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Interplay between protease and reverse transcriptase dimerization in a model HIV-1 polyprotein.

Brisa Caroline Alves Chagas1, Xiaohong Zhou1, Michel Guerrero1

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

Protein Science : a Publication of the Protein Society
|June 19, 2024
PubMed
Summary
This summary is machine-generated.

The reverse transcriptase (RT) dimer interface is key for human immunodeficiency virus type I (HIV-1) Gag-Pol polyprotein dimerization. Mutations disrupting the RT interface prevent dimerization, unlike those affecting the protease (PR) interface.

Keywords:
Gag‐Pol polyproteinHIV‐1darunavirdimerizationinhibitorproteasereverse transcriptase

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Area of Science:

  • Virology
  • Structural Biology
  • Biochemistry

Background:

  • The Gag-Pol polyprotein of human immunodeficiency virus type I (HIV-1) is essential for viral replication, encoding key enzymes like protease (PR), reverse transcriptase (RT), and integrase (IN).
  • The mature forms of these enzymes exist as dimers or tetramers, but the mechanisms of their precursor dimerization remain unclear.
  • Understanding precursor dimerization is crucial for developing novel antiviral strategies targeting HIV-1 replication.

Purpose of the Study:

  • To investigate the dimerization mechanisms of protease (PR) and reverse transcriptase (RT) within the precursor polyprotein.
  • To elucidate the roles of the PR and RT dimer interfaces in the dimerization of the PR-RT precursor.
  • To assess the impact of specific mutations on PR-RT dimerization and its functional implications.

Main Methods:

  • Construction and characterization of a model PR-RT precursor with an inactivating mutation in the PR active site (D25A) and SUMO-tag.
  • Preparation of PR-RT mutants with dimer dissociation mutations in the PR (PR(T26A)-RT) or RT (PR-RT(W401A)) regions.
  • Size exclusion chromatography (SEC) to analyze monomer-dimer states and determine dissociation constants.
  • Reverse transcriptase and RT maturation assays to evaluate functional consequences.

Main Results:

  • SEC analysis revealed that the PR-RT(W401A) mutant, with a mutation in the RT region, existed solely as a monomer, indicating the critical role of the RT interface.
  • The PR-RT and PR(T26A)-RT mutants showed both monomer and dimer fractions, suggesting a lesser contribution of the PR interface.
  • Dimerization of PR-RT was significantly enhanced by the protease inhibitor darunavir.
  • The estimated dissociation constant for PR-RT dimer was comparable to mature RT forms, suggesting its relevance in viral assembly.

Conclusions:

  • The reverse transcriptase (RT) dimer interface is the primary determinant of dimerization in the PR-RT precursor.
  • The protease (PR) dimer interface plays a secondary role in the dimerization of the PR-RT precursor.
  • These findings provide insights into the structural organization of HIV-1 Gag-Pol and potential targets for antiviral drug development.