One of the most challenging goals of hepatitis C virus (HCV) research is to develop well-tolerated regimens with high cure rates across a variety of patient populations. Such a regimen will likely require a combination of at least two distinct direct-acting antivirals (DAAs). Combining two or more DAAs with different resistance profiles increases the number of mutations required for viral breakthrough. Currently, most DAAs inhibit HCV replication. We recently reported that the combination of two distinct classes of HCV inhibitors, entry inhibitors and replication inhibitors, prolonged reductions in extracellular HCV in persistently infected cells. We therefore sought to identify new inhibitors targeting aspects of the HCV replication cycle other than RNA replication. We report here the discovery of the first small-molecule HCV infectivity inhibitor, GS-563253, also called HCV infectivity inhibitor 1 (HCV II-1). HCV II-1 is a substituted tetrahydroquinoline that selectively inhibits genotype 1 and 2 HCVs with low-nanomolar 50% effective concentrations. It was identified through a high-throughput screen and subsequent chemical optimization. HCV II-1 only permits the production and release of noninfectious HCV particles from cells. Moreover, infectious HCV is rapidly inactivated in its presence. HCV II-1 resistance mutations map to HCV E2. In addition, HCV-II prevents HCV endosomal fusion, suggesting that it either locks the viral envelope in its prefusion state or promotes a viral envelope conformation change incapable of fusion. Importantly, the discovery of HCV II-1 opens up a new class of HCV inhibitors that prolong viral suppression by HCV replication inhibitors in persistently infected cell cultures.O ne of the most challenging goals of hepatitis C virus (HCV) research is to develop well-tolerated regimens with high cure rates. The standard of care for HCV patients over the past decade has been to treat with pegylated interferon combined with ribavirin. Relatively recently, the HCV NS3-4A protease inhibitors teleprevir and boceprevir were added to this standard of care and have improved the sustained virologic response (1). However, poor tolerability to treatments containing pegylated interferon has motivated researchers to attempt to develop a variety of other interferon-free treatments (2). An interferon-free cure will likely require a combination of at least two antivirals with differing modes of action (3, 4). Combining multiple antivirals with different resistance profiles increases the number of resistance mutations required for viral breakthrough (5). Studies to determine optimal antiviral combinations which take into account the probability of emergence of specific resistance mutations as well as the subsequent viral fitness (3, 6, 7) are under way.We recently demonstrated that the in vitro combination of HCV entry inhibitors with HCV replication inhibitors could prolong the efficacy compared to a single treatment with either inhibitor class. Specifically, we studied the entry inhibitor anti-CD81 anti...