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 (<i>Areca catechu</i> 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 <i>Arepavirus</i> in the family <i>Potyviridae</i>. 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 (<i>NbeIF4A</i>, <i>NbSAMS1α</i>, <i>NbTEF1α</i>, <i>NbUEP1,</i> and <i>NbRan2</i>) was upregulated under the condition of viral infection, while the expression of another five genes (<i>NbpsbS1</i>, <i>NbPGK</i>, <i>NbchIP</i>, <i>NbClpC1A</i>, and <i>NbCysPrx</i>) was downregulated. Functional assays showed that silencing <i>NbeIF4A</i> or <i>NbPGK</i> significantly reduced viral accumulation <i>in Nicotiana benthamiana</i>. 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.