Table S1 - Novel Small-Molecule Inhibitors of Hepatitis C Virus Entry Block Viral Spread and Promote Viral Clearance in Cell Culture

HCV cell cultures representing various drug-induced and natural amino acid polymorphisms were established as described in the legend to Figure 7. Once peak viral titers were achieved, HCV RNA was isolated from each culture and the E1/E2 glycoprotein coding sequence was amplified by RT-PCR and subjected to direct DNA sequencing. Amino acid substitutions identified in the E1 or E2 glycoproteins are summarized in Table S1. The time to peak titers and associated sequencing data are also denoted in Table S1. Following electroporation of naïve target cells, GT 1a/2a-V719A HCV initially exhibited 1–2 log lower Renilla luciferase activity compared to the parent GT 1a/2a HCV background. At day 40, however, the GT 1a-V719A HCV cell culture experienced a burst in luciferase activity. Sequencing of the E1/E2 envelope glycoproteins from day 76 cultures revealed the presence of one additional amino acid variant, D263E which is localized to the N-terminal region of a hydrophobic domain in E1 (aa262–290) that houses a putative fusion peptide sequence. No changes were observed in the E1/E2 envelope sequences from either the parent GT 1a/2a HCV or the GT 1a/2a-V719G HCV variant cultures as determined by sequencing of day 69 and 76 cultures, respectively. In contrast to the GT 1a/2a HCV cell culture, the parent GT 1b/2a HCV showed a distinct pattern of infectivity in cell culture. In this case, Renilla luciferase activity rapidly reached a maximum level at day 9–10, tapering off over the following 67 days in culture. The GT 1b/2a-V719I and GT 1b/2a-V719G HCV variants exhibited 6- and 20-fold reduced levels of Renilla luciferase activity compared to the parent GT 1b/2a HCV background, respectively, while the GT 1b/2a-V719G and GT 1b/2a-V719L HCV variants exhibited a more dramatic, 2–3 log, reduction in viral titers. Sequencing of day 10 cultures revealed the presence of two additional amino acid variations in the GT 1b/2a-V719I HCV sequence. One substitution, A357T, was localized to the transmembrane domain of E1 (aa353–381) while the second substitution, A746P, was located at the immediate C-terminus of the E2 protein near the transmembrane domain. No other changes were identified by sequencing of the GT 1b/2a-V719L (day 51), GT 1b/2a-V719A HCV (day 9) variants or the parent GT 1b/2a HCV background (day 9). The GT 1b/2a-V719G HCV variant exhibited low levels of infectivity compared to the parent GT 1b/2a HCV at day 9–10. A significant burst in Renilla luciferase activity was observed between days 43–50. Sequencing at day 69 revealed two additional amino acid substitutions including: A217E and A457G located in the E1 and E2 glycoproteins, respectively. Surprisingly, viral titers of HCV GT 1b/2a reporter constructs bearing these amino acid substitutions reached and surpassed the peak levels observed for the parental GT1 b/2a HCV strain. Additional experiments are necessary to determine whether these amino acid variants represent adaptive or compensatory mutations or are simply the result of genetic drift in the long-term HCV cell cultures. Moreover, the potential role of these amino acid positions on viral entry, gene expression, and virus assembly/release requires additional study. (DOC)