Interaction of Human Immunodeficiency Virus Type 1 Reverse Transcriptase with Primer tRNA^Lys3 and Affinity Modification of The Enzyme by tRNA^Lys3 Derivatives

Abstract

The recognition of primer tRNA by retroviral reverse transcriptase is a crucial step in the replication of retroviruses. In the complex formed by HIV-1 reverse transcriptase and its natural primer tRNALy", the heterodimeric enzyme, p66/p51, binds two molecules of tRNALy" with different affinities. The same complex but in the presence of a non-complementary template, poly(A), gave higher Kd values. Preincubation of the reverse transcriptase with tRNA at concentrations comparable to the Kd2 value results in different levels of stimulation of the DNA polymerase activity: 300% in the absence and 70-80% in the presence of poly(A). The activation of the catalytically active p66 subunit is most probably mediated through tRNA interaction with the site of reverse transcriptase presenting the lower affinity.In this article, we describe the results obtained with new chemically reactive derivatives of tRNA bearing three or seven hydrophobic residues. Incubation of reverse transcriptase with tRNA derivatives, in the presence or absence of poly(A), leads to covalent binding of the reagents and inactivation of the enzymatic activity. However, during the initial step of the modification reaction, in the absence of poly(A), a slight stimulation of reverse transcriptase by tRNA derivatives took place, followed by a decrease in the enzymatic activity due to the covalent binding of tRNA derivatives to reverse transcriptase. In the presence of poly(A), enzyme inactivation occurs according to pseudo-first-order reaction kinetics. The affinities of tRNA derivatives for the p66/p51 heterodimer estimated from affinity modification data (K,, values) and from the inhibition of polymerization reaction (K, values) were determined. Each analog of tRNA presented two Kd and two K , values.

Structural Constraints in the HIV-1 Reverse Trancriptase−Primer/Template Complex for the Initiation of DNA Synthesis from Primer tRNALys3

“…We have previously demonstrated that the binding of a template poly(A) does not prevent the interaction of HIV-1 RT with tRNA Lys3 (41). Here we showed that, in the presence of poly(A), the tRNA derivatives were used as primers by HIV-1 RT.…”