by Wanfeng Liu, Yu Shi, Xudong Guo, Chenyu Lou, Zhangyi Xie, Yitian Shen, Liang Zhang, Sunzhong Mao, Xiangyang Xue, Shiyu FengHuman cytomegalovirus (HCMV) is a major pathogen affecting immunocompromised individuals. Current antiviral drugs target the mid-to-late stages of viral replication but are limited by toxicity and drug resistance. The IE1 protein serves as a key early regulator of HCMV replication; however, it has long been considered undruggable due to the lack of conventional drug-binding sites. In this study, by integrating molecular docking-based virtual screening with in-vitro surface plasmon resonance (SPR) validation, we discovered for the first time that the HIV protease inhibitor ritonavir may bind to the IE1 protein. Further experiments demonstrated that ritonavir significantly reduces IE1 protein levels in a time- and dose-dependent manner without affecting its mRNA expression. Mechanistic investigations revealed that ritonavir promotes selective degradation of IE1 through activation of the ubiquitin-proteasome pathway. Functional assays confirmed that ritonavir effectively inhibits HCMV replication, markedly reducing viral DNA loads and the production of infectious viral particles. Notably, ritonavir exhibited synergistic antiviral effects when combined with the first-line drug ganciclovir (GCV). In summary, this study successfully repurposes the approved drug ritonavir as a regulator of IE1 protein expression, demonstrating not only its anti-HCMV activity and synergistic effects with GCV but also providing a new therapeutic strategy for HCMV infection.