Nucleotide Sugar Pucker Preference Mitigates Excision by HIV-1 RT

Area of Science:

• Molecular Biology
• Virology
• Biochemistry

Background:

• HIV-1 reverse transcriptase (RT) is a critical enzyme for retroviral replication.
• AZT resistance (AZTr) in HIV-1 RT poses a significant challenge in antiviral therapy.
• The catalytic activity of RT, including nucleotide excision, is crucial for its function.

Purpose of the Study:

• To investigate the impact of 3'-terminal nucleotide sugar pucker conformation on HIV-1 RT excision.
• To determine if altered sugar conformations affect both wild-type (WT) and AZT-resistant (AZTr) RT activity.
• To elucidate the relationship between nucleotide sugar conformation, RT positioning, and catalytic function.

Main Methods:

• Chemical synthesis of DNA primers with varied 3'-terminal nucleotide sugar pucker conformations.
• Solid-phase synthesis techniques were employed for primer preparation.
• Assays were conducted to assess the excision of 3'-terminal nucleotides by WT-RT and AZTr-RT.

Main Results:

• Nucleosides favoring the North conformation were significantly more resistant to excision by both WT-RT and AZTr-RT.
• DNA primers with North puckered nucleotides at the 3'-terminus altered the translocation status of the RT/template/primer complex.
• This altered translocation provides a mechanism to prevent nucleotide excision.

Conclusions:

• A direct correlation exists between the 3'-terminal nucleotide sugar conformation and HIV-1 RT's susceptibility to phosphorolytic excision.
• Nucleotide sugar conformation influences the precise positioning of HIV-1 RT on its nucleic acid substrate.
• Sugar conformation is a key determinant of RT catalytic function and resistance to excision.