Defective Strand-Displacement DNA Synthesis Due to Accumulation of Thymidine Analogue Resistance Mutations in HIV-2...

Area of Science:

• Molecular Biology
• Virology
• Enzymology

Background:

• Retroviral reverse transcriptases (RTs) possess inherent strand displacement DNA synthesis capabilities.
• The molecular mechanisms governing RT strand displacement activity, particularly the role of the fingers subdomain, remain incompletely understood.
• Thymidine analogue resistance-associated mutations (TAMs) confer drug resistance in HIV-1 but are less common in HIV-2.

Purpose of the Study:

• To investigate the molecular determinants of strand displacement activity in human immunodeficiency virus type 2 (HIV-2) reverse transcriptase (RT).
• To compare the strand displacement activity of HIV-1 and HIV-2 RT mutants, particularly those associated with drug resistance.
• To elucidate the role of the fingers subdomain in RT-mediated DNA synthesis.

Main Methods:

• Screening of purified HIV-1 and HIV-2 RT enzymes, including drug-resistant mutants.
• Assessing strand displacement DNA synthesis activity using nicked and gapped DNA substrates.
• Quantifying nucleotide incorporation rates in strand displacement assays.

Main Results:

• Specific HIV-2 RT mutants (D67N/K70R/S215Y and M41L/D67N/K70R/S215Y) exhibited significantly reduced strand displacement activity.
• These defective HIV-2 RT mutants showed the lowest nucleotide incorporation rates on strand displacement substrates.
• In contrast, similar mutations had no significant impact on the strand displacement activity of HIV-1 RT.

Conclusions:

• The fingers subdomain of HIV-2 RT plays a crucial role in strand displacement DNA synthesis.
• The reduced strand displacement efficiency of certain HIV-2 RT mutants correlates with lower viral replication capacity.
• These findings may inform the development of novel antiviral strategies targeting strand-displacement defective RTs.