Cellular Growth Kinetics Distinguish a Cyclophilin Inhibitor from an HSP90 Inhibitor as a Selective Inhibitor of Hepatitis C Virus

During antiviral drug discovery, it is critical to distinguish molecules that selectively interrupt viral replication from those that reduce virus replication by adversely affecting host cell viability. In this report we investigate the selectivity of inhibitors of the host chaperone proteins cyclophilin A (CypA) and heat-shock protein 90 (HSP90) which have each been reported to inhibit replication of hepatitis C virus (HCV). By comparing the toxicity of the HSP90 inhibitor, 17-(Allylamino)-17-demethoxygeldanamycin (17-AAG) to two known cytostatic compounds, colchicine and gemcitabine, we provide evidence that 17-AAG exerts its antiviral effects indirectly through slowing cell growth. In contrast, a cyclophilin inhibitor, cyclosporin A (CsA), exhibited selective antiviral activity without slowing cell proliferation. Furthermore, we observed that 17-AAG had little antiviral effect in a non-dividing cell-culture model of HCV replication, while CsA reduced HCV titer by more than two orders of magnitude in the same model. The assays we describe here are useful for discriminating selective antivirals from compounds that indirectly affect virus replication by reducing host cell viability or slowing cell growth

Hepatitis C viral entry inhibitors prolong viral suppression by replication inhibitors in persistently-infected Huh7 cultures.

Efforts to treat HCV patients are focused on developing antiviral combinations that lead to the eradication of infection. Thus, it is important to identify optimal combinations from the various viral inhibitor classes. Based on viral dynamic models, HCV entry inhibitors are predicted to reduce viral load in a monophasic manner reflecting the slow death rate of infected hepatocytes (t1/2 = 2-70 days) and the protection of naïve, un-infected cells from HCV infection. In contrast, replication inhibitors are predicted to reduce viral load in a biphasic manner. The initial rapid reduction phase is due to the inhibition of virus production and elimination of plasma virus (t1/2∼3 hours). The second, slower reduction phase results from the elimination of infected hepatocytes. Here we sought to compare the ability of HCV entry and replication inhibitors as well as combinations thereof to reduce HCV infection in persistently-infected Huh7 cells. Treatment with 5 × EC50 of entry inhibitors anti-CD81 Ab or EI-1 resulted in modest (≤ 1 log10 RNA copies/ml), monophasic declines in viral levels during 3 weeks of treatment. In contrast, treatment with 5 × EC50 of the replication inhibitors BILN-2016 or BMS-790052 reduced extracellular virus levels more potently (~2 log10 RNA copies/ml) over time in a biphasic manner. However, this was followed by a slow rise to steady-state virus levels due to the emergence of resistance mutations. Combining an entry inhibitor with a replication inhibitor did not substantially enhance the rate of virus reduction. However, entry/replication inhibitor and replication/replication inhibitor combinations reduced viral levels further than monotherapies (up to 3 log10 RNA copies/ml) and prolonged this reduction relative to monotherapies. Our results demonstrated that HCV entry inhibitors combined with replication inhibitors can prolong antiviral suppression, likely due to the delay of viral resistance emergence

Hepatitis C Virus NS2 Protein Contributes to Virus Particle Assembly via Opposing Epistatic Interactions with the E1-E2 Glycoprotein and NS3-NS4A Enzyme Complexes ▿

The hepatitis C virus NS2 protein has been recently implicated in virus particle assembly. To further understand the role of NS2 in this process, we conducted a reverse genetic analysis of NS2 in the context of a chimeric genotype 2a infectious cell culture system. Of 32 mutants tested, all were capable of RNA replication and 25 had moderate-to-severe defects in virus assembly. Through forward genetic selection for variants capable of virus spread, we identified second-site mutations in E1, E2, NS2, NS3, and NS4A that suppressed NS2 defects in assembly. Two suppressor mutations, E1 A78T and NS3 Q221L, were further characterized by additional genetic and biochemical experiments. Both mutations were shown to suppress other NS2 defects, often with mutual exclusivity. Thus, several NS2 mutants were enhanced by NS3 Q221L and inhibited by E1 A78T, while others were enhanced by E1 A78T and inhibited by NS3 Q221L. Furthermore, we show that the NS3 Q221L mutation lowers the affinity of native, full-length NS3-NS4A for functional RNA binding. These data reveal a complex network of interactions involving NS2 and other viral structural and nonstructural proteins during virus assembly

Characterization of HCV persistently-infected cultures.

<p>(A) Examples of the level of HCV(1b/2a)-infected cells <i>vs</i>. uninfected cells after 21 days with or without replication inhibitor/entry inhibitor combination therapy. The yellow cells are infected (<i>i.e.</i> stained with anti-NS5A Ab as described in the Materials and Methods) and the blue cells are uninfected. The pluses signify relative quantifications of the percentage of infected cells in each culture (see Materials and Methods). (B) Bar graph depicting quantification of infected <i>vs</i>. uninfected cells in the untreated HCV persistently-infected culture shown in Figure. 1A (see Materials and Methods). (C) Viability of HCV persistently-infected cells after 25 days of incubation (see Materials and Methods). (D) Extracellular HCV levels (log₁₀ RNA copies/ml) during an 18-day time course initiated 7 days post infection (average of 3 assays) (HCV(2a) levels (solid circles), HCV(1b/2a) levels (solid squares, dashed line)).</p

Summary of HCV(1b/2a) Levels After 21 Days of Treatment +/− Anti-CD81 Ab.

a<p>The percentage of infected cells was estimated after anti-NS5A Ab staining (see Materials and Methods).</p

Summary of Antiviral Assay Results for the HCV Inhibitors Used in this Work.

a<p>All values are reported in nM unless otherwise indicated.</p>b<p>The HCV(1b/2a) chimeric virus expresses HCV(1b) envelope proteins and HCV(2a) replicative proteins <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065273#pone.0065273-Chan1" target="_blank">[26]</a>.</p>c<p>Not determined.</p

Reduction of HCV(1b/2a) levels by EI-1 alone and in combination with BILN-2061 or BMS-790052.

<p>HCV(1b/2a) persistently-infected cell cultures were treated with 5×EC₅₀ concentrations of the indicated HCV inhibitors for 20 days (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065273#pone-0065273-t001" target="_blank">Table 1</a> and Materials and Methods). HCV levels were normalized relative to the level of the DMSO control at each time point. This data is the average of 3 assays. Error bars represent standard deviation. Asterisks indicate statistically significant differences at day 20 from the DMSO day 20 time point (<i>t</i> test P≤0.05). DMSO (solid circles and solid line), EI-1 (pierced diamonds and dashed line), BILN-2061 (solid squares and dashed line), BMS-790052 (pierced squares and solid line), BILN-2061/EI-1 (diamonds and solid line),BMS-790052/EI-1 (solid hexagons and solid line), and BILN 2061/BMS-790052 (pierced diamonds and dotted line).</p

Reduction of HCV(1b/2a) levels by anti-CD81 Ab alone and in combination with BILN-2061 or BMS-790052.

<p>HCV(1b/2a) persistently-infected cell cultures were treated with 5×EC₅₀ concentrations of the indicated HCV inhibitors for 21 days (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065273#pone-0065273-t001" target="_blank">Table 1</a> and Materials and Methods). HCV levels were normalized relative to the level of the DMSO control at each time point. This data is the average of 3 assays. Error bars represent standard deviation. Asterisks indicate statistically significant differences at day 21 from the DMSO day 21 time point (<i>t</i> test P≤0.05). (A) DMSO (solid circles and solid line), anti-CD81 Ab (pierced circles and dashed line), BILN-2061 (solid squares and dashed line), BMS-790052 (pierced squares and solid line), BILN-2061/anti-CD81 Ab (solid diamonds and solid line), BMS-790052/anti-CD81 Ab (solid hexagons and solid line), and BILN 2061/BMS-790052 (pierced diamonds and dashed line).</p

Summary of HCV(2a) Levels After 21 Days of Treatment +/− Anti-CD81 Ab.

a<p>The percentage of infected cells was estimated after anti-NS5A Ab staining (see Materials and Methods).</p

Summary of the Resistance Mutations Observed After 3 weeks of HCV Inhibitor Treatment.

a<p>These resistance mutations were observed in 1/5 sequenced clones.</p>b<p>The HCV(1b/2a) chimeric virus expresses HCV(1b) envelope proteins and HCV(2a) replicative proteins <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065273#pone.0065273-Chan1" target="_blank">[26]</a>.</p>c<p>Not determined.</p