Structural Insight into Molecular Inhibitory Mechanism of InsP₆ on African Swine Fever Virus mRNA-Decapping Enzyme...

Area of Science:

• Structural biology
• Virology
• Biochemistry

Background:

• African swine fever virus (ASFV) infection poses a significant threat to domestic pigs, with no effective vaccines or treatments currently available.
• The ASFV g5R protein (g5Rp) is a unique viral enzyme essential for viral gene expression by removing the 5' cap from cellular mRNAs.
• g5Rp belongs to the Nudix hydrolase superfamily and plays a dual role in cellular mRNA degradation and diphosphoinositol polyphosphate hydrolysis.

Purpose of the Study:

• To elucidate the structural basis of g5Rp function, including its dimeric form and interaction with inositol hexakisphosphate (InsP6).
• To identify key residues involved in RNA binding and decapping activity.
• To understand the inhibitory mechanism of InsP6 on g5Rp-mediated mRNA decapping.

Main Methods:

• X-ray crystallography was used to determine the structures of dimeric g5Rp and its complex with InsP6.
• Structure-based mutagenesis was employed to identify critical residues for RNA binding and enzyme activity.
• Biochemical assays were performed to assess the inhibitory effect of InsP6 on g5Rp activity.

Main Results:

• The crystal structures revealed that g5Rp forms a dimer through extensive polar and nonpolar interactions, with each protomer comprising a unique N-terminal helical domain and a C-terminal Nudix domain.
• Key residues (K8, K94, K95, K98, K175, R221, and K243) were identified at the substrate RNA binding interface, crucial for RNA binding and decapping activity.
• InsP6 was found to inhibit g5Rp-mediated mRNA decapping by competitively occupying the same binding site as the substrate RNA.

Conclusions:

• The study provides the first structural insights into the dimeric g5Rp enzyme and its interaction with InsP6.
• The findings highlight InsP6 as a competitive inhibitor of g5Rp, elucidating its role in regulating viral mRNA decapping.
• These structure-function insights offer a foundation for designing novel antiviral inhibitors targeting g5Rp to combat ASFV infections.