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High-throughput screening and rapid inhibitor triage using an infectious chimeric Hepatitis C virus.

Wichroski MJ, Fang J, Eggers BJ, Rose RE, Mazzucco CE, Pokornowski KA, Baldick CJ, Anthony MN, Dowling CJ, Barber LE, Leet JE, Beno BR, Gerritz SW, Agler ML, Cockett MI, Tenney DJ - PLoS ONE (2012)

Bottom Line: The assay was validated using known HCV antivirals and through a large-scale, high-throughput screening campaign that identified novel and selective entry, replication and late-stage inhibitors.Selection and characterization of resistant viruses provided information regarding inhibitor target and mechanism.Leveraging results from this robust whole-virus assay represents a critical first step towards identifying inhibitors of novel targets to broaden the spectrum of antivirals for the treatment of HCV.

View Article: PubMed Central - PubMed

Affiliation: Bristol-Myers Squibb Research and Development, Wallingford, Connecticut, United States of America.

ABSTRACT
The recent development of a Hepatitis C virus (HCV) infectious virus cell culture model system has facilitated the development of whole-virus screening assays which can be used to interrogate the entire virus life cycle. Here, we describe the development of an HCV growth assay capable of identifying inhibitors against all stages of the virus life cycle with assay throughput suitable for rapid screening of large-scale chemical libraries. Novel features include, 1) the use of an efficiently-spreading, full-length, intergenotypic chimeric reporter virus with genotype 1 structural proteins, 2) a homogenous assay format compatible with miniaturization and automated liquid-handling, and 3) flexible assay end-points using either chemiluminescence (high-throughput screening) or Cellomics ArrayScan™ technology (high-content screening). The assay was validated using known HCV antivirals and through a large-scale, high-throughput screening campaign that identified novel and selective entry, replication and late-stage inhibitors. Selection and characterization of resistant viruses provided information regarding inhibitor target and mechanism. Leveraging results from this robust whole-virus assay represents a critical first step towards identifying inhibitors of novel targets to broaden the spectrum of antivirals for the treatment of HCV.

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Related in: MedlinePlus

Late Stage Inhibitors.Inhibitors that showed enhanced potency in the multi-cycle virus replication assay (Inhs 14–16) were tested for their ability to block the production of infectious virus. A. Control inhibitors that block virus entry (EI) and genome replication (BMS-339) inhibited Renilla luciferase expression in both producer (black bars) and target (gray bars) cells while a control late-stage inhibitor (LY411575) only affected Renilla luciferase expression in target cells. Similar to LY411575, Inhs 14–17 exhibited less than 20% inhibition of Renilla luciferase expression in producer cells but >75% inhibition in target cells suggesting a block in the production of infectious virus. B. Genotype coverage was assessed by comparing the potency (EC50) of Inhs 14–17 and LY411575 against HCVcc with genotype 1a, 1b or 2a structural proteins.
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pone-0042609-g008: Late Stage Inhibitors.Inhibitors that showed enhanced potency in the multi-cycle virus replication assay (Inhs 14–16) were tested for their ability to block the production of infectious virus. A. Control inhibitors that block virus entry (EI) and genome replication (BMS-339) inhibited Renilla luciferase expression in both producer (black bars) and target (gray bars) cells while a control late-stage inhibitor (LY411575) only affected Renilla luciferase expression in target cells. Similar to LY411575, Inhs 14–17 exhibited less than 20% inhibition of Renilla luciferase expression in producer cells but >75% inhibition in target cells suggesting a block in the production of infectious virus. B. Genotype coverage was assessed by comparing the potency (EC50) of Inhs 14–17 and LY411575 against HCVcc with genotype 1a, 1b or 2a structural proteins.

Mentions: To confirm that Inhs 14–17 targeted a late stage in the virus life cycle, their ability to block production of infectious virus was analyzed. Huh-7.5 cells were infected with HCVcc (MOI = 1) in the presence of 2× EC90 of each compound and supernatant from these cells (virus producer cells) was harvested at 48 h pi and transferred to naïve target cells. Luciferase expression was measured in both producer and target cells at 48 h pi. As expected, viral replication was inhibited in producer cells by both the control entry (EI) and replication (BMS-339) inhibitors (Fig. 8A). On the contrary, LY411575 and Inhs 14–17 had no effect on genome replication in infected producer cells but rather inhibited the infection of naïve target cells (Fig. 8A) suggesting that these inhibitors target a late stage of the virus life cycle that affects the production of infectious virus. LY411575, as well as Inhs 14–17, exhibited similar potency against genotype 1a, 1b and 2a HCVcc chimeras (Fig. 8B).


High-throughput screening and rapid inhibitor triage using an infectious chimeric Hepatitis C virus.

Wichroski MJ, Fang J, Eggers BJ, Rose RE, Mazzucco CE, Pokornowski KA, Baldick CJ, Anthony MN, Dowling CJ, Barber LE, Leet JE, Beno BR, Gerritz SW, Agler ML, Cockett MI, Tenney DJ - PLoS ONE (2012)

Late Stage Inhibitors.Inhibitors that showed enhanced potency in the multi-cycle virus replication assay (Inhs 14–16) were tested for their ability to block the production of infectious virus. A. Control inhibitors that block virus entry (EI) and genome replication (BMS-339) inhibited Renilla luciferase expression in both producer (black bars) and target (gray bars) cells while a control late-stage inhibitor (LY411575) only affected Renilla luciferase expression in target cells. Similar to LY411575, Inhs 14–17 exhibited less than 20% inhibition of Renilla luciferase expression in producer cells but >75% inhibition in target cells suggesting a block in the production of infectious virus. B. Genotype coverage was assessed by comparing the potency (EC50) of Inhs 14–17 and LY411575 against HCVcc with genotype 1a, 1b or 2a structural proteins.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3412796&req=5

pone-0042609-g008: Late Stage Inhibitors.Inhibitors that showed enhanced potency in the multi-cycle virus replication assay (Inhs 14–16) were tested for their ability to block the production of infectious virus. A. Control inhibitors that block virus entry (EI) and genome replication (BMS-339) inhibited Renilla luciferase expression in both producer (black bars) and target (gray bars) cells while a control late-stage inhibitor (LY411575) only affected Renilla luciferase expression in target cells. Similar to LY411575, Inhs 14–17 exhibited less than 20% inhibition of Renilla luciferase expression in producer cells but >75% inhibition in target cells suggesting a block in the production of infectious virus. B. Genotype coverage was assessed by comparing the potency (EC50) of Inhs 14–17 and LY411575 against HCVcc with genotype 1a, 1b or 2a structural proteins.
Mentions: To confirm that Inhs 14–17 targeted a late stage in the virus life cycle, their ability to block production of infectious virus was analyzed. Huh-7.5 cells were infected with HCVcc (MOI = 1) in the presence of 2× EC90 of each compound and supernatant from these cells (virus producer cells) was harvested at 48 h pi and transferred to naïve target cells. Luciferase expression was measured in both producer and target cells at 48 h pi. As expected, viral replication was inhibited in producer cells by both the control entry (EI) and replication (BMS-339) inhibitors (Fig. 8A). On the contrary, LY411575 and Inhs 14–17 had no effect on genome replication in infected producer cells but rather inhibited the infection of naïve target cells (Fig. 8A) suggesting that these inhibitors target a late stage of the virus life cycle that affects the production of infectious virus. LY411575, as well as Inhs 14–17, exhibited similar potency against genotype 1a, 1b and 2a HCVcc chimeras (Fig. 8B).

Bottom Line: The assay was validated using known HCV antivirals and through a large-scale, high-throughput screening campaign that identified novel and selective entry, replication and late-stage inhibitors.Selection and characterization of resistant viruses provided information regarding inhibitor target and mechanism.Leveraging results from this robust whole-virus assay represents a critical first step towards identifying inhibitors of novel targets to broaden the spectrum of antivirals for the treatment of HCV.

View Article: PubMed Central - PubMed

Affiliation: Bristol-Myers Squibb Research and Development, Wallingford, Connecticut, United States of America.

ABSTRACT
The recent development of a Hepatitis C virus (HCV) infectious virus cell culture model system has facilitated the development of whole-virus screening assays which can be used to interrogate the entire virus life cycle. Here, we describe the development of an HCV growth assay capable of identifying inhibitors against all stages of the virus life cycle with assay throughput suitable for rapid screening of large-scale chemical libraries. Novel features include, 1) the use of an efficiently-spreading, full-length, intergenotypic chimeric reporter virus with genotype 1 structural proteins, 2) a homogenous assay format compatible with miniaturization and automated liquid-handling, and 3) flexible assay end-points using either chemiluminescence (high-throughput screening) or Cellomics ArrayScan™ technology (high-content screening). The assay was validated using known HCV antivirals and through a large-scale, high-throughput screening campaign that identified novel and selective entry, replication and late-stage inhibitors. Selection and characterization of resistant viruses provided information regarding inhibitor target and mechanism. Leveraging results from this robust whole-virus assay represents a critical first step towards identifying inhibitors of novel targets to broaden the spectrum of antivirals for the treatment of HCV.

Show MeSH
Related in: MedlinePlus