Targeting cellular cathepsins inhibits hepatitis E virus infection

Background and Aims The hepatitis E virus (HEV) is estimated to be responsible for 70,000 deaths annually, yet therapy options remain limited. In the pursuit of effective antiviral therapies, targeting viral entry holds promise and has proven effective for other hepatotropic viruses. However, the precise mechanisms and host factors required during HEV entry remain unclear. Cellular proteases have emerged as a class of host factors required for viral surface protein activation and productive cell entry by many viruses. Hence, we investigated the functional requirement and therapeutic potentials of cellular proteases during HEV infection. Approach and Results Using our recently established HEV cell culture model and subgenomic HEV replicons, we found that blocking lysosomal cathepsins (CTS) with small molecule inhibitors, impedes HEV infection without affecting replication. Most importantly, the pan-cathepsin inhibitor K11777 robustly suppressed HEV infections with an EC 50 of ∼ 0.01 nM. Inhibition by K11777, devoid of notable toxicity in hepatoma cells until micromolar concentrations, was also observed in differentiated HepaRG and ex vivo in primary human hepatocytes. Furthermore, through time-of-addition experiments, we confirmed that HEV entry is potently blocked by inhibition of cathepsins and cathepsin L (CTSL) knockout cells were less permissive to HEV suggesting that CTSL is critical for HEV infection. Conclusions In summary, our study highlights the pivotal role of lysosomal cathepsins, especially CTSL, in the HEV entry process. The profound anti-HEV efficacy of the pan-cathepsin inhibitor, K11777, especially with its notable safety profile in primary cells, further underscores its potential as a promising therapeutic candidate.