Mechanism of Inhibition of the Human Immunodeficiency Virus Type 1 Reverse Transcriptase by d4TTP: an Equivalent Incorporation Efficiency Relative to the Natural Substrate dTTP†

Among the clinically used nucleoside analogue inhibitors that target human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT), there is little detailed mechanistic information on the interactions of 2*,3*-didehydro-2*,3*-dideoxythymidine-5*-triphosphate (d4TTP) with the enzyme z primer-template complex and how these interactions compare with those of the natural substrate, dTTP. Using a pre-steady-state kinetic analysis, we found that d4TTP was incorporated by HIV-1 RT just as efficiently as dTTP during both DNA- and RNA-dependent DNA synthesis. To our knowledge, these results represent the first observation of a 3*-modified nucleoside triphosphate analogue that has an incorporation efficiency comparable to that observed for the natural substrate during DNA synthesis by HIV-1 RT. This information provides a mechanistic basis for understanding the inhibition of HIV-1 RT by d4TTP as well as insight into the clinically observed lack of d4T resistance mutations in HIV-1 RT isolated from AIDS patients. The replication of human immunodeficiency virus (HIV), which causes AIDS, requires the virally encoded enzyme reverse transcriptase (RT). RT converts the single-stranded HIV RNA genome to a double-stranded DNA copy by catalyzing both DNA-dependent and RNA-dependent DNA polymerization as well as RNase H cleavage activity to remove the RNA template once the DNA has been synthesized. Because of its unique catalytic properties, RT has been the target enzyme for many antiviral therapeutic agents used in the treatment of AIDS, including nucleoside and nonnucleoside analogues (2‐4, 8, 26). The nucleoside analogues that are used clinically