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

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 tRNAprimed (؊)-strand DNA synthesis.

The Pyrophosphate Analogue Foscarnet Traps the Pre-translocational State of HIV-1 Reverse Transcriptase in a Brownian Ratchet Model of Polymerase Translocation

“…1). The substrate was derived from the polypurine tract (PPT) of HIV-1 as previously described (30). Reactions with PFA show distinct bands at positions ϩ2, ϩ3, ϩ7, ϩ8, ϩ12, ϩ14, ϩ16, and ϩ25 (Fig.…”

“…Steady-state Kinetics-Steady-state kinetic parameters K m , k cat , and K i for single nucleotide incorporation events in the presence or absence of PFA were determined through gelbased assays (30). We used three different primers (PPTϩ4, PPTϩ6, and PPTϩ16) that allow the study of single nucleotide incorporation events.…”

Site-specific Footprinting Reveals Differences in the Translocation Status of HIV-1 Reverse Transcriptase

“…Mutant enzymes were generated through site-directed mutagenesis using the Stratagene Quick-change kit according to the manufacturer's protocol. Substrates used in this study were derived from the HIV-1 polypurine tract (PPT) as described in our previous studies (24,25): 5Ј-TTAAAAGAAAAGGGGGGA (PPT-1/18D); 5Ј-TTA-AAAGAAAAGGGGGG (PPT-1/17D); and 5Ј-ACTGGAAGGGCTGAT-TCA (PPT-2/18D). The model template used in this study provides complementarity to these primers: 5Ј-CGTTGTCAGTGAATCAGCC-CTTCCAGTCCCCCCTTTTCTTTTAAAAAGTGGCAAGA.…”

Kinetics of Inhibition of HIV Type 1 Reverse Transcriptase-Bearing NRTI-associated Mutations by Apricitabine Triphosphate

“…To study the rescue of chain-terminated DNA synthesis and the efficiency of chain-termination, both a DNA primer (PPT-17) and DNA template (PPT-57) were derived from the polypurine tract (PPT) of the HIV-1 genome (Gotte et al, 2001). To study steady-state kinetics, an HIV-1 RNA (pHIV-PBS) containing the region from the 5′ UTR to the primer binding site (PBS) was in vitro transcribed and purified as previously described (Liang et al, 1998).…”