Limits...
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.

Show MeSH

Related in: MedlinePlus

The gt 1a/2a-Rluc virus is capable of multi-cycle virus growth amenable to unbiased inhibitor detection.A. Schematic of the genotype 1a/2a-Rluc virus which harbors Core-NS2 from the genotype 1a H77 isolate fused to NS3-NS5B of the genotype 2a JFH1 isolate with an Rluc reporter gene cloned between NS5A and NS5B. B–E. Huh-7.5 cells were infected with gt 1a/2a-Rluc (MOI = 0.05) and processed for HCV Core immunofluorescence or Renilla Luciferase expression 48, 72 and 96 h post-infection (pi). Virus spread was inhibited using an HCV entry inhibitor (EI; 1 µM) added 16 h post-entry [EI (Post-Entry)] or with an SPP inhibitor (LY411575; 0.5 µM). Virus infectivity and spread were assessed directly using immunofluorescence microscopy of HCV Core (green) and Huh-7.5 nuclei (red) to calculate the number of infected cells per viral foci (B, C, & D) or indirectly with Renilla Luciferase (D & E). Results are expressed as the mean and standard deviation of at least two independent assays.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3412796&req=5

pone-0042609-g001: The gt 1a/2a-Rluc virus is capable of multi-cycle virus growth amenable to unbiased inhibitor detection.A. Schematic of the genotype 1a/2a-Rluc virus which harbors Core-NS2 from the genotype 1a H77 isolate fused to NS3-NS5B of the genotype 2a JFH1 isolate with an Rluc reporter gene cloned between NS5A and NS5B. B–E. Huh-7.5 cells were infected with gt 1a/2a-Rluc (MOI = 0.05) and processed for HCV Core immunofluorescence or Renilla Luciferase expression 48, 72 and 96 h post-infection (pi). Virus spread was inhibited using an HCV entry inhibitor (EI; 1 µM) added 16 h post-entry [EI (Post-Entry)] or with an SPP inhibitor (LY411575; 0.5 µM). Virus infectivity and spread were assessed directly using immunofluorescence microscopy of HCV Core (green) and Huh-7.5 nuclei (red) to calculate the number of infected cells per viral foci (B, C, & D) or indirectly with Renilla Luciferase (D & E). Results are expressed as the mean and standard deviation of at least two independent assays.

Mentions: An intergenotypic (1a/2a) reporter (Renilla luciferase; Rluc) virus, engineered and adapted for high-titer replication and spread [8] was used to develop a multi-cycle virus growth assay. The gt 1a/2a-Rluc virus harbors genotype 1a (H77) Core-NS2, genotype 2a (JFH1) NS3-NS5B and a stable Rluc cassette inserted intergenically between NS5A and NS5B (Fig. 1A) and typically yields titers of 1–5×105 focus forming units (ffu)/ml (data not shown). As a first step in developing a screening assay capable of detecting inhibitors at all stages of the virus life cycle, the spreading kinetics of the gt 1a/2a-Rluc virus was characterized in Huh-7.5 cells. To discriminate between enlarging foci due to virus spread and those due to cell division alone, control cultures included inhibitors that block either virus entry or assembly. A virus entry inhibitor capable of blocking both entry and cell-cell spread (EI [8]) was added to cultures 16 h following the initial infection (EI added post-entry; EIPE) to simulate a late-stage inhibitor through its ability to block virus spread. A signal peptide peptidase (SPP) inhibitor, LY411575, which blocks the SPP-mediated processing/maturation of Core, was used to block the release of infectious virus [29], [30], [31], [32], [33], [34], [35], [36], [37], [38], [39], [40]. The results showed that HCV Core-positive virus foci were detectable within 48 h post-infection (pi) and their size (2.2±0.8 mean cells/foci) was indistinguishable from cultures in which spread was inhibited with EIPE (1.9±0.8) or LY411575 (2.0±0.6; Figs. 1B & C), suggesting that virus spread was not detectable at this time point. Expansion of virus foci was typically observed within 72 h (17.6±9.4) and large foci (88.0±29) were observed at 96 h pi (Figs. 1B & C). The expansion of virus foci was blocked by both EIPE and LY411575 with >20 fold inhibition observed at the 96 h time point (Figs. 1B & C). Importantly, the expansion of virus foci correlated with an increase in Renilla luciferase expression (Fig. 1D). Consistent with the results above, EIPE and LY411575 exhibited only modest inhibition (<20%) of luciferase expression at 48 h pi but achieved >80% at 72 h and >90% inhibition at 96 pi (Fig. 1E). As expected, addition of the entry inhibitor or an NS3 protease inhibitor (BMS-339) at the time of infection inhibited luciferase expression at all time points (Fig. 1E). Taken together, these results suggested that a 96 h incubation period was necessary and sufficient for unbiased identification of inhibitors of all phases of viral replication using the gt 1a/2a-Rluc virus. Our goal was to develop an assay where >90% of the Luciferase signal was due to virus spread so as to avoid bias towards early or genome replication inhibitors. While it has been reported that earlier time points are optimal for spreading of fully genotype 2a viruses, Figure 1 shows that a 96 h incubation period was required to achieve this goal. These results further demonstrated that single and multi-cycle virus replication could be delineated by monitoring luciferase expression at either the 48 or 96 h pi time points, respectively.


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)

The gt 1a/2a-Rluc virus is capable of multi-cycle virus growth amenable to unbiased inhibitor detection.A. Schematic of the genotype 1a/2a-Rluc virus which harbors Core-NS2 from the genotype 1a H77 isolate fused to NS3-NS5B of the genotype 2a JFH1 isolate with an Rluc reporter gene cloned between NS5A and NS5B. B–E. Huh-7.5 cells were infected with gt 1a/2a-Rluc (MOI = 0.05) and processed for HCV Core immunofluorescence or Renilla Luciferase expression 48, 72 and 96 h post-infection (pi). Virus spread was inhibited using an HCV entry inhibitor (EI; 1 µM) added 16 h post-entry [EI (Post-Entry)] or with an SPP inhibitor (LY411575; 0.5 µM). Virus infectivity and spread were assessed directly using immunofluorescence microscopy of HCV Core (green) and Huh-7.5 nuclei (red) to calculate the number of infected cells per viral foci (B, C, & D) or indirectly with Renilla Luciferase (D & E). Results are expressed as the mean and standard deviation of at least two independent assays.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0042609-g001: The gt 1a/2a-Rluc virus is capable of multi-cycle virus growth amenable to unbiased inhibitor detection.A. Schematic of the genotype 1a/2a-Rluc virus which harbors Core-NS2 from the genotype 1a H77 isolate fused to NS3-NS5B of the genotype 2a JFH1 isolate with an Rluc reporter gene cloned between NS5A and NS5B. B–E. Huh-7.5 cells were infected with gt 1a/2a-Rluc (MOI = 0.05) and processed for HCV Core immunofluorescence or Renilla Luciferase expression 48, 72 and 96 h post-infection (pi). Virus spread was inhibited using an HCV entry inhibitor (EI; 1 µM) added 16 h post-entry [EI (Post-Entry)] or with an SPP inhibitor (LY411575; 0.5 µM). Virus infectivity and spread were assessed directly using immunofluorescence microscopy of HCV Core (green) and Huh-7.5 nuclei (red) to calculate the number of infected cells per viral foci (B, C, & D) or indirectly with Renilla Luciferase (D & E). Results are expressed as the mean and standard deviation of at least two independent assays.
Mentions: An intergenotypic (1a/2a) reporter (Renilla luciferase; Rluc) virus, engineered and adapted for high-titer replication and spread [8] was used to develop a multi-cycle virus growth assay. The gt 1a/2a-Rluc virus harbors genotype 1a (H77) Core-NS2, genotype 2a (JFH1) NS3-NS5B and a stable Rluc cassette inserted intergenically between NS5A and NS5B (Fig. 1A) and typically yields titers of 1–5×105 focus forming units (ffu)/ml (data not shown). As a first step in developing a screening assay capable of detecting inhibitors at all stages of the virus life cycle, the spreading kinetics of the gt 1a/2a-Rluc virus was characterized in Huh-7.5 cells. To discriminate between enlarging foci due to virus spread and those due to cell division alone, control cultures included inhibitors that block either virus entry or assembly. A virus entry inhibitor capable of blocking both entry and cell-cell spread (EI [8]) was added to cultures 16 h following the initial infection (EI added post-entry; EIPE) to simulate a late-stage inhibitor through its ability to block virus spread. A signal peptide peptidase (SPP) inhibitor, LY411575, which blocks the SPP-mediated processing/maturation of Core, was used to block the release of infectious virus [29], [30], [31], [32], [33], [34], [35], [36], [37], [38], [39], [40]. The results showed that HCV Core-positive virus foci were detectable within 48 h post-infection (pi) and their size (2.2±0.8 mean cells/foci) was indistinguishable from cultures in which spread was inhibited with EIPE (1.9±0.8) or LY411575 (2.0±0.6; Figs. 1B & C), suggesting that virus spread was not detectable at this time point. Expansion of virus foci was typically observed within 72 h (17.6±9.4) and large foci (88.0±29) were observed at 96 h pi (Figs. 1B & C). The expansion of virus foci was blocked by both EIPE and LY411575 with >20 fold inhibition observed at the 96 h time point (Figs. 1B & C). Importantly, the expansion of virus foci correlated with an increase in Renilla luciferase expression (Fig. 1D). Consistent with the results above, EIPE and LY411575 exhibited only modest inhibition (<20%) of luciferase expression at 48 h pi but achieved >80% at 72 h and >90% inhibition at 96 pi (Fig. 1E). As expected, addition of the entry inhibitor or an NS3 protease inhibitor (BMS-339) at the time of infection inhibited luciferase expression at all time points (Fig. 1E). Taken together, these results suggested that a 96 h incubation period was necessary and sufficient for unbiased identification of inhibitors of all phases of viral replication using the gt 1a/2a-Rluc virus. Our goal was to develop an assay where >90% of the Luciferase signal was due to virus spread so as to avoid bias towards early or genome replication inhibitors. While it has been reported that earlier time points are optimal for spreading of fully genotype 2a viruses, Figure 1 shows that a 96 h incubation period was required to achieve this goal. These results further demonstrated that single and multi-cycle virus replication could be delineated by monitoring luciferase expression at either the 48 or 96 h pi time points, respectively.

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