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Correction: Mofed et al. Construction of a Macrophage-Tropic Subtype C HIV-1-mGreenLantern Reporter Virus for Studies on HIV-1 Replication and the Impact of Methamphetamine. <i>Viruses</i> 2024, <i>16</i>, 1859.

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Nucleoside Triphosphates - From Synthesis to Biochemical Characterization
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Published on: April 3, 2014

Utilization of a deoxynucleoside diphosphate substrate by HIV reverse transcriptase.

Scott J Garforth1, Michael A Parniak, Vinayaka R Prasad

  • 1Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York, United States of America.

Plos One
|May 1, 2008
PubMed
Summary

HIV-1 reverse transcriptase (RT) can incorporate deoxynucleoside diphosphates (dNDPs) into DNA and remove AZT via inorganic phosphate (Pi). These findings reveal new RT functions and explain HIV-1 resistance complexity.

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Determining 3'-Termini and Sequences of Nascent Single-Stranded Viral DNA Molecules during HIV-1 Reverse Transcription in Infected Cells
13:07

Determining 3'-Termini and Sequences of Nascent Single-Stranded Viral DNA Molecules during HIV-1 Reverse Transcription in Infected Cells

Published on: January 30, 2019

Area of Science:

  • Molecular Biology
  • Virology
  • Biochemistry

Background:

  • Deoxynucleoside triphosphates (dNTPs) are standard DNA synthesis substrates for enzymes like HIV-1 reverse transcriptase (RT).
  • Cells contain significant levels of deoxynucleoside diphosphates (dNDPs), and their potential use by HIV-1 RT could impact antiviral drug efficacy.
  • Previous research suggested HIV-1 RT might incorporate dNDPs due to non-essential interactions in its active site.

Purpose of the Study:

  • To investigate the ability of wild-type and mutant HIV-1 RT to utilize dNDP substrates for DNA synthesis.
  • To determine if HIV-1 RT can perform inorganic phosphate-dependent phosphorolysis of chain-terminating nucleoside analogs.
  • To assess the contribution of these newly identified functions to HIV-1 drug resistance.

Main Methods:

  • Recombinant wild-type (wt) and K65 mutant HIV-1 RTs were used in primer extension assays with dNDP substrates.
  • The phosphorolytic activity of wt and thymidine analog mutation (TAM)-containing RTs on 3'-terminal AZT was evaluated at physiological inorganic phosphate (Pi) levels.

Main Results:

  • Wild-type HIV-1 RT successfully incorporated dNDP substrates, while K65 mutant RTs failed to do so.
  • Wild-type HIV-1 RT exhibited inorganic phosphate-dependent phosphorolysis of 3'-terminal AZT.
  • Both wt and TAM-containing RTs efficiently catalyzed Pi-mediated phosphorolysis of 3'-terminal AZT, comparable to ATP-dependent excision.

Conclusions:

  • HIV-1 RT possesses novel catalytic functions: utilizing dNDPs for DNA synthesis and employing inorganic phosphate for phosphorolytic removal of primer termini.
  • The ability to use dNDPs is rare among DNA polymerases, and inorganic phosphate-dependent phosphorolysis has not been previously documented for any DNA polymerase.
  • Pi-mediated phosphorolysis contributes to AZT resistance, indicating a more complex mechanism of HIV-1 drug resistance than previously understood.