HCV Replicon Systems: Workhorses of Drug Discovery and Resistance

Khan, Shaheen; Soni, Shalini; Veerapu, Naga Suresh

doi:10.3389/fcimb.2020.00325

Cited by 17 publications

(13 citation statements)

References 136 publications

(217 reference statements)

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“…While the use of replicon DNA to transfect BHK-21 cells streamlines the workflow relative to the use of transcribed RNA, the workflow can be simplified further through the use stable replicon harboring cell lines; similar tools have proven extremely beneficial in drug development for HCV (58,59). Surprisingly, while a stable replicon harboring cell line was generated for SARS-CoV (60), none of the published SARS-CoV-2 replicon systems have led to the production of stable replicon harboring cell lines.…”

Section: Generation Of Sars-cov-2 Replicon-expressing Cell Linesmentioning

confidence: 99%

Subgenomic SARS-CoV-2 replicon and reporter replicon cell lines enable ultrahigh throughput antiviral screening and mechanistic studies with antivirals, viral mutations or host factors that affect COVID-19 replication

Lan

Tedbury

Ong

et al. 2021

Preprint

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Replicon-based technologies were used to develop reagents and assays for advanced drug discovery efforts against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and for examining all facets of the SARS-CoV-2 replication cycle at reduced biocontainment level. Specifically: a) 21 replicons were cloned in bacterial artificial chromosomes (BACs) and delivered as transfectable plasmid DNA or transcribed RNA in various cell types. Replicons carrying mutations that affect the activity or antiviral susceptibility of SARS-CoV-2 enzymes were used to establish utility for mechanistic studies while reducing the community risks associated with gain-of-function studies in fully infectious virus. b) A BHK-21 stable cell line harboring SARS-CoV-2 replicon was generated and characterized in robust high/ultra-high throughput assays of antiviral efficacy with orthogonal SARS-CoV-2 replication reporter genes (Nano luciferase and enhanced green fluorescent protein-eGFP); the estimated antiviral potencies in the fully infectious SARS-CoV-2 system and in the transient or stable replicon systems were similar. HEK293 and Calu1 stable cell lines expressing SARS-CoV-2 replicon have also been prepared. Finally, c) we generated trans-encapsidated replicons by co-expression with SARS-CoV-2 structural proteins, thus producing single-round infectious SARS-CoV-2 virus-like particles able to transduce susceptible cell types, thus expanding utility to enable study of virion assembly and entry into target cells. Hence, these SARS-CoV-2 replicon-based reagents include a novel approach to replicon-harboring cell line generation and are valuable tools that can be used at lower biosafety level (BSL2) for drug discovery efforts, characterization of SARS-CoV-2 and variant evolution in the COVID-19 pandemic, mechanisms of inhibition and resistance, and studies on the role of SARS-CoV-2 genes and host dependency factors.

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Section: Generation Of Sars-cov-2 Replicon-expressing Cell Linesmentioning

confidence: 99%

Subgenomic SARS-CoV-2 replicon and reporter replicon cell lines enable ultrahigh throughput antiviral screening and mechanistic studies with antivirals, viral mutations or host factors that affect COVID-19 replication

Lan

Tedbury

Ong

et al. 2021

Preprint

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show abstract

“…However, stable cell lines harboring EV71 SGR were not successful in our hands even with EMCV IRES to enhance viral RNA replication, suggesting some adaptive mutations are required for efficient replication. Indeed, stable cell lines harboring SGR are successful only in several viruses, mainly in the flaviviridae family, like Hepatitis C virus ( Lohmann et al, 1999 ; Bartenschlager, 2002 ), Dengue virus ( Ng et al, 2007 ), etc., and most of them are associated with adaptive mutations ( Bartenschlager, 2002 ; Khan et al, 2020 ).…”

Section: Discussionmentioning

confidence: 99%

Reverse Genetic Approaches for the Generation of Full Length and Subgenomic Replicon of EV71 Virus

et al. 2021

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Enterovirus 71 (EV71) is a neurotropic pathogen that causes hand, foot, and mouth disease (HFMD) and it has been consistently associated with severe neurological, cardiac, and respiratory complications. Yet there is no specific treatment for this virus and we still know little about the viral pathogenesis. In this study, we first generated an infectious cDNA clone of EV71 virus from a patient virus strain and made a full-length virus with a NanoLuc reporter gene through reverse genetic approaches. The reporter gene of this virus is genetically stable when passaging in cells and could be used for antiviral testing. In addition, we also made subgenomic replicons (SGRs) of EV71, which lacks part of the structural genes dispensable for viral replication and showed that SGR can be used for viral replication study. Overall, these reporter viral systems are useful tools for EV71 pathogenesis study and antiviral screening.

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“…Drug discovery and resistance studies on HCV become more and more important in the era of DAAs. The HCV replicon systems have become indispensable in the development of effective DAAs and the investigation of drug resistance (24), requiring a large number of infectious particles for high throughput anti-viral library assessments. These two new purification methods of HCV particles have shown potential application in this field.…”

Section: Discussionmentioning

confidence: 99%

Two New Purification Methods of Hepatitis C Virus Particles from Serum-Free Culture System

Zhu

Liu

et al. 2021

Hepat Mon

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Background: The traditional ultracentrifugation purification method of hepatitis C virus (HCV) particles requires special equipment, limiting its wide application. Therefore, more effective and convenient methods for HCV are needed. Objectives: The present study aimed to establish simple and effective purification methods for HCV. Methods: The infectious clone of the HCV genome (JFH-1) was transfected to the human hepatoma cell line (Huh7.5.1) and cultured in Dulbecco’s modified eagle medium/nutrient mixture F-12. The infectivity of JFH-1 culture was determined by reverse transcription-quantitative polymerase chain reaction and immunofluorescence. After concentration by centrifugal filter devices, HCV particles were purified by heparin-affinity chromatography and magnetic separation technique. The purified viruses were detected by the western blot and immune-electron microscopy. Results: The infectious titer of JFH-1 transfected Huh7.5.1 in the serum-free culture medium was 4.5 × 104 FFU/mL, and HCV ribonucleic acid load was 3.946 × 106 IU/mL in 30 days of cell culture post-transfection. After purification by heparin-affinity chromatography or magnetic separation method, viral particles were visualized with spherical morphology and an average diameter of 55 nm assessed by electron microscopy. The viruses were confirmed by the western blot and immune-electron microscopy with specific antibodies to HCV. Conclusions: The heparin-affinity chromatography and magnetic separation methods were established for the purification of HCV, which were simple and efficient methods for the stable purification of HCV particles on a large scale.

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HCV Replicon Systems: Workhorses of Drug Discovery and Resistance

Cited by 17 publications

References 136 publications

Subgenomic SARS-CoV-2 replicon and reporter replicon cell lines enable ultrahigh throughput antiviral screening and mechanistic studies with antivirals, viral mutations or host factors that affect COVID-19 replication

Subgenomic SARS-CoV-2 replicon and reporter replicon cell lines enable ultrahigh throughput antiviral screening and mechanistic studies with antivirals, viral mutations or host factors that affect COVID-19 replication

Reverse Genetic Approaches for the Generation of Full Length and Subgenomic Replicon of EV71 Virus

Two New Purification Methods of Hepatitis C Virus Particles from Serum-Free Culture System

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