Structural Insight into Molecular Inhibitory Mechanism of InsP6 on African Swine Fever Virus mRNA-Decapping Enzyme g5Rp
The removal of the 5′ cap from cellular mRNAs by the African swine fever virus (ASFV) decapping enzyme, the g5R protein (g5Rp), supports viral gene expression during early infection stages. As the sole Nudix decapping enzyme encoded in the ASFV genome, g5Rp facilitates both the breakdown of cellular mRNA and the hydrolysis of diphosphoinositol polyphosphates. In this study, we present the structures of dimeric g5Rp and its complex with inositol hexakisphosphate (InsP6). The two g5Rp protomers form a dimer through head-to-head interaction, stabilized by extensive polar and nonpolar interactions. Each protomer includes a unique N-terminal helical domain and a C-terminal Nudix domain. We identified critical residues, including K8, K94, K95, K98, K175, R221, and K243, on the RNA binding interface of g5Rp, essential for RNA binding and decapping activity. Additionally, InsP6 was found to inhibit g5Rp-mediated mRNA decapping by occupying regions that overlap with the primary RNA binding sites, revealing InsP6’s regulatory role in mRNA degradation by g5Rp. Together, these findings provide structural insights into RNA-g5Rp interactions and highlight InsP6’s inhibitory mechanism on mRNA decapping by g5Rp.
Significance: African swine fever is a highly contagious, often fatal hemorrhagic disease in pigs, with no effective vaccines or specific treatments available. The g5Rp protein, the only viral mRNA-decapping enzyme, plays a crucial role in mRNA regulation and translation initiation during ASFV infection. This study presents crystal TH5427 structures of the g5Rp dimer and its complex with InsP6. Structure-based mutagenesis identified key residues for RNA binding and InsP6 interaction, demonstrating how InsP6 competitively inhibits g5Rp by blocking mRNA binding. These structure-function insights could aid in designing inhibitors targeting this essential viral enzyme, offering potential anti-ASFV strategies.