Abstract
We have recently demonstrated that HIV-1 RT mutants characterized by low dNTP binding affinity display significantly reduced dNTP incorporation kinetics in comparison to wild-type RT. This defect is particularly emphasized at low dNTP concentrations where WT RT remains capable of efficient synthesis. Kinetic interference in DNA synthesis can induce RT pausing and slow down the synthesis rate. RT stalling and slow synthesis rate can enhance RNA template cleavage by RT-RNase H, facilitating transfer of the primer to a homologous template. We therefore hypothesized that reduced dNTP binding RT mutants can promote template switching during minus strand synthesis more efficiently than WT HIV-1 RT at low dNTP concentrations. To test this hypothesis, we employed two dNTP binding HIV-1 RT mutants, Q151N and V148I. Indeed, as the dNTP concentration was decreased, the template switching frequency progressively increased for both WT and mutant RTs. However, as predicted, the RT mutants promoted more transfers compared with WT RT. The WT and mutant RTs were similar in their intrinsic RNase H activity, supporting that the elevated template switching efficiency of the mutants was not the result of the mutations enhancing RNase H activity. Rather, kinetic interference leading to stalled DNA synthesis likely enhanced transfers. These results suggest that the RT-dNTP substrate interaction mechanistically influences strand transfer and recombination of HIV-1 RT.
Highlights
Mechanisms involved in the interaction of DNA polymerases with dNTP substrates have been extensively studied
We recently demonstrated that Human immunodeficiency virus type 1 (HIV-1) variants harboring reduced dNTP binding mutant RTs fail to infect macrophage (ϳ0.05 M dNTP) even though these mutant viruses are capable of infecting T cells (ϳ5 M dNTP) [9]
In contrast to previous studies that have examined template features that promote pausing, in this study we address how restricted DNA polymerization kinetics, resulting from disruption of RT-dNTP interactions, can lead to stalling of synthesis and increased strand transfer
Summary
Plasmids, and Chemicals—Escherichia coli XL-1 Blue (Stratagene) was used during the construction of NL4-3 WT and mutant RT expression plasmids; E. coli Rosetta II (DE3) pLysS (Novagen, WI) was used for the overexpression of all RT proteins. RNAs were generated using an in vitro run-off transcription reaction using T7 RNA polymerase with BamHI-linearized plasmids as templates These templates were gel purified on 6% polyacrylamide-urea denaturing gel. RNAs labeled in this manner were gel-purified on either 6% (for donor and acceptor RNAs) or 12% (for 38-mer RNA, previously described, [28]) polyacrylamide-urea denaturing gel. Final reactions contained 6 nM primer, 4 nM donor template (strand transfer assays contained in addition, 16 nM acceptor), RT protein (p66 homodimer, concentrations described in figure legends), 16 M oligo(dT), 50 nM TrisHCl (pH 8.0), 6 mM MgCl2, 50 mM KCl, 10 mM NaCl, 1 mM dithiothreitol, 1 mM EDTA, and 50, 5, or 2 M dNTP in a final reaction volume of 12.5 l. Products were analyzed on 14% polyacrylamide-urea denaturing gel and visualized and quantitated as above
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