Abstract
During HIV replication, reverse transcriptase (RT), assisted by the nucleocapsid protein (NC), converts the genomic RNA into proviral DNA. This process appears to be the major source of genetic variability, as RT can misincorporate nucleotides during minus and plus strand DNA synthesis. To investigate nucleotide addition or substitution by RT, we set up in vitro models containing HIV-1 RNA, cDNA, NC, and various RTs. We used the wild type RT and azidothymidine- and didanosine-resistant RTs, because they represent the major forms of resistant RTs selected in patients undergoing therapies. Results show that all RTs can add nucleotides in a non-template fashion at the cDNA 3'-end, a reaction stimulated by NC. Nucleotide substitutions were examined using in vitro systems where 3'-mutated cDNAs were extended by RT on an HIV-1 RNA template. With NC, RT extension of the mutated cDNAs was efficient, and surprisingly, mutations were frequently corrected. These results suggest for the first time that RT has excision-repair activity that is triggered by NC. Chaperoning of RT by NC might be explained by the fact that NC stabilizes an RT-DNA binary complex. In conclusion, RT-NC interactions appear to play critical roles in HIV-1 variability.
Highlights
From the ‡LaboRetro, Unitede Virologie Humaine, INSERM U412, Ecole Normale Superieure de Lyon, 46 Allee d’Italie, Institut Federatif de Recherche 128, 69364 Lyon Cedex 07, France, §HIV-Drug Resistance Program, NCI, National Institutes of Health, Frederick, Maryland 21702, ¶Centre de Recherche de Biochimie Macromoleculaire-CNRS-Formation de Recherche en Evolution2593, 1919 Route de Mende, 36293 Montpellier Cedex 05, France, and ʈAIDS Vaccine Program, Science Applications International Corporation-Frederick, Inc., NCI, National Institutes of Health, Frederick, Maryland 21702
To study non-template nucleotide additions at the cDNA 3Ј-end by HIV-1 reverse transcriptase (RT), we derived an in vitro system formed of an RNA template, a primer DNA corresponding to the newly synthesized cDNA, nucleocapsid protein (NC), HIV-1 RT, and dNTPs (Fig. 1)
We have revealed new roles for NC in the synthesis of HIV-1 proviral DNA
Summary
RNA and DNA—RNAs were either HIV-1 RNA-generated in vitro by T7 phage RNA polymerase [10] or synthetic oligoribonucleotides. Template-independent Addition of nt, Nucleotide Excision, and Primer Extension Assays—Once nucleoprotein complexes were formed, the reaction volume was increased to 25 l by adding HIV-1 RT at 0.25 M, one dNTP (Invitrogen) at 0.25 mM, or an equimolar mixture or AZTTP (PerkinElmer Life Sciences) at 0.1 mM, 30 mM NaCl, and 3 mM MgCl2. The reaction mixture contained 25 nM template-primer and 37 nM HIV-1 RT in 12.5 mM Tris-HCl, pH 7.8, 40 mM NaCl, 9 mM MgCl2, 5 mM dithiothreitol, and 0.01% (v/v) Triton X-100. To assess the concentration-dependent effect of NC on single nucleotide extension, the reaction was modified as follows: a mixture containing 25 nM template-primer and 25 nM HIV-1 RT was preincubated with varying concentrations of NC (0.1–1.2 M NC-(1–71) or 0.2–3.2 M NC-(1–55)rec in 12.5 mM Tris-HCl, pH 7.8, 40 mM NaCl, 9 mM MgCl2, 5 mM dithiothreitol, and 0.01% (v/v) Triton X-100. Plasmids were sequenced and sequences analyzed using the Edit View software
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