A Comprehensive Screening of the Interactors of Areca Palm Necrotic Ringspot Virus (ANRSV) HCPro2 Highlights the Proviral Roles of eIF4A and PGK in Viral Infection.

The areca palm ( L.), a medicinal tropical crop, hosts three novel viruses, areca palm necrotic ringspot virus (ANRSV), areca palm necrotic spindle-spot virus (ANSSV), and ANRSV2, which form a new genus in the family . Both viruses feature a unique tandem leader protease arrangement (HCPro1-HCPro2). To elucidate HCPro2's role, this study identified its interaction partners in infected cells using affinity purification coupled with liquid chromatography-tandem mass spectrometry, a yeast two-hybrid system, and co-immunoprecipitation. Thirteen host proteins and five viral factors (HCPro1, 6K2, VPg, NIa-Pro, NIb) were validated as HCPro2 interactors. Among the host proteins interacting with HCPro2, the expression of five genes (, , , and ) was upregulated under the condition of viral infection, while the expression of another five genes (, , , , and ) was downregulated. Functional assays showed that silencing or significantly reduced viral accumulation . These findings reveal HCPro2's network of virus-host interaction, highlighting its critical role in viral pathogenesis. Further exploration of these interactions may clarify the evolutionary significance of tandem leader proteases and inform novel plant antiviral strategies.