Analysis of Efficiency and Fidelity of HIV-1 (+)-Strand DNA Synthesis Reveals a Novel Rate-limiting Step during Retroviral Reverse Transcription

Matthias Götte,Masanori Kameoka,Nathan Mclellan,Luciano Cellai,Mark A Wainberg

doi:10.1074/jbc.m009097200

Matthias Götte, Masanori Kameoka + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m009097200

Copy DOI

Abstract

We have analyzed the efficiency and accuracy of polymerization at several different stages during the initiation of human immunodeficiency virus type 1 (HIV-1) (+)-strand DNA synthesis. This reaction is of particular interest, as it involves the recruitment by reverse transcriptase of an RNA primer that serves as substrate for both the polymerase and RNase H activities of the enzyme. We found that the correct incorporation of the first two nucleotides was severely compromised and that formation of mismatches was completely absent at this stage of initiation. Although the fidelity of incorporations decreased concomitantly with ensuing polymerization, the elongation of mispaired primers was literally blocked. Instead, mispaired primer strands initiated a switch from active synthesis of DNA to premature RNase H-mediated primer removal. These findings suggest the existence of a fragile equilibrium between these two enzymatic activities that is shifted toward RNase H cleavage once the polymerization process is aggravated. Our data show that the initiation of HIV-1 (+)-strand DNA synthesis differs significantly from reactions involving other primer/template combinations, including tRNA-primed (-)-strand DNA synthesis.

Highlights

Alterations in the availability of deoxynucleoside triphosphates1 can profoundly affect retroviral reverse transcription in several ways [1, 2]
Experimental Design—We recently devised an in vitro system that allows the study of structure-function relationships of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) at various stages of initiation of (ϩ)-strand DNA synthesis through use of select primer/template combinations [23]
Reactions were monitored with 5Ј-end-labeled primer strands; and unless otherwise indicated, the primer/template substrate was used at a 10-fold excess over RT to ensure that reaction conditions fulfilled steady-state criteria

Summary

EXPERIMENTAL PROCEDURES

Nucleic Acids and HIV-1 RT—The oligonucleotides used in this study were chemically synthesized using the phosphoramidite method. 3Ј-End labeling was performed with [␣-32P]dATP using the RNase H-deficient RT-E478Q mutant For this purpose, 100 ng of the pure RNA primer, pre-annealed to the template, was incubated at 37 °C for 30 min with [␣-32P]dATP and 1 ␮g of enzyme. 50 ␮M was required for efficient dATP incorporation at position ϩ1 using the pure RNA primer, whereas 10 ␮M was sufficient in the case of the DNA primer These conditions allowed us to monitor correct and incorrect nucleotide insertions at position ϩ2 using varying concentrations of dCTP or dTTP (see Fig. 1A). Pyrophosphorolysis—To monitor pyrophosphorolysis, the pre-annealed RNA/DNA or DNA/DNA substrates (100 nM) were preincubated for 5 min at 37 °C with the RNase H-deficient mutant enzyme (30 nM) in buffer containing 50 mM Tris-HCl (pH 7.8), 50 mM NaCl, and 6 mM MgCl2.

RESULTS

DNA synthesis in absence of variable dNTP

DISCUSSION