High‐Affinity Interaction of Human Immunodeficiency Virus Type‐1 Reverse Transcriptase with Partially Complementary Primers

Abstract

The comparison of K,, and V,,,,, values for various primers in the reaction of polymerization catalyzed by the human immunodeficiency virus type-I (HIV-1) reverse transcriptase was carried out. The primers were : (a) complementary to the template, (b) partially complementary with mismatched nucleotides at different positions from the 3' end or (c) non-complementary. Non-complementary primers were not elongated by HIV-1 reverse transcriptase. However, if they contained only one residue complementary to the template or an abasic unit at the 3' end, they could serve as primers. The most effective discrimination between matched and mismatched primers, due to a decrease in the affinity and V,,,,,, was found in the case of oligonucleotides containing non-complementary bases at the second or third position from the 3' end of the primer. The efficiency of discrimination by HIV-1 reverse transcriptase between matched and mismatched base-paired primers was about 1 -1.5 orders of magnitude lower than that of procaryotic, eucaryotic and archaebacterial DNA polymerases and avian myeloblastosis virus reverse transcriptase. Oligonucleotides such as (dT),(dCdG),(dT), showed higher affinity for the enzyme than (dT), or (dT), primers. These data suggest that HIV-1 reverse transcriptase, in contrast to procaryotic, eucaryotic and archaebacterial DNA polymerases, forms additional contacts with the 5'-end region of the non-complementary primer. In addition, using tRNAy', the natural primer of HIV-1, it was shown that the p66 subunit of reverse transcriptase can be crosslinked, in the presence of a platinum derivative, to the 5' end of tRNA. Thus. besides the normal binding site for the 3' end of tRNA, which is crucial for the initiation of cDNA synthesis, the 5' end of the tRNA also interacts with a specific site on the enzyme. K~J N Y I~~T :human immunodeficiency virus type-1 reverse transcriptase ; mismatched primer elongation ; kinetic analysis ; discrimination ; tRNAiy' Studies of enzyme-kinetic mechanisms responsible for the accuracy of DNA replication are essential in understanding the molecular basis of mutagenesis. The ability of DNA polymera s e~ from bacteria, bacteriophages and eucaryotic organisms to copy DNA templates with high fidelity has been ascribed, in part, to the associated 3'-5' exonuclease proofreading activity of these DNA polymerases (Brutiag and Kornberg;Muzycska et al., 1972;Fry and Loeb, 1986). The human immunodeficiency virus type-1 (HIV-1) has been found to exhibit extensive penomic heterogeneity. An explanation for generating sequence diversity derives from the fact that reverse transcriptase (RT) lacks 3'-5' exonuclease proofreading activity for Corre.\L.sponrlence to G. A.

Interaction of Human Immunodeficiency Virus Type 1 Reverse Transcriptase with Primer tRNALys3 and Affinity Modification of The Enzyme by tRNALys3 Derivatives

“…We have shown, by platinum-induced crosslinking, that the 5' end of tRNALys' interacts mainly with the p66 subunit of reverse transcriptase (Zakharova et al, 1995b). When higher concentrations of tRNA were used, both subunits became labeled, although the label was found mainly associated to the p66 subunit (our unpublished results).…”

Interaction of Oligonucleotides Conjugated to Substituted Chromones and Coumarins with HIV-1 Reverse Transcriptase

“…Concerning the second possibility, we have already shown that HIV-1 RT, besides interacting with the 3' end of the primer, is also able to interact with the 5'-terminal part of partially complementary primers (Zakharova et al, 1995c). We hypothesized that the 5' end of primers may interact with the tRNA-binding site of RT.…”

“…1 RT, when their 3'-terminal base is not complementary to the template (Zakharova et al, 1995c). At the same time, the elimination of the base from the 3'-terminal unit of the noncomplementary d(pA)l0 restores the ability of the 3' abasic ODN, d[(pA)9p(rib)], to function as an effective primer in the polymerization reaction catalyzed by HIV-1 RT.…”

“…INTRODUCTION tures (Nevinsky et al, 1992;Zakharova et al, 1995c). We showed that HIV-1 can bind the primer either in the presence or Human immunodeficiency virus type 1 (HIV-1) reverse in the absence of the template (Andreola et al, 1993).…”

Inhibition of HIV-1 Replication Using a Mutated tRNALys-3 Primer

“…Incorporation is also not dependent on the primer-binding site. Interactions have been described between the p66 as well as the p51 subunits of RT and the 5Ј end of tRNA by platinum cross-linking (10). Preincubation of heterodimeric RT (p66/p51) was noted to increase catalytic activity on a poly(A)-oligo(dT) template (11) suggesting that tRNA binding may also lead to "activation" of the p66/p51 heterodimer.…”