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Updated: Sep 25, 2025

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

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Relative domain orientation of the L289K HIV-1 reverse transcriptase monomer

Zhaoyong Xi1, Tatiana V Ilina1, Michel Guerrero1

  • 1Department of Structural Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.

Protein Science : a Publication of the Protein Society
|April 28, 2022
PubMed

Insights

The L289K mutation in the p51 subunit of HIV-1 reverse transcriptase (RT) disrupts p66/p51 heterodimer formation, impacting enzyme function. This finding clarifies the mechanism of RT assembly and potential therapeutic targets.

Area of Science:

  • Biochemistry
  • Structural Biology
  • Virology

Background:

  • HIV-1 reverse transcriptase (RT) is a key enzyme for viral replication, existing as a p66/p51 heterodimer.
  • Single amino acid substitutions, like L289K, can significantly reduce RT dimer affinity and enzymatic activity.

Purpose of the Study:

  • To investigate the structural and functional impact of the L289K mutation on HIV-1 RT heterodimer formation.
  • To elucidate the role of specific domains within the RT monomer and their interactions in heterodimer assembly.

Main Methods:

  • Small-angle X-ray scattering (SAXS) and proton paramagnetic relaxation enhancement (PRE) to study monomer structure.
  • Site-specific 19F NMR to probe domain interactions.
  • Size exclusion chromatography (SEC) to analyze heterodimer formation.

Main Results:

  • The L289K mutation in the p66 monomer (p66L289K) revealed that the thumb and RNH domains are not rigidly interacting but spatially close.
  • Structural modeling predicted that p66L289K/p51 could form a heterodimer, but p66/p51L289K could not.
  • SEC experiments confirmed that L289K substitution in the p51 subunit, not p66, significantly reduces p66/p51 heterodimer formation.

Conclusions:

  • The L289K mutation in the p51 subunit is critical for disrupting p66/p51 heterodimer formation.
  • The inter-subunit interaction between RNH and thumb domains is crucial for p66/p51 assembly.
  • Understanding these mechanisms can inform the development of novel HIV-1 therapies targeting RT assembly.
Keywords:
HIV-1NMRSAXSparamagnetic relaxation enhancementreverse transcriptasestructure

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