Hepatitis C virus (HCV) infection causes 500,000 deaths annually, associated with end-stage liver diseases. Investigations of the HCV life cycle widened the knowledge in virology, and here we discovered that two piperazinylbenzenesulfonamides inhibit HCV entry into liver cells. The entry process of HCV into host cells is a complex process, not fully understood, but characterized by multiple spatially and temporally regulated steps involving several known host factors. Through a high-content virus-infection screening analysis with a library of 1,120 biologically active chemical compounds, we identified SB258585, an antagonist of the serotonin receptor 6 (5-HT6), as a new inhibitor of HCV entry in liver-derived cell lines, as well as in primary hepatocytes. A functional characterization suggested a role for this compound, as well as for the compound SB399885 sharing a similar structure, as inhibitors of a late HCV entry step, modulating the localization of the co-receptor tight junction protein claudin 1 (CLDN1), in a 5-HT6 independent manner. Both chemical compounds induced an intracellular accumulation of CLDN1, reflecting export impairment. This regulation correlated with the modulation of protein kinase A (PKA) activity. The PKA inhibitor H89 fully reproduced these phenotypes. Furthermore, PKA activation resulted in increased CLDN1 accumulation at the cell surface. Interestingly, increase of CLDN1 recycling did not correlate with an increased interaction with CD81 or HCV entry. These findings reinforce the hypothesis of a common pathway shared by several viruses, which involves G-protein coupled receptor -dependent signaling in late steps of viral entry.

IMPORTANCE The HCV entry process is highly complex and important details of this structured event are poorly understood. By screening a library of biologically active chemical compounds, we identified two piperazinylbenzenesulfonamides as inhibitors of HCV entry. The mechanism of inhibition was not through previously described activity of these inhibitors as antagonists of the serotonin receptor 6, but instead through modulation of PKA activity in a 5-HT6 independent manner, as proven by the lack of 5-HT6 in liver. We thus highlighted the involvement of PKA pathway in modulating HCV post-binding step entry and in the recycling of the tight junction protein claudin-1 (CLDN1) towards the cell surface. Our work underscores once more the complexity of HCV entry steps and suggests a role for PKA pathway as regulator of CLDN1 recycling, having an impact on both cell biology and virology.