The African swine fever virus g5R protein possesses mRNA decapping activity

Area of Science:

• Virology
• Molecular Biology
• Enzymology

Background:

• African Swine Fever Virus (ASFV) possesses a unique Nudix enzyme, g5R protein (g5Rp).
• Nudix enzymes are known to hydrolyze various nucleotide substrates and diphosphoinositol polyphosphates.
• Previous studies indicated ASFV g5Rp hydrolyzes diphosphoinositol polyphosphates and GTP, but not methylated mRNA cap analogues.

Purpose of the Study:

• To investigate the mRNA decapping activity of ASFV g5Rp.
• To determine the substrate specificity and mechanism of ASFV g5Rp in relation to mRNA capping.

Main Methods:

• Recombinant ASFV g5Rp was expressed and purified.
• Enzymatic assays were performed using methylated mRNA cap analogues and RNA-tethered substrates.
• Mutagenesis of the Nudix motif was conducted to assess its role in activity.
• Inhibition assays were performed using uncapped RNA and methylated cap analogues.

Main Results:

• Recombinant ASFV g5Rp demonstrated mRNA decapping activity when the cap was tethered to an RNA moiety, producing m(7)GDP.
• Mutations within the Nudix motif abolished the decapping function, confirming g5Rp's role.
• The enzyme's activity was potently inhibited by excess uncapped RNA, but not by methylated cap analogues.

Conclusions:

• ASFV g5Rp possesses mRNA decapping activity, dependent on RNA tethering.
• Substrate recognition by g5Rp appears to involve RNA binding rather than direct recognition of methylated cap structures.
• These findings provide new insights into the enzymatic functions of viral Nudix proteins and their potential roles in viral replication or host-pathogen interactions.