Mg2+ dependency of HIV-1 reverse transcription, inhibition by nucleoside analogues and resistance.
Metal ions are essential for DNA polymerase and RNase H activities of HIV-1 reverse transcriptase (RT). RT studies are routinely performed at 6-8 mM Mg2+, despite the fact that the in vivo concentration might be as low as 0.2 mM. We studied the influence of MgCl2 and ATP, which likely binds a significant fraction of the magnesium pool in vivo, on the DNA polymerase and RNase H activities of HIV-1 RT, its inhibition by nucleoside RT inhibitors (NRTIs) and primer unblocking by AZT-resistant RT. At low Mg2+ concentration, reverse transcription of a natural template strongly increased despite a dramatically reduced intrinsic polymerase activity under such conditions. Low Mg2+ concentrations affected the RNA stability and indirectly decreased its degradation by the RNase H activity. The reduced RNA degradation prevented premature dissociation of the template and primer strands that otherwise generated dead-end DNA products. In addition, low Mg2+ dramatically decreased the incorporation of NRTIs into DNA and increased nucleotide excision by AZT-resistant RT. The latter effect is also most likely owing to the diminished cleavage of the RNA template. Thus, differences in the free Mg2+ concentration between different cell types or during the cell cycle might strongly affect HIV-1 replication and its inhibition.
Goldschmidt
Valérie
V
Unité Propre de Recherche 9002 du CNRS conventionnée à l'Université Louis Pasteur, IBMC, 15 rue René Descartes, 67084 Strasbourg cedex, France.
Didierjean
Joël
J
Ehresmann
Bernard
B
Ehresmann
Chantal
C
Isel
Catherine
C
Marquet
Roland
R
D000255:Adenosine Triphosphate / Q000494:pharmacology; D019380:Anti-HIV Agents / Q000494:pharmacology*; D004247:DNA / Q000096:biosynthesis; D017931:DNA Primers; D004277:DNA, Single-Stranded / Q000096:biosynthesis; D024882:Drug Resistance, Viral; D054303:HIV Reverse Transcriptase / Q000378:metabolism*; D008274:Magnesium / Q000494:pharmacology*; D009705:Nucleosides / Q000494:pharmacology; D018894:Reverse Transcriptase Inhibitors / Q000494:pharmacology*; D048348:Reverse Transcription / Q000187:drug effects; D016914:Ribonuclease H / Q000378:metabolism; D015215:Zidovudine / Q000494:pharmacology
D019380:Anti-HIV Agents; D017931:DNA Primers; D004277:DNA, Single-Stranded; D009705:Nucleosides; D018894:Reverse Transcriptase Inhibitors; D015215:Zidovudine; D000255:Adenosine Triphosphate; D004247:DNA; D054303:HIV Reverse Transcriptase; D016914:Ribonuclease H; D008274:Magnesium