Yongzhe Zhu

Background: Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, remains a leading cause of viral encephalitis. Current management is largely supportive, with no specific antivirals. This study evaluated the antiviral efficacy and mechanism of action of CC-90009 against JEV in vitro and in vivo. Methods: Five targeted protein degraders (TPDs) were screened for anti-JEV activity in the human neuroblastoma cell line SH-SY5Y. Time-of-addition, binding, and endocytosis assays were used to delineate the phase of action of CC-90009, a cereblon (CRBN) E3 ligase modulator (CELMoD) and molecular glue degrader. Small interfering RNA knockdown and co-immunoprecipitation (Co-IP) confirmed targets essential for its antiviral effects. The broad-spectrum activity of CC-90009 against other mosquito-borne viruses was also evaluated. In vivo efficacy was tested in a murine JEV model. Results: Of the five TPDs tested, only CC-90009 significantly inhibited JEV infection in SH-SY5Y cells, acting during both viral entry and post-entry phases without reducing adsorbed or internalised virions. CC-90009 reduced JEV RNA and non-structural protein accumulation. Knockdown of G1-to-S phase transition 1 (GSPT1), a key target of CC-90009, suppressed JEV infection and translation; Co-IP confirmed GSPT1 interaction with JEV non-structural protein 5 (NS5). CC-90009 disrupted JEV translation and replication by inducing proteasomal degradation of the GSPT1/NS5 complex, further demonstrating its broad-spectrum antiviral activity through the effective inhibition of West Nile virus and chikungunya virus. In vivo, it protected mice from JEV-induced mortality, reducing viral load, antigen levels, and brain pathology. Conclusions: CC-90009 exerts potent anti-JEV activity both in vitro and in vivo by inducing proteasomal degradation of the GSPT1/NS5 complex, thereby disrupting viral translation and replication. This targeted protein degradation strategy represents a novel host-directed antiviral approach with promising therapeutic potential against mosquito-borne viral encephalitis.

Enterovirus A71 (EV‐A71) causes hand, foot, and mouth disease, which can lead to further infections and spreads via the oral cavity; however, the mechanism of how EV‐A71 infects the human oral cavity remains unclear. Screening and validation using small‐interfering RNAs and chemical inhibitors showed that EV‐A71 entry into human oral epidermoid carcinoma KB cells was independent of clathrin‐, caveolin‐, endophilin‐, dynamin‐, and macropinocytosis‐mediated pathways. However, the clathrin‐independent carriers/GPI‐anchored‐protein‐enriched endosomal compartment (CLIC/GEEC) pathway is crucial for EV‐A71 entry into KB cells and normal human oral epithelial cells (NHOEC), which requires the entire actin cytoskeleton and membrane cholesterol. Inhibition of GBF1, ARF1, CDC42, PICK1, GRAF1, and IRSp53, the key molecules of the CLIC/GEEC pathway, significantly suppressed EV‐A71 entry and infection. Confocal microscopy showed that internalized EV‐A71 colocalized with CDC42 and GRAF1. Knockdown of CDC42 or GRAF1 reduced the number of internalized viral particles, which were predominantly localized at the plasma membrane. By using the sucrose density gradient centrifugation, EV‐A71 and glycosylphosphatidylinositol‐anchored GFP (GPI‐GFP) were observed within the same low‐density components. Furthermore, endocytosed EV‐A71 was colocalized with GRAF1 and GPI‐GFP and transported to the late endosomes. Mouse experiments demonstrated that the GBF1 inhibitor, Golgicide A, significantly reduced EV‐A71 infection‐induced mortality. Immunohistochemical staining and histopathological section analysis revealed that Golgicide A markedly decreased the viral load in brain tissue and oral epithelium, and alleviated the pathological damage induced by the virus in brain tissue. Our findings reveal a novel pathway for EV‐A71 entry into KB cells and provide a new target for drug development.

Infection of the central nervous system caused by enterovirus 71 (EV71) remains the main cause of death in hand‐foot‐and‐mouth disease. However, the mechanism responsible for how EV71 breaks through the blood‐brain barrier to infect brain cells has yet to be elucidated. By performing a high‐throughput small interfering RNA (siRNA) screening and validation, we found that the infection of human brain microvascular endothelial cells (HBMECs) by EV71 was independent of the endocytosis pathways mediated by caveolin, clathrin, and macropinocytosis but dependent on ADP‐ribosylation factor 6 (ARF6), a small guanosinetriphosphate (GTP)‐binding protein of the Ras superfamily. The specific siRNA targeting ARF6 markedly inhibited HBMECs susceptibility to EV71. EV71 infectivity was inhibited by NAV‐2729, a specific inhibitor of ARF6, in a dose‐dependent manner. The subcellular analysis demonstrated the co‐localization of the endocytosed EV71 and ARF6, while knockdown of ARF6 with siRNA remarkably influenced EV71 endocytosis. By immunoprecipitation assays, we found a direct interaction of ARF6 with EV71 viral protein. Furthermore, ARF1, another small GTP‐binding protein, was also found to participate in ARF6‐mediated EV71 endocytosis. Murine experiments demonstrated that NAV‐2729 significantly alleviated mortality caused by EV71 infection. Our study revealed a new pathway by which EV71 enters the HBMECs and provides new targets for drug development.

Culex mosquitoes are the primary vectors of the Japanese encephalitis virus (JEV). Since its discovery in 1935, Japanese encephalitis (JE), caused by JEV, has posed a significant threat to human health. Despite the widespread implementation of several JEV vaccines, the transmission chain of JEV in the natural ecosystem has not changed, and the vector of transmission cannot be eradicated. Therefore, JEV is still the focus of attention for flaviviruses. At present, there is no clinically specific drug for JE treatment. JEV infection is a complex interaction between the virus and the host cell, which is the focus of drug design and development. An overview of antivirals that target JEV elements and host factors is presented in this review. In addition, drugs that balance antiviral effects and host protection by regulating innate immunity, inflammation, apoptosis, or necrosis are reviewed to treat JE effectively.