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1.
J Med Virol ; 96(3): e29547, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38511574

RESUMO

We have previously developed a bacterial artificial chromosome (BAC)-vectored SARS-CoV-2 replicon, namely BAC-CoV2-Rep, which, upon transfection into host cells, serves as a transcription template for SARS-CoV-2 replicon mRNA to initiate replicon replication and produce nanoluciferase (Nluc) reporter from the subgenomic viral mRNA. However, an inherent issue of such DNA-launched replicon system is that the nascent full-length replicon transcript undergoes process by host RNA splicing machinery, which reduces replicon replication and generates spliced mRNA species expressing NLuc reporter independent of replicon replication. To mitigate this problem, we employed Isoginkgetin, a universal eukaryotic host splicing inhibitor, to treat cells transfected with BAC-CoV2-Rep. Isoginkgetin effectively increased the level of full-length replicon transcripts while concurrently reducing the level of Nluc signal derived from spliced replicon mRNA, making the Nluc reporter signal more correlated with replicon replication, as evidenced by treatment with known SARS-CoV-2 replication inhibitors including Remdesivir, GC376, and EIDD-1931. Thus, our study emphasizes that host RNA splicing is a confounding factor for DNA-launched SARS-CoV-2 replicon systems, which can be mitigated by Isoginkgetin treatment.


Assuntos
Biflavonoides , COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/genética , Replicon , RNA Mensageiro , Replicação Viral
2.
Ann Clin Microbiol Antimicrob ; 23(1): 19, 2024 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-38402160

RESUMO

OBJECTIVE: In our study, K. pneumoniae strains (non-susceptible to carbapenem) (n = 60) were obtained from various clinical samples from Rize State Hospital between 2015 and 2017 and it is aimed to identify antibiotic resistance genes and replicon typing. METHODS: Antibiotic susceptibility tests of the strains were performed with Kirby-Bauer disk diffusion test and the Vitek-2 automated system (BioMerieux, France). Antibiotic resistance genes and replicon typing was characterized by PCR method. RESULTS: It was determined that K. pneumaniae isolates were mostly isolated from the samples of the intensive care unit. All of the K. pneumoniae strains examined in this study were found to be ampicillin/sulbactam and ertapenem resistant but colistin susceptible. Amoxacillin/clavulonic acid resistance was detected at 98.14% of strains. The blaOXA-48 gene was mostly detected in isolates. The most common type of plasmid was I1 and 3 different plasmid types were found in five different strains together. CONCLUSION: This study also shows that the distribution of NDM-1 and OXA-48 carbapenemases has increased since the first co-display in Türkiye and that IncHI1 is the first record in our country. This study provides an overview of the major plasmid families occurring in multiple antibiotic-resistant strains of K. pneumoniae. To our knowledge, this study represents the first report of IncHI1 record in Türkiye.


Assuntos
Enterobacteriáceas Resistentes a Carbapenêmicos , Infecções por Klebsiella , Humanos , Klebsiella pneumoniae , Testes de Sensibilidade Microbiana , Proteínas de Bactérias/genética , Antibacterianos/farmacologia , beta-Lactamases/genética , Carbapenêmicos/farmacologia , Enterobacteriáceas Resistentes a Carbapenêmicos/genética , Replicon
3.
BMC Microbiol ; 24(1): 64, 2024 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-38373913

RESUMO

BACKGROUND: Multi-drug-resistant organisms (MDROs) in gram-negative bacteria have caused a global epidemic, especially the bacterial resistance to carbapenem agents. Plasmid is the common vehicle for carrying antimicrobial resistance genes (ARGs), and the transmission of plasmids is also one of the important reasons for the emergence of MDROs. Different incompatibility group plasmid replicons are highly correlated with the acquisition, dissemination, and evolution of resistance genes. Based on this, the study aims to identify relevant characteristics of various plasmids and provide a theoretical foundation for clinical anti-infection treatment. METHODS: 330 gram-negative strains with different antimicrobial phenotypes from a tertiary hospital in Henan Province were included in this study to clarify the difference in incompatibility group plasmid replicons. Additionally, we combined the information from the PLSDB database to elaborate on the potential association between different plasmid replicons and ARGs. The VITEK mass spectrometer was used for species identification, and the VITEK-compact 2 automatic microbial system was used for the antimicrobial susceptibility test (AST). PCR-based replicon typing (PBRT) detected the plasmid profiles, and thirty-three different plasmid replicons were determined. All the carbapenem-resistant organisms (CROs) were tested for the carbapenemase genes. RESULTS: 21 plasmid replicon types were detected in this experiment, with the highest prevalence of IncFII, IncFIB, IncR, and IncFIA. Notably, the detection rate of IncX3 plasmids in CROs is higher, which is different in strains with other antimicrobial phenotypes. The number of plasmid replicons they carried increased with the strain resistance increase. Enterobacterales took a higher number of plasmid replicons than other gram-negative bacteria. The same strain tends to have more than one plasmid replicon type. IncF-type plasmids tend to be associated with MDROs. Combined with PLSDB database analysis, IncFII and IncX3 are critical platforms for taking blaKPC-2 and blaNDM. CONCLUSIONS: MDROs tend to carry more complex plasmid replicons compared with non-MDROs. The plasmid replicons that are predominantly prevalent and associated with ARGs differ in various species. The wide distribution of IncF-type plasmids and their close association with MDROs should deserve our attention. Further investigation into the critical role of plasmids in the carriage, evolution, and transmission of ARGs is needed.


Assuntos
Antibacterianos , Anti-Infecciosos , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Plasmídeos/genética , beta-Lactamases/genética , Bactérias Gram-Negativas/genética , Carbapenêmicos/farmacologia , Fenótipo , Replicon , Testes de Sensibilidade Microbiana , Klebsiella pneumoniae/genética
4.
Science ; 383(6681): 421-426, 2024 Jan 26.
Artigo em Inglês | MEDLINE | ID: mdl-38271510

RESUMO

The evolution of new function in living organisms is slow and fundamentally limited by their critical mutation rate. Here, we established a stable orthogonal replication system in Escherichia coli. The orthogonal replicon can carry diverse cargos of at least 16.5 kilobases and is not copied by host polymerases but is selectively copied by an orthogonal DNA polymerase (O-DNAP), which does not copy the genome. We designed mutant O-DNAPs that selectively increase the mutation rate of the orthogonal replicon by two to four orders of magnitude. We demonstrate the utility of our system for accelerated continuous evolution by evolving a 150-fold increase in resistance to tigecycline in 12 days. And, starting from a GFP variant, we evolved a 1000-fold increase in cellular fluorescence in 5 days.


Assuntos
Replicação do DNA , Evolução Molecular Direcionada , Proteínas de Escherichia coli , Escherichia coli , Evolução Molecular , Replicon , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Evolução Molecular Direcionada/métodos , Proteínas de Fluorescência Verde/genética , Tigeciclina/farmacologia , Antibacterianos/farmacologia , Farmacorresistência Bacteriana/genética , Fluorescência
5.
J Med Virol ; 96(1): e29376, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-38235850

RESUMO

Semliki Forest virus (SFV) viral replicon particles (VRPs) have been frequently used in various animal models and clinical trials. Chimeric replicon particles offer different advantages because of their unique biological properties. We here constructed a novel three-plasmid packaging system for chimeric SFV/SIN VRPs. The capsid and envelope of SIN structural proteins were generated using two-helper plasmids separately, and the SFV replicon contained the SFV replicase gene, packaging signal of SIN, subgenomic promoter followed by the exogenous gene, and 3' UTR of SIN. The chimeric VRPs carried luciferase or eGFP as reporter genes. The fluorescence and electron microscopy results revealed that chimeric VRPs were successfully packaged. The yield of the purified chimeric VRPs was approximately 2.5 times that of the SFV VRPs (1.38 × 107 TU/ml vs. 5.41 × 106 TU/ml) (p < 0.01). Furthermore, chimeric VRPs could be stored stably at 4°C for at least 60 days. Animal experiments revealed that mice immunized with chimeric VRPs (luciferase) had stronger luciferase expression than those immunized with equivalent amount of SFV VRPs (luciferase) (p < 0.01), and successfully expressed luciferase for approximately 12 days. Additionally, the chimeric VRPs expressed the RBD of SARS-CoV-2 efficiently and induced robust RBD-specific antibody responses in mice. In conclusion, the chimeric VRPs constructed here met the requirements of a gene delivery tool for vaccine development and cancer therapy.


Assuntos
Vírus da Floresta de Semliki , Vírus Sindbis , Camundongos , Animais , Vírus da Floresta de Semliki/genética , Vírus Sindbis/genética , Plasmídeos/genética , Replicon , Luciferases/genética , Vetores Genéticos
6.
Science ; 383(6681): 372-373, 2024 Jan 26.
Artigo em Inglês | MEDLINE | ID: mdl-38271527

RESUMO

Orthogonal replication enables rapid continuous biomolecular evolution in Escherichia coli.


Assuntos
Replicação do DNA , Evolução Molecular Direcionada , Escherichia coli , Escherichia coli/genética , Replicon
7.
Gene ; 896: 147990, 2024 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-37977321

RESUMO

Temperature-sensitive plasmids are useful for genome engineering and several synthetic biology applications. There are only limited reports on temperature-sensitive plasmids for Rhodococcus and none for Gordonia. Here, we report the construction of a temperature-sensitive pRC4 replicon that is functional in Rhodococcus and Gordonia. The amino acid residues were predicted for the temperature-sensitive phenotype in the pRC4 replicon using in silico methods and molecular simulation of the DNA-binding replication protein with the origin of replication. The amino acid residues were mutated, and the temperature-sensitive phenotype was validated in Gordonia sp. IITR100. Similar results were also observed in Rhodococcus erythropolis, suggesting that the temperature-sensitive phenotype was exhibited across genera.


Assuntos
Vetores Genéticos , Rhodococcus , Temperatura , Plasmídeos/genética , Replicon/genética , Proteínas de Ligação a DNA/genética , Rhodococcus/genética , Aminoácidos/genética
8.
Biotechnol J ; 19(1): e2300254, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-37750498

RESUMO

The novel mosquito-borne Tembusu virus (TMUV, family Flaviviridae) was discovered as the cause of a severe outbreak of egg-drop syndrome affecting ducks in Southeast Asia in 2010. TMUV infection can also lead to high mortality in various additional avian species such as geese, pigeons, and chickens. This study describes the construction of an infectious cDNA clone of a contemporary duck-isolate (TMUV WU2016). The virus recovered after transfection of BHK-21 cells shows enhanced virus replication compared to the mosquito-derived MM1775 strain. Next, the WU2016 cDNA clone was modified to create a SP6 promoter-driven, self-amplifying mRNA (replicon) capable of expressing a range of different reporter genes (Renilla luciferase, mScarlet, mCherry, and GFP) and viral (glyco)proteins of avian influenza virus (AIV; family Orthomyxoviridae), infectious bursal disease virus (IDBV; family Bunyaviridae) and infectious bronchitis virus (IBV; family Coronaviridae). The current study demonstrates the flexibility of the TMUV replicon system, to produce different heterologous proteins over an extended period of time and its potential use as a platform technology for novel poultry vaccines.


Assuntos
Culicidae , Infecções por Flavivirus , Flavivirus , Doenças das Aves Domésticas , Animais , Infecções por Flavivirus/veterinária , Infecções por Flavivirus/genética , Aves Domésticas/genética , Genes Reporter/genética , DNA Complementar , Antígenos Heterófilos , Doenças das Aves Domésticas/genética , Galinhas , Flavivirus/genética , Patos/genética , Células Clonais , Replicon
9.
J Med Virol ; 95(12): e29290, 2023 12.
Artigo em Inglês | MEDLINE | ID: mdl-38102947

RESUMO

Hepatitis C virus (HCV) infection is a major cause of chronic liver disease worldwide. Among its 8 genotypes (GT), GT3 has a relatively lower sustained virological response to highly effective direct-acting antiviral agents (DAA). Sofosbuvir (SOF), an anti-NS5B polymerase inhibitor, is a core component of many anti-HCV DAA cocktail regimens, and its resistant mutations are rare in clinics because these mutations usually severely impair the NS5B polymerase activity, including a mutation S282T in NS5B, the most frequently reported SOF-resistant mutation. In this study, we selected SOF-resistant variants of a previously developed GT3 subgenomic replicon (PR87A7). Two mutations were identified in the viral genome of SOF-resistant PR87A7 variants, Q606R in nontargeted NS3 and S282T in targeted N5SB. We demonstrated that Q606R could rescue the replication defect of S282T in PR87A7, and the resulting double mutant confers the SOF resistance. Finally, we showed that NS3-606R could not compensate for the replication defect of S282T in other GTs. In conclusion, we identified a novel GT3-specific combination of two mutations that confers SOF resistance. Our result calls for attention to potential mutations that may arise in nontargeted viral proteins during the SOF-based DAA treatment of chronic HCV.


Assuntos
Hepatite C Crônica , Hepatite C , Humanos , Sofosbuvir/farmacologia , Sofosbuvir/uso terapêutico , Antivirais/farmacologia , Antivirais/uso terapêutico , Hepacivirus/genética , Hepatite C/tratamento farmacológico , Mutação , Proteínas não Estruturais Virais/genética , Genótipo , Replicon , Farmacorresistência Viral/genética
10.
Front Cell Infect Microbiol ; 13: 1268227, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37942479

RESUMO

Engineering of reverse genetics systems for newly emerged viruses allows viral genome manipulation, being an essential tool for the study of virus life cycle, virus-host interactions and pathogenesis, as well as for the development of effective antiviral strategies. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an emergent human coronavirus that has caused the coronavirus disease (COVID-19) pandemic. The engineering of a full-length infectious cDNA clone and a fluorescent replicon of SARS-CoV-2 Wuhan-Hu-1, using a bacterial artificial chromosome, is reported. Viral growth and genetic stability in eleven cell lines were analyzed, showing that both VeroE6 cells overexpressing transmembrane serin protease 2 (TMPRSS2) and human lung derived cells resulted in the optimization of a cell system to preserve SARS-CoV-2 genetic stability. The recombinant SARS-CoV-2 virus and a point mutant expressing the D614G spike protein variant were virulent in a mouse model. The RNA replicon was propagation-defective, allowing its use in BSL-2 conditions to analyze viral RNA synthesis. The SARS-CoV-2 reverse genetics systems developed constitute a useful tool for studying the molecular biology of the virus, the development of genetically defined vaccines and to establish systems for antiviral compounds screening.


Assuntos
COVID-19 , SARS-CoV-2 , Camundongos , Animais , Humanos , SARS-CoV-2/genética , COVID-19/genética , Virulência/genética , RNA Viral/genética , Antivirais , Replicon , Replicação Viral
11.
J Virol ; 97(11): e0097923, 2023 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-37902397

RESUMO

IMPORTANCE: Our study highlights the mechanisms behind the cell's resistance to stress granule (SG) formation after infection with Old World alphaviruses. Shortly after infection, the replication of these viruses hinders the cell's ability to form SGs, even when exposed to chemical inducers such as sodium arsenite. This resistance is primarily attributed to virus-induced transcriptional and translational shutoffs, rather than interactions between the viral nsP3 and the key components of SGs, G3BP1/2, or the ADP-ribosylhydrolase activity of nsP3 macro domain. While interactions between G3BPs and nsP3 are essential for the formation of viral replication complexes, their role in regulating SG development appears to be small, if any. Cells harboring replicating viruses or replicons with lower abilities to inhibit transcription and/or translation, but expressing wild-type nsP3, retain the ability for SG development. Understanding these mechanisms of regulation of SG formation contributes to our knowledge of viral replication and the intricate relationships between alphaviruses and host cells.


Assuntos
Alphavirus , DNA Helicases , Interações entre Hospedeiro e Microrganismos , Biossíntese de Proteínas , Grânulos de Estresse , Transcrição Gênica , Alphavirus/fisiologia , DNA Helicases/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Replicon , RNA Helicases/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Grânulos de Estresse/metabolismo , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismo , Replicação Viral
12.
J Virol ; 97(11): e0122523, 2023 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-37877718

RESUMO

IMPORTANCE: Alphavirus replicons are being developed as self-amplifying RNAs aimed at improving the efficacy of mRNA vaccines. These replicons are convenient for genetic manipulations and can express heterologous genetic information more efficiently and for a longer time than standard mRNAs. However, replicons mimic many aspects of viral replication in terms of induction of innate immune response, modification of cellular transcription and translation, and expression of nonstructural viral genes. Moreover, all replicons used in this study demonstrated expression of heterologous genes in cell- and replicon's origin-specific modes. Thus, many aspects of the interactions between replicons and the host remain insufficiently investigated, and further studies are needed to understand the biology of the replicons and their applicability for designing a new generation of mRNA vaccines. On the other hand, our data show that replicons are very flexible expression systems, and additional modifications may have strong positive impacts on protein expression.


Assuntos
Alphavirus , Regulação Viral da Expressão Gênica , Interações entre Hospedeiro e Microrganismos , Replicon , Proteínas Virais , Alphavirus/genética , Alphavirus/metabolismo , Vacinas de mRNA/genética , Replicon/genética , Replicação Viral/genética , RNA Viral/biossíntese , RNA Viral/genética , Interações entre Hospedeiro e Microrganismos/genética , Proteínas Virais/biossíntese , Proteínas Virais/genética
13.
Sheng Wu Gong Cheng Xue Bao ; 39(7): 2634-2643, 2023 Jul 25.
Artigo em Chinês | MEDLINE | ID: mdl-37584120

RESUMO

The antigen gene expression level of a DNA vaccine is the key factor influencing the efficacy of the DNA vaccine. Accordingly, one of the ways to improve the antigen gene expression level of a DNA vaccine is to utilize a plasmid vector that is replicable in eukaryotic cells. A replicative DNA vaccine vector pCMVori was constructed based on the non-replicative pcDNA3.1 and the replicon of porcine circovirus 2 (PCV2) in this study. An EGFP gene was cloned into pCMVori and the control plasmid pcDNA3.1. The two recombinant vectors were transfected into PK-15 cell, and the plasmid DNA and RNA were extracted from the transfected cells. Real-time PCR was used to determine the plasmid replication efficiency of the two plasmids using plasmid before and after Bcl Ⅰ digestion as templates, and the transcription level of the Rep gene in PCV2 replicon was detected by RT-PCR. The average fluorescence intensity of cells transfected with the two plasmids was analyzed with software Image J, and the transcription level of EGFP was determined by means of real-time RT-PCR. The results showed that the replication efficiency of pCMVori in PK-15 cells incubated for 48 h was 136%, and the transcriptions of Rep and Rep' were verified by RT-PCR. The average fluorescence intensity of the cells transfected with pCMVori-EGFP was 39.14% higher than that of pcDNA3.1-EGFP, and the transcription level of EGFP in the former was also 40% higher than that in the latter. In conclusion, the DNA vaccine vector pCMVori constructed in this study can independently replicate in eukaryotic cells. As a result, the expression level of cloned target gene was elevated, providing a basis for developing the pCMVori-based DNA vaccine.


Assuntos
Circovirus , Vacinas de DNA , Animais , Suínos , Circovirus/genética , Vacinas de DNA/genética , Replicon/genética , Vetores Genéticos/genética , Plasmídeos/genética
14.
PLoS Genet ; 19(8): e1010829, 2023 08.
Artigo em Inglês | MEDLINE | ID: mdl-37535631

RESUMO

The emergence of antibiotic resistance under treatment depends on the availability of resistance alleles and their establishment in the population. Novel resistance alleles are encoded either in chromosomal or extrachromosomal genetic elements; both types may be present in multiple copies within the cell. However, the effect of polyploidy on the emergence of antibiotic resistance remains understudied. Here we show that the establishment of resistance alleles in microbial populations depends on the ploidy level. Evolving bacterial populations under selection for antibiotic resistance, we demonstrate that resistance alleles in polyploid elements are lost frequently in comparison to alleles in monoploid elements due to segregational drift. Integrating the experiments with a mathematical model, we find a remarkable agreement between the theoretical and empirical results, confirming our understanding of the allele segregation process. Using the mathematical model, we further show that the effect of polyploidy on the establishment probability of beneficial alleles is strongest for low replicon copy numbers and plateaus for high replicon copy numbers. Our results suggest that the distribution of fitness effects for mutations that are eventually fixed in a population depends on the replicon ploidy level. Our study indicates that the emergence of antibiotic resistance in bacterial pathogens depends on the pathogen ploidy level.


Assuntos
Poliploidia , Replicon , Humanos , Ploidias , Cromossomos , Resistência Microbiana a Medicamentos/genética , Alelos
15.
Sci Adv ; 9(31): eadh4057, 2023 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-37540755

RESUMO

Nipah virus (NiV) causes a highly lethal disease in humans who present with acute respiratory or neurological signs. No vaccines against NiV have been approved to date. Here, we report on the clinical impact of a novel NiV-derived nonspreading replicon particle lacking the fusion (F) protein gene (NiVΔF) as a vaccine in three small animal models of disease. A broad antibody response was detected that included immunoglobulin G (IgG) and IgA subtypes with demonstrable Fc-mediated effector function targeting multiple viral antigens. Single-dose intranasal vaccination up to 3 days before challenge prevented clinical signs and reduced virus levels in hamsters and immunocompromised mice; decreases were seen in tissues and mucosal secretions, critically decreasing potential for virus transmission. This virus replicon particle system provides a vital tool to the field and demonstrates utility as a highly efficacious and safe vaccine candidate that can be administered parenterally or mucosally to protect against lethal Nipah disease.


Assuntos
Infecções por Henipavirus , Vírus Nipah , Vacinas Virais , Cricetinae , Humanos , Animais , Camundongos , Infecções por Henipavirus/prevenção & controle , Infecções por Henipavirus/genética , Vacinação , Modelos Animais de Doenças , Vírus Nipah/genética , Replicon
16.
PLoS Genet ; 19(7): e1010857, 2023 07.
Artigo em Inglês | MEDLINE | ID: mdl-37494383

RESUMO

Borrelia burgdorferi, a causative agent of Lyme disease, contains the most segmented bacterial genome known to date, with one linear chromosome and over twenty plasmids. How this unusually complex genome is organized, and whether and how the different replicons interact are unclear. We recently demonstrated that B. burgdorferi is polyploid and that the copies of the chromosome and plasmids are regularly spaced in each cell, which is critical for faithful segregation of the genome to daughter cells. Regular spacing of the chromosome is controlled by two separate partitioning systems that involve the protein pairs ParA/ParZ and ParB/Smc. Here, using chromosome conformation capture (Hi-C), we characterized the organization of the B. burgdorferi genome and the interactions between the replicons. We uncovered that although the linear chromosome lacks contacts between the two replication arms, the two telomeres are in frequent contact. Moreover, several plasmids specifically interact with the chromosome oriC region, and a subset of plasmids interact with each other more than with others. We found that Smc and the Smc-like MksB protein mediate long-range interactions on the chromosome, but they minimally affect plasmid-chromosome or plasmid-plasmid interactions. Finally, we found that disruption of the two partition systems leads to chromosome restructuring, correlating with the mis-positioning of chromosome oriC. Altogether, this study revealed the conformation of a complex genome and analyzed the contribution of the partition systems and SMC family proteins to this organization. This work expands the understanding of the organization and maintenance of multipartite bacterial genomes.


Assuntos
Borrelia burgdorferi , Borrelia burgdorferi/genética , Plasmídeos/genética , Replicon/genética , Genoma Bacteriano , Telômero , Proteínas de Bactérias/genética , DNA Bacteriano/genética
17.
Virus Res ; 334: 199176, 2023 09.
Artigo em Inglês | MEDLINE | ID: mdl-37473963

RESUMO

The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), has necessitated the global development of countermeasures since its outbreak. However, current therapeutics and vaccines to stop the pandemic are insufficient and this is mainly because of the emergence of resistant variants, which requires the urgent development of new countermeasures, such as antiviral drugs. Replicons, self-replicating RNAs that do not produce virions, are a promising system for this purpose because they safely recreate viral replication, enabling antiviral screening in biosafety level (BSL)-2 facilities. We herein constructed three pCC2Fos-based RNA replicons lacking some open reading frames (ORF) of SARS-CoV-2: the Δorf2-8, Δorf2.4, and Δorf2 replicons, and validated their replication in Huh-7 cells. The functionalities of the Δorf2-8 and Δorf2.4 replicons for antiviral drug screening were also confirmed. We conducted puromycin selection following the construction of the Δorf2.4-puro replicon by inserting a puromycin-resistant gene into the Δorf2.4 replicon. We observed the more sustained replication of the Δorf2.4-puro replicon by puromycin pressure. The present results will contribute to the establishment of a safe and useful replicon system for analyzing SARS-CoV-2 replication mechanisms as well as the development of novel antiviral drugs in BSL-2 facilities.


Assuntos
Antivirais , COVID-19 , Humanos , Antivirais/farmacologia , SARS-CoV-2/genética , COVID-19/genética , Avaliação Pré-Clínica de Medicamentos , Contenção de Riscos Biológicos , Replicação Viral , Replicon , Puromicina/farmacologia
18.
Biotechnol Lett ; 45(8): 1029-1038, 2023 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-37266878

RESUMO

Self-replicating RNA (repRNA) derived from Venezuelan equine encephalitis (VEE) virus is a promising platform for gene therapy and confers prolonged gene expression due to its self-replicating capability, but repRNA suffers from a suboptimal transgene expression level due to its induction of intracellular innate response which may result in inhibition of translation. To improve transgene expression of repRNA, we introduced point mutations in the non-structural protein 1-4 (nsP1-4) coding region of VEE replicon vectors. As a proof of concept, inflammatory cytokines served as genes of interest and were cloned in their wild type and several mutant replicon vectors, followed by transfection in mammalian cells. Our data show that VEE replicons bearing nsP1GGAC-nsP2T or nsP1GGAC-nsP2AT mutations in the nsP1-4 coding region could significantly reduce the recognition by innate immunity as evidenced by the decreased production of type I interferon, and enhance transgene expression in host cells. Thus, the newly discovered mutant VEE replicon vectors could serve as promising gene expression platforms to advance VEE-derived repRNA-based gene therapies.


Assuntos
Vírus da Encefalite Equina Venezuelana , Animais , Vírus da Encefalite Equina Venezuelana/genética , Linhagem Celular , Fases de Leitura Aberta , RNA/metabolismo , Replicon/genética , Mutação , Expressão Gênica , Mamíferos/genética
19.
Microbiol Spectr ; 11(4): e0042323, 2023 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-37378517

RESUMO

Dengue virus (DENV) is the most widespread arbovirus, causing symptoms ranging from dengue fever to severe dengue, including hemorrhagic fever and shock syndrome. Four serotypes of DENV (DENV-1 to -4) can infect humans; however, no anti-DENV drug is available. To facilitate the study of antivirals and viral pathogenesis, here we developed an infectious clone and a subgenomic replicon of DENV-3 strains for anti-DENV drug discovery by screening a synthetic compound library. The viral cDNA was amplified from a serum sample from a DENV-3-infected individual during the 2019 epidemic; however, fragments containing the prM-E-partial NS1 region could not be cloned until a DENV-3 consensus sequence with 19 synonymous substitutions was introduced to reduce putative Escherichia coli promoter activity. Transfection of the resulting cDNA clone, plasmid DV3syn, released an infectious virus titer of 2.2 × 102 focus-forming units (FFU)/mL. Through serial passages, four adaptive mutations (4M) were identified, and addition of 4M generated recombinant DV3syn_4M, which produced viral titers ranging from 1.5 × 104 to 6.7 × 104 FFU/mL and remained genetically stable in transformant bacteria. Additionally, we constructed a DENV-3 subgenomic replicon and screened an arylnaphthalene lignan library, from which C169-P1 was identified as exhibiting inhibitory effects on viral replicon. A time-of-drug addition assay revealed that C169-P1 also impeded the internalization process of cell entry. Furthermore, we demonstrated that C169-P1 inhibited the infectivity of DV3syn_4M, as well as DENV-1, DENV-2, and DENV-4, in a dose-dependent manner. This study provides an infectious clone and a replicon for the study of DENV-3 and a candidate compound for future development against DENV-1 to -4 infections. IMPORTANCE Dengue virus (DENV) is the most prevalent mosquito-transmitted virus, and there is no an anti-dengue drug. Reverse genetic systems representative of different serotype viruses are invaluable tools for the study of viral pathogenesis and antiviral drugs. Here, we developed an efficient infectious clone of a clinical DENV-3 genotype III isolate. We successfully overcame the instability of flavivirus genome-length cDNA in transformant bacteria, an unsolved issue for construction of cDNA clones of flaviviruses, and adapted this clone to efficiently produce infectious viruses following plasmid transfection of cell culture. Moreover, we constructed a DENV-3 subgenomic replicon and screened a compound library. An arylnaphthalene lignan, C169-P1, was identified as an inhibitor of virus replication and cell entry. Finally, we demonstrated that C169-P1 exhibited a broad-spectrum antiviral effect against the infections with DENV-1 to -4. The reverse genetic systems and the compound candidate described here facilitate the study of DENV and related RNA viruses.


Assuntos
Vírus da Dengue , Flavivirus , Animais , Humanos , Vírus da Dengue/genética , Sorogrupo , DNA Complementar/farmacologia , Antivirais/farmacologia , Flavivirus/genética , Replicon , Células Clonais
20.
Proc Natl Acad Sci U S A ; 120(26): e2304082120, 2023 06 27.
Artigo em Inglês | MEDLINE | ID: mdl-37339222

RESUMO

A synthetic biology approach toward constructing an RNA-based genome expands our understanding of living things and opens avenues for technological advancement. For the precise design of an artificial RNA replicon either from scratch or based on a natural RNA replicon, understanding structure-function relationships of RNA sequences is critical. However, our knowledge remains limited to a few particular structural elements intensively studied so far. Here, we conducted a series of site-directed mutagenesis studies of yeast narnaviruses ScNV20S and ScNV23S, perhaps the simplest natural autonomous RNA replicons, to identify RNA elements required for maintenance and replication. RNA structure disruption corresponding to various portions of the entire narnavirus genome suggests that pervasive RNA folding, in addition to the precise secondary structure of genome termini, is essential for maintenance of the RNA replicon in vivo. Computational RNA structure analyses suggest that this scenario likely applies to other "narna-like" viruses. This finding implies selective pressure on these simplest autonomous natural RNA replicons to fold into a unique structure that acquires both thermodynamic and biological stability. We propose the importance of pervasive RNA folding for the design of RNA replicons that could serve as a platform for in vivo continuous evolution as well as an interesting model to study the origin of life.


Assuntos
Vírus de RNA , RNA Viral , RNA Viral/genética , RNA Viral/química , Dobramento de RNA , Genoma Viral/genética , Vírus de RNA/genética , Sequência de Bases , Replicon/genética , Replicação Viral
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