Structure-Substrate Binding Relationships of HIV-1 Reverse Transcriptase
Human Immunodeficiency Virus, type 1 (HIV-1), is the causative agent of the Acquired Immunodeficiency Syndrome (AIDS). HIV-1 reverse transcriptase (RT), a heterodimer p66/p51, has been the major target for treatment of AIDS. The significance of the p51 subunit and the RNase H domain of p66 in terms of their influence on the RNA-dependent DNA synthesis was investigated. Clones of the wildtype HIV-1 RT subunits, p66 and p51, and a recombinant C-terminal deletion mutant, p64, [Barr, P. J. (1987) Bio/Technoloav 5, 486-489] were employed to study the structure-substrate binding relationships of HIV-1 RT. The activity assays of RNA-dependent DNA synthesis on both poly(rA)(dT) and a random base RNA template hybridized with a DNA oligomer showed that p51 significantly affects the enzyme activity. The increase in processivity by p51 in the p66/p51 heterodimer was also demonstrated. These observations suggested that the integrity of p51 is important in subunit-interactions for maintaining a favorable conformation of the enzyme for optimal function. C-terminal deletion in p66 was seen to decrease the processivity. The dissociation constant (Kd) for poly(rA)(dT) obtained by nitrocellulose binding assays suggested that the processivity of HIV 1 RT on poly(rA)(dT) correlated with the affinity for the substrate. The processivity of RT on RNA335-DNA20 was seen to be affected by the pause sites observed on the autoradiograms. The pauses of DNA synthesis tended to occur at positions of template containing poly G-C sequences. The order of processivity observed on RNA335-DNA20 was p64/p64, p66/p66 < p64/p51 < p66/p51. The C-terminal deletion in p66 was shown to affect the ability to extend the DNA strand on RNA template. In those non-wildtype forms of HIV-1 RT (p66/p66, p64/p64, and p64/p51), the affinity for primer-template seemed to be sensitive to the structure of the RNA template as seen when comparing Kds between poly(rA)(dT) and RNA335-DNA20. The wildtype enzyme, p66/p51, appeared to have a similar affinity for both substrates.
HIV-1, AIDS, Reverse transcriptase, Biochemistry, Molecular Biology
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