on virus replication, (ii) to identify the effects of mutations on viral replication during viral epidemics, and (iii) to perform high-throughput screening of antiviral drugs. Collectively, this replicon system would be useful for virologists to study SARS-CoV-2 virology, for epidemiologists to monitor virus mutations, and for industry to develop antiviral drugs.
Asunto(s)
Antivirales/farmacología , COVID-19/virología , ARN Viral/biosíntesis , Replicón/efectos de los fármacos , SARS-CoV-2/efectos de los fármacos , SARS-CoV-2/genética , Evaluación Preclínica de Medicamentos/métodos , Femenino , Ingeniería Genética , Células HEK293 , Ensayos Analíticos de Alto Rendimiento , Humanos , Concentración 50 Inhibidora , Mutación , Pandemias , ARN Viral/genética , Replicón/genética , SARS-CoV-2/metabolismo , Replicación Viral/efectos de los fármacosRESUMEN
Vaccines and antiviral agents are in urgent need to stop the COVID-19 pandemic. To facilitate antiviral screening against SARS-CoV-2 without requirement for high biosafety level facility, we developed a bacterial artificial chromosome (BAC)-vectored replicon of SARS-CoV-2, nCoV-SH01 strain, in which secreted Gaussia luciferase (sGluc) was encoded in viral subgenomic mRNA as a reporter gene. The replicon was devoid of structural genes spike (S), membrane (M), and envelope (E). Upon transfection, the replicon RNA replicated in various cell lines, and was sensitive to interferon alpha (IFN-α), remdesivir, but was resistant to hepatitis C virus inhibitors daclatasvir and sofosbuvir. Replication of the replicon was also sensitive overexpression to zinc-finger antiviral protein (ZAP). We also constructed a four-plasmid in-vitro ligation system that is compatible with the BAC system, which makes it easy to introduce desired mutations into the assembly plasmids for in-vitro ligation. This replicon system would be helpful for performing antiviral screening and dissecting virus-host interactions.
Asunto(s)
Antivirales/farmacología , COVID-19/virología , Cromosomas Artificiales Bacterianos , Replicón/efectos de los fármacos , SARS-CoV-2/efectos de los fármacos , SARS-CoV-2/genética , Adenosina Monofosfato/análogos & derivados , Adenosina Monofosfato/farmacología , Alanina/análogos & derivados , Alanina/farmacología , Animales , Línea Celular , Chlorocebus aethiops , Células HEK293 , Hepacivirus/efectos de los fármacos , Hepatitis C/tratamiento farmacológico , Hepatitis C/virología , Humanos , Interferón-alfa/farmacología , Proteínas de Unión al ARN/biosíntesis , Proteínas de Unión al ARN/genética , Sofosbuvir/farmacología , Células Vero , Replicación Viral/efectos de los fármacos , Tratamiento Farmacológico de COVID-19RESUMEN
Despite the introduction of directly acting antivirals (DAAs), for the treatment of hepatitis C virus (HCV) infection, their cost, patient compliance, and viral resistance are still important issues to be considered. Here, we describe the generation of a novel JFH1-based HCV subgenomic replicon double reporter cell line suitable for testing different antiviral drugs and therapeutic interventions. This cells line allowed a rapid and accurate quantification of cell growth/viability and HCV RNA replication, thus discriminating specific from unspecific antiviral effects caused by DAAs or cytotoxic compounds, respectively. By correlating cell number and virus replication, we could confirm the inhibitory effect on the latter of cell over confluency and characterize an array of lentiviral vectors expressing single, double, or triple cassettes containing different combinations of short hairpin (sh)RNAs, targeting both highly conserved viral genome sequences and cellular factors crucial for HCV replication. While all vectors were effective in reducing HCV replication, the ones targeting viral sequences displayed a stronger antiviral effect, without significant cytopathic effects. Such combinatorial platforms as well as the developed double reporter cell line might find application both in setting-up anti-HCV gene therapy approaches and in studies aimed at further dissecting the viral biology/pathogenesis of infection.
Asunto(s)
Antivirales/farmacología , Vectores Genéticos , Lentivirus/genética , ARN Interferente Pequeño/genética , Replicación Viral/efectos de los fármacos , Línea Celular Tumoral , Terapia Genética , Genoma Viral , Células HEK293 , Hepacivirus/genética , Hepatitis C/virología , Humanos , ARN Interferente Pequeño/metabolismo , Replicón/efectos de los fármacos , Proteínas no Estructurales Virales/genéticaRESUMEN
BACKGROUND & AIMS: HCV is a positive-strand RNA virus that primarily infects human hepatocytes. Recent studies have reported that C19orf66 is expressed as an interferon (IFN)-stimulated gene; however, the intrinsic regulation of this gene within the liver as well as its antiviral effects against HCV remain elusive. METHODS: Expression of C19orf66 was quantified in both liver biopsies and primary human hepatocytes, with or without HCV infection. Mechanistic studies of the potent anti-HCV phenotype mediated by C19orf66 were conducted using state-of-the-art virological, biochemical and genetic approaches, as well as correlative light and electron microscopy and transcriptome and proteome analysis. RESULTS: Upregulation of C19orf66 mRNA was observed in both primary human hepatocytes upon HCV infection and in the livers of patients with chronic hepatitis C (CHC). In addition, pegIFNα/ribavirin therapy induced C19orf66 expression in patients with CHC. Transcriptomic profiling and whole cell proteomics of hepatoma cells ectopically expressing C19orf66 revealed no induction of other antiviral genes. Expression of C19orf66 restricted HCV infection, whereas CRIPSPR/Cas9 mediated knockout of C19orf66 attenuated IFN-mediated suppression of HCV replication. Co-immunoprecipitation followed by mass spectrometry identified a stress granule protein-dominated interactome of C19orf66. Studies with subgenomic HCV replicons and an expression system revealed that C19orf66 expression impairs HCV-induced elevation of phosphatidylinositol-4-phosphate, alters the morphology of the viral replication organelle (termed the membranous web) and thereby targets viral RNA replication. CONCLUSION: C19orf66 is an IFN-stimulated gene, which is upregulated in hepatocytes within the first hours post IFN treatment or HCV infection in vivo. The encoded protein possesses specific antiviral activity against HCV and targets the formation of the membranous web. Our study identifies C19orf66 as an IFN-inducible restriction factor with a novel antiviral mechanism that specifically targets HCV replication. LAY SUMMARY: Interferon-stimulated genes are thought to be important to for antiviral immune responses to HCV. Herein, we analysed C19orf66, an interferon-stimulated gene, which appears to inhibit HCV replication. It prevents the HCV-induced elevation of phosphatidylinositol-4-phosphate and alters the morphology of HCV's replication organelle.
Asunto(s)
Antivirales/uso terapéutico , Hepacivirus/genética , Hepatitis C Crónica/tratamiento farmacológico , Hepatitis C Crónica/metabolismo , Interferones/uso terapéutico , Orgánulos/virología , Proteínas de Unión al ARN/metabolismo , Compartimentos de Replicación Viral/efectos de los fármacos , Replicación Viral/efectos de los fármacos , Adulto , Línea Celular Tumoral , Femenino , Técnicas de Inactivación de Genes , Genotipo , Células HEK293 , Hepatitis C Crónica/patología , Hepatitis C Crónica/virología , Hepatocitos/metabolismo , Humanos , Hígado/patología , Masculino , Persona de Mediana Edad , Orgánulos/efectos de los fármacos , Orgánulos/metabolismo , ARN Viral/metabolismo , Proteínas de Unión al ARN/genética , Replicón/efectos de los fármacos , Replicón/genética , Ribavirina/uso terapéutico , Resultado del Tratamiento , Replicación Viral/genéticaRESUMEN
RNA viruses can cause severe diseases such as dengue, Lassa, chikungunya and Ebola. Many of these viruses can only be propagated under high containment levels, necessitating the development of low containment surrogate systems such as subgenomic replicons and minigenome systems. Replicons are self-amplifying recombinant RNA molecules expressing proteins sufficient for their own replication but which do not produce infectious virions. Replicons can persist in cells and are passed on during cell division, enabling quick, efficient and high-throughput testing of drug candidates that act on viral transcription, translation and replication. This review will explore the history and potential for drug discovery of hepatitis C virus, dengue virus, respiratory syncytial virus, Ebola virus and norovirus replicon and minigenome systems.
Asunto(s)
Antivirales/farmacología , Virus ARN/efectos de los fármacos , ARN Viral/genética , Replicón/efectos de los fármacos , Replicación Viral/efectos de los fármacos , Animales , Descubrimiento de Drogas/métodos , Genoma Viral/efectos de los fármacos , HumanosRESUMEN
Whole-genome sequence analysis was performed on a multidrug-resistant Providencia rettgeri PR002 clinical strain isolated from the urine of a hospitalized patient in Pretoria, South Africa, in 2013. The resistome, mobilome, pathogenicity island(s), as well as virulence and heavy-metal resistance genes of the isolate, were characterized using whole-genome sequencing and bioinformatic analysis. PR002 had a genome assembly size of 4,832,624 bp with a GC content of 40.7%, an A/C2 plasmid replicase gene, four integrons/gene cassettes, 17 resistance genes, and several virulence and heavy metal resistance genes, confirming PR002 as a human pathogen. A novel integron, In1483, harboring the gene blaOXA-2 , was identified, with other uncharacterized class 1 integrons harboring aacA4cr and dfrA1. Aac(3')-IIa and blaSCO-1 , as well as blaPER-7 , sul2, and tet(B), were found bracketed by composite Tn3 transposons, and IS91, IS91, and IS4 family insertion sequences, respectively. PR002 was resistant to all antibiotics tested except amikacin, carbapenems, cefotaxime-clavulanate, ceftazidime-clavulanate, cefoxitin, and fosfomycin. PR002 was closely related to PR1 (USA), PRET_2032 (SPAIN), DSM_1131, and NCTC7477 clinical P. rettgeri strains, but not close enough to suggest it was imported into South Africa from other countries. Multidrug resistance in P. rettgeri is rare, particularly in clinical settings, making this case an important incident requiring urgent attention. This is also the first report of an A/C plasmid in P. rettgeri. The array, multiplicity, and diversity of resistance and virulence genes in this strain are concerning, necessitating stringent infection control, antibiotic stewardship, and periodic resistance surveillance/monitoring policies to preempt further horizontal and vertical spread of these resistance genes.
Asunto(s)
Farmacorresistencia Bacteriana Múltiple/genética , Genoma Bacteriano/genética , Integrones/genética , Plásmidos/genética , Providencia/genética , Replicón/genética , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Farmacorresistencia Bacteriana Múltiple/efectos de los fármacos , Infecciones por Enterobacteriaceae/tratamiento farmacológico , Infecciones por Enterobacteriaceae/genética , Genoma Bacteriano/efectos de los fármacos , Genómica/métodos , Humanos , Integrones/efectos de los fármacos , Masculino , Pruebas de Sensibilidad Microbiana/métodos , Persona de Mediana Edad , Filogenia , Providencia/efectos de los fármacos , Replicón/efectos de los fármacosRESUMEN
The multiresistance plasmid, pZM3, from a 1970 Salmonella enterica serovar Wien isolate from Algeria represents the multiresistance FIme-type plasmids conferring resistance to ampicillin, chloramphenicol, kanamycin, neomycin, sulfonamides, streptomycin, spectinomycin, tetracycline, and mercuric ions circulating in the Middle East in the 1970s. pZM3 was sequenced to determine the relationship between IS1936, the IS26-like insertion sequence it carries, and IS26. IS1936 is identical to IS26. pZM3 is a 166.8-kb plasmid with three replicons typed as FIA-1, FIB-1, and FII-1, consistent with other FIme plasmids. However, Tn3, containing the blaTEM-1a ampicillin resistance gene, disrupts the FII repA gene. pZM3 also contains an IS1-flanked virulence region, including the sit and aerobactin operons, shared with many other FIB-1 virulence plasmids. The remaining resistance genes are located in a 44.7-kb complex resistance island that includes the Tn21-like transposon, Tn1935, identified previously. Relative to Tn21, Tn1935 includes an additional gene cassette, oxa1, and Tn4352 in tniA. Tn1935 is in the same Tn2670 context as Tn21 in NR1, and identity to NR1 extends beyond the IS1 flanking the catA1 gene. On the other side, IS1-mediated events have brought in a Tn10 remnant and inverted part of it, highlighting the role of IS1 in resistance region evolution. The backbone of pZM3 was found to be almost identical to that of pRSB225, recovered in Germany in 2013, and their resistance islands are in the same position. The pRSB225 resistance island has evolved in situ from the pZM3 configuration through an insertion, a replacement, and an inversion.
Asunto(s)
Antibacterianos/farmacología , Farmacorresistencia Bacteriana Múltiple/efectos de los fármacos , Plásmidos/genética , Salmonella enterica/efectos de los fármacos , Virulencia/genética , Argelia , Secuencia de Bases , Elementos Transponibles de ADN , ADN Bacteriano/genética , Replicón/efectos de los fármacosRESUMEN
Dengue virus (DENV) is the causative agent of dengue fever (DF), dengue haemorrhagic fever (DHF), and dengue shock syndrome (DSS) and continues to be a public health problem in the tropical and subtropical areas. However, there is currently no antiviral treatment for DENV infection. In this study, our aim was to develop a stable reporter replicon cell system that supports constant viral RNA replication in cultured cells. The isolated replicon cells exhibited high levels of luciferase activity in the culture supernatant concomitant with expression of virus-encoded NS1, NS3 and NS5 proteins in the cells. The NS1, NS3 proteins and dsRNA were detected in the replicon cells by immunofluorescence analysis. Furthermore, the anti-DENV inhibitors ribavirin and bromocriptine significantly reduced the luciferase activity in a dose-dependent manner. High-throughput screening with a compound library using the stably-transfected replicon cells showed a Z' factor value of 0.57. Our screening yielded several candidates including one compound that has already shown anti-DENV activity. Taken together, our results demonstrate that this DENV subgenomic replicon cell system expressing a secretory luciferase gene can be useful for the high-throughput screening of anti-DENV compounds and the analysis of the replication mechanism of the DENV RNA.
Asunto(s)
Antivirales/farmacología , Virus del Dengue , Luciferasas , Bromocriptina/farmacología , Línea Celular , Virus del Dengue/efectos de los fármacos , Virus del Dengue/genética , Genes Reporteros , Ensayos Analíticos de Alto Rendimiento/métodos , Humanos , Luciferasas/genética , Luciferasas/metabolismo , ARN Viral/genética , Replicón/efectos de los fármacos , Ribavirina/farmacología , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/metabolismo , Replicación Viral/efectos de los fármacosRESUMEN
Oligostilbenoid compounds, a group of resveratrol multimers, display several anti-microbial activities through the neutralization of cytotoxic oxidants, and by inhibiting essential host and viral enzymes. In our previous study, we identified a series of oligostilbenoid compounds as potent hepatitis C virus (HCV) replication inhibitors. In particular, vitisin B, a resveratrol tetramer, exhibited the most dramatic anti-HCV activity (EC50 = 6 nM and CC50 > 10 µM) via the disruption of the viral helicase NS3 (IC50 = 3 nM). However, its further development as an HCV drug candidate was halted due to its intrinsic drawbacks, such as poor stability, low water solubility, and restricted in vivo absorption. In order to overcome these limitations, we focused on (+)-ε-viniferin, a resveratrol dimer, as an alternative. We prepared three different versions of (+)-ε-viniferin, including one which was extracted from the grapevine root (EVF) and two which were chemically synthesized with either penta-acetylation (SVF-5Ac) or no acetylation (SVF) using a newly established synthesis method. We confirmed their anti-HCV replication activities and minimal cytotoxicity by using genotype 1b and 2a HCV replicon cells. Their anti-HCV replication action also translated into a significant reduction of viral protein expression. Anti-HCV NS3 helicase activity by EVF was also verified in vitro. Finally, we demonstrated that SVF has improved pharmacokinetic properties over vitisin B. Overall, the favorable antiviral and pharmacokinetic properties of these three versions of viniferin warrant their further study as members of a promising new class of anti-HCV therapeutics.
Asunto(s)
Antivirales/farmacología , Benzofuranos/farmacología , Hepacivirus/efectos de los fármacos , Resveratrol/química , Estilbenos/farmacología , Replicación Viral/efectos de los fármacos , Animales , Antivirales/síntesis química , Antivirales/química , Antivirales/aislamiento & purificación , Benzofuranos/síntesis química , Benzofuranos/química , Benzofuranos/aislamiento & purificación , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Genotipo , Hepacivirus/enzimología , Hepacivirus/genética , Humanos , Ratones , Estructura Molecular , Replicón/efectos de los fármacos , Estilbenos/síntesis química , Estilbenos/química , Estilbenos/aislamiento & purificación , Proteínas no Estructurales Virales/antagonistas & inhibidores , Vitis/químicaRESUMEN
Cellular autophagy (Macrophagy) is a self-degradative process, executed through the network of autophagy associated genes (ATGs) encoded proteins. Both in vitro and in vivo studies suggest that dengue virus (DENV) induces autophagy and supports the viral genome replication and translation. Therefore, the cellular autophagy induced by dengue virus can be a good target for antiviral drug development. The action of mycophenolic acid (MPA), a specific inhibitor of DENV replication, was investigated in the stable BHK-21/DENV2 replicon cells. The inhibition was mediated by enhanced degradation of autophagic substrates in stable BHK-21/DENV2 replicon cells as evidenced by a decrease in lapidated LC3 (LC3II) and p62 expression in the presence of MPA. In contrast, the results indicated that four gene sets, namely Transmembrane protein 74 (TMEM74), Unc-51-like kinase 2 (ULK2), Cathepsin D (CTSD) and Estrogen receptor 1 (ESR1) were upregulated in stable BHK-21/DENV2 replicon cells, due to the sustained dynamic replication of DENV2 genome. These ATGs involved in the pre-autophagosomal structure (PAS) formation, were suppressed in the presence MPA. Instead, MPA induced the expression of different set of autophagy genes such as ATG4, AKT1, APP, ATG16L1, ATG16L2, B2M and HPRT1. An enzyme involved in the nucleotide salvage pathway, HPRT1, was highly expressed in the presence of MPA. The study shows that DENV2 replication is dependent on PAS formation and is inhibited in the presence of MPA by enhancing the degradation of autophagic substrates and suppression of PAS formation. This study provides impetus in designing MPA analogues to effectively inhibit dengue viral replication.
Asunto(s)
Antivirales/farmacología , Autofagia/efectos de los fármacos , Virus del Dengue/efectos de los fármacos , Ácido Micofenólico/farmacología , Replicón/efectos de los fármacos , Replicación Viral/efectos de los fármacos , Precursor de Proteína beta-Amiloide/metabolismo , Autofagia/genética , Proteínas Relacionadas con la Autofagia/metabolismo , Catepsina D/metabolismo , Línea Celular , Cisteína Endopeptidasas/metabolismo , Dengue , Virus del Dengue/genética , Receptor alfa de Estrógeno/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Proteínas de la Membrana/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas de Unión al ARN/metabolismo , Microglobulina beta-2/metabolismoRESUMEN
Hepatitis E, which is caused by hepatitis E virus (HEV), is generally a self-limiting, acute, and rarely fatal disease. It is sometimes fulminant and lethal, especially during pregnancy. Indeed, it occasionally takes a chronic course in immunocompromised individuals. To cure hepatitis E patients, the broad-spectrum antivirals (ribavirin and pegylated interferon α) are used. However, this treatment is insufficient and unsafe in some patients due to embryoteratogenic effects, leukopenia, and thrombocytopenia. In this study, we constructed an HEV replication reporter system with Gaussia luciferase for comprehensively screening anti-HEV drug candidates, and developed a cell-culture system using cells robustly producing HEV to validate the efficacy of anti-HEV drug candidates. We screened anti-HEV drug candidates from United States Food and Drug Administration-approved drugs using the established HEV replication reporter system, and investigated the selected candidates and type III interferons (interferon λ1-3) using the cell-culture system. In conclusion, we constructed an HEV replicon system for anti-HEV drug screening and a novel cell-culture system to strictly evaluate the replication-inhibitory activities of the obtained anti-HEV candidates. Our findings suggested that interferon λ1-3 might be effective for treating hepatitis E.
Asunto(s)
Antivirales/farmacología , Evaluación Preclínica de Medicamentos/métodos , Virus de la Hepatitis E/efectos de los fármacos , Interferones/farmacología , Técnicas de Cultivo de Célula , Línea Celular , Genes Reporteros , Virus de la Hepatitis E/fisiología , Humanos , Replicón/efectos de los fármacos , Replicación Viral/efectos de los fármacos , Interferón lambdaRESUMEN
We report the synthesis and biological evaluation of a series of 4'-fluoro-2'- C-substituted uridines. Triphosphates of the uridine analogues exhibited a potent inhibition of hepatitis C virus (HCV) NS5B polymerase with IC50 values as low as 27 nM. In an HCV subgenomic replicon assay, the phosphoramidate prodrugs of these uridine analogues demonstrated a very potent activity with EC50 values as low as 20 nM. A lead compound AL-335 (53) demonstrated high levels of the nucleoside triphosphate in vitro in primary human hepatocytes and Huh-7 cells as well as in dog liver following a single oral dose. Compound 53 was selected for the clinical development where it showed promising results in phase 1 and 2 trials.
Asunto(s)
Alanina/análogos & derivados , Antivirales/farmacología , Hepacivirus/efectos de los fármacos , Profármacos/farmacología , Nucleótidos de Uracilo/farmacología , Uridina/análogos & derivados , Alanina/síntesis química , Alanina/farmacología , Animales , Antivirales/síntesis química , Línea Celular Tumoral , Perros , Hepacivirus/enzimología , Hepatitis C/tratamiento farmacológico , Humanos , Inhibidores de la Síntesis del Ácido Nucleico/síntesis química , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Fosforamidas , Profármacos/síntesis química , Replicón/efectos de los fármacos , Nucleótidos de Uracilo/síntesis química , Nucleótidos de Uracilo/metabolismo , Uridina/síntesis química , Uridina/farmacología , Proteínas no Estructurales Virales/antagonistas & inhibidoresRESUMEN
Recently, in a program designed to improve the metabolic stability of the HCV inhibitor GSK5852, N-benzoxaborole benzofuran (GSK8175) emerged as a clinical candidate that not only retains the broad-spectrum activity against HCV subgenomic replicons but is free of the N-benzylboronic acid structure, which is a metabolic liability, and probably the cause of low in vivo clearance in preclinical species. This Viewpoint discusses some medicinal chemistry issues involved in the identification of GSK8175.