Selective excision of chain-terminating nucleotides by HIV-1 reverse transcriptase with phosphonoformate as substrate

Area of Science:

• Biochemistry
• Virology
• Drug Discovery

Background:

• HIV-1 reverse transcriptase (RT) resistance to nucleoside analogs often involves phosphorolytic removal of chain-terminating nucleotides.
• Understanding this resistance mechanism is crucial for developing effective antiviral therapies.

Purpose of the Study:

• To investigate the effects of phosphonoformate (PFA) and other pyrophosphate (PP(i)) analogs on the phosphorolysis activity of HIV-1 RT.
• To elucidate the mechanism by which PFA and related compounds interact with HIV-1 RT and affect nucleoside analog resistance.

Main Methods:

• Enzyme kinetics assays were performed using purified HIV-1 RT.
• The effects of PFA, phosphonoacetate, and pamidronate on PP(i)- and ATP-dependent phosphorolysis were analyzed.
• Experiments involved both terminated and non-terminated DNA primers, as well as AZT-triphosphate.

Main Results:

• PFA acted as an alternative substrate, facilitating AZT removal from terminated primers, but competitively inhibited phosphorolysis on non-terminated primers.
• Phosphonoacetate was a substrate for excision on both primer types, while pamidronate competitively inhibited RT activity.
• Pamidronate, but not PFA, showed synergistic inhibition with AZT-triphosphate in the presence of PP(i) or ATP.

Conclusions:

• Only specific pyrophosphate analogs can enhance nucleosidic inhibitors by preventing chain-terminator excision by HIV-1 RT.
• PFA's mechanism involves acting as a substrate for chain-terminator removal under certain conditions.
• These findings offer insights into combined chemotherapy strategies against HIV-1.