Combination indices of EI-1 with HCV replication inhibitors.

a<p>NS5A, BMS-790052; NS3, BMS-605339.</p>b<p>Mean of 8 independent experiments.</p>c<p>Loewe combination index at the combined EC₅₀ level.</p

Activity of EI-1 in infectious virus assays.

a<p>Mean of ≥4 independent experiments.</p

Effect of EI-1 on HCV cell-to-cell spread.

<p>(A) Huh-7.5 cells were infected with 0.001 ffu/cell HCVcc-1a/2a at 37°C. At 12 hrs post infection, the inoculum was removed and replaced with medium +1% agarose overlay containing EI-1 (0.5 µM) or DMSO and the cultures were incubated at 37°C for 2, 3 or 4 days. Infected cells were labeled by indirect immunofluorescence using an anti-HCV core monoclonal antibody (green) and nuclei were stained with Hoechst 3325 (red). Images were captured using a Nikon Eclipse TE300 inverted epi-fluorescence microscope. (B) The mean number and standard deviation of infected cells/focus was determined from visual counting of infected cells in ≥100 foci for each time point. (C) The mean number and standard deviation of foci/well was determined at 2 and 4 days post infection.</p

Structure-activity-relationship of the EI-1 chemotype.

a<p>Mean of ≥3 independent experiments.</p>b<p>R, aniline ring substituent (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1001086#ppat-1001086-g001" target="_blank">Fig 1A</a>).</p

Schematic diagram of the HCV E2 protein and sequences of the region encompassing the EI-1 resistance residue.

<p>Previously defined regions of the protein are indicated by the shaded boxes. Numbers correspond to the HCV polyprotein amino acid positions in E2. HVR1, hypervariable region 1. HVR2, hypervariable region 2. pFP₁ and pFP₂, putative fusion peptide regions. igVR, intergenotypic variability region. HR, heptad repeat. TMD, transmembrane domain. The asterisk indicates the position of residue 719 that is involved in EI-1 resistance. The protein sequence alignment from amino acids 709–729 for each of the HCVpp genotype isolates from <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1001086#ppat-1001086-g002" target="_blank">Figure 2</a> is shown. The shaded residues are part of the TMD.</p

Kinetics of inhibition by HCV entry inhibitors.

<p>(A) Time course of inhibition. HCVpp (1b) was added to Huh-H1 cells at 4°C and incubated for 1.5 hr. Unadsorbed virus was removed by 2 washes with cold media, fresh media was added, and the cells were shifted to 37°C to allow synchronous infection to proceed. At the indicated time points, the media was removed and replaced with media containing 0.125 µM EI-1 (triangles and solid line) or 10 nM bafilomycin A1 (squares and dashed line) and incubated at 37° for 3 days. Inhibition was calculated as a % relative to control infections containing inhibitor throughout the experiment (100%) and those lacking inhibitor (0%). The −1.5 hr time point, in which inhibitor was present throughout the infection, represents maximum inhibition. Mean values and standard deviations were calculated from triplicate wells and the results presented are from a representative of multiple experiments. (B) Attachment and post-attachment efficacy of inhibitors. Infections were performed as described in (A). Heparin (200 µg/ml), anti-CD81 monoclonal antibody (2 µg/ml), EI-1 (0.125µM), or bafilomycin A1 (10 nM) were present in the media either continuously, during the 4°C incubation only (attachment), or during the 37°C incubation phase only (post-attachment). Inhibition was calculated as % relative to control infections lacking inhibitor. Results are expressed as mean and standard deviation from 3 independent experiments.</p

E1 and E2 amino acid changes that emerged during selection with EI-1.

a<p>Fold increase in EC₅₀ compared to wild-type virus. Values represent the mean of ≥3 experiments.</p

Compound structure and activity in the HCVpp assay.

<p>(A) Structure of EI-1. (B) Dose dependent inhibition of HCVpp-1b infectivity by EI-1. Huh-H1 cells were infected with HCVpp-1b or VSVpp in the presence of various concentrations of EI-1, incubated at 37°C, and luciferase activity was measured 3 days post infection. Data are presented as percent inhibition relative to control infections lacking compound. Results are expressed as mean and standard deviation from triplicate assays. EI-1 EC₅₀ values are 0.016±0.001 and 33±2.1µM for HCVpp and VSVpp, respectively.</p

HCV genotype coverage of EI-1.

<p>Huh-H1 cells were mixed with HCVpp or VSVpp in the presence of various concentrations of EI-1. Infected cells were incubated at 37°C and luciferase activity was determined 3 days post infection. The average EC₅₀ (≥2 experiments) of each of the 40 isolates representing HCV genotypes 1–5 (triangles), as well as VSVpp (square), is shown.</p

EI-1 resistance levels of site-directed changes engineered into HCVcc or HCVpp.

a<p>Fold increase in EC₅₀ compared to wild-type virus. Values represent the mean of ≥3 experiments.</p