Binding interface and impact on protease cleavage for an RNA aptamer to HIV-1 reverse transcriptase

Area of Science:

• Biochemistry
• Molecular Biology
• Virology

Background:

• RNA aptamers targeting HIV-1 reverse transcriptase (RT) show potential for antiviral therapy.
• The specific molecular interactions governing aptamer recognition and inhibition of HIV-1 RT remain poorly understood.
• Investigating the biophysical determinants of aptamer specificity is crucial for developing effective broad-spectrum inhibitors.

Purpose of the Study:

• To elucidate the structural and biophysical basis of recognition between a broad-spectrum UCAA-family RNA aptamer and HIV-1 RT.
• To identify critical aptamer structural elements and RT-binding sites involved in inhibition.
• To explore the aptamer's effect on HIV-1 RT maturation processes.

Main Methods:

• Structure-activity relationship (SAR) studies and hydroxyl radical probing to map critical aptamer regions.
• Hydrogen-deuterium exchange (HDX) footprinting to identify RT-aptamer contact sites.
• Alanine scanning mutagenesis of HIV-1 RT to assess the impact of specific residues on aptamer binding and inhibition.
• 2D proton nuclear magnetic resonance (NMR) and small-angle X-ray scattering (SAXS) for aptamer structure determination.
• Computational modeling to predict the docked complex structure.

Main Results:

• Key aptamer structural elements, including the UCAA bulge motif, were identified as critical for RT interaction and inhibition.
• HDX footprinting revealed extensive contacts between the aptamer and both subunits of HIV-1 RT, particularly the p51 subunit's C-terminus.
• Mutagenesis studies pinpointed specific RT residues (P420, L422, K424) essential for aptamer-mediated inhibition.
• The aptamer was found to enhance the proteolytic cleavage of the p66/p66 precursor by HIV-1 protease, suggesting stabilization of a mature conformation.
• A computational model of the docked complex provided structural insights into the RT-aptamer interface.

Conclusions:

• The study illuminates the molecular features governing the specific recognition of HIV-1 RT by a broad-spectrum antiviral aptamer.
• The findings provide a mechanistic understanding of aptamer inhibition and suggest a novel role in promoting RT maturation.
• These insights open avenues for designing advanced aptamer-based therapeutics to combat HIV replication by targeting RT recognition and maturation.