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Related Experiment Videos

Dimerization kinetics of HIV-1 and HIV-2 reverse transcriptase: a two step process

G Divita1, K Rittinger, C Geourjon

  • 1Max-Planck-Institut für Medizinische Forschung, Abteilung Biophysik, Heidelberg, Germany.

Journal of Molecular Biology
|February 3, 1995
PubMed
Summary

The dimerization of human immunodeficiency virus (HIV) reverse transcriptase (RT) involves two steps: subunit association and slow isomerization. This process impacts enzyme activity and structure, with HIV-2 RT maturing faster than HIV-1 RT.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Virology

Background:

  • Human immunodeficiency virus (HIV) reverse transcriptase (RT) is crucial for viral replication.
  • Understanding the dimerization process of HIV RT subunits (p66 and p51) is key to enzyme function.
  • Previous studies have explored HIV RT structure and activity, but the precise dimerization mechanism requires further elucidation.

Purpose of the Study:

  • To investigate the dimerization mechanisms of human immunodeficiency virus (HIV) types 1 and 2 reverse transcriptase (RT).
  • To characterize the kinetics and structural implications of the two-step heterodimer formation.
  • To compare the maturation rates and potential structural elements involved in HIV-1 and HIV-2 RT dimerization.

Main Methods:

  • Utilized complementary approaches including fluorescence spectroscopy, size exclusion-HPLC, and polymerase activity assays.

Related Experiment Videos

  • Analyzed concentration-dependent association kinetics and subunit interactions.
  • Employed iodide quenching and 1-anilino-8-naphthalenesulphonate binding to probe hydrophobic cluster accessibility.
  • Investigated primer/template binding using fluorescently labeled probes.

    • Main Results:

    • HIV-1 and HIV-2 RT heterodimer formation occurs in a two-step process: rapid association forming an intermediate, followed by slow isomerization to a mature form.
    • The initial association is a second-order reaction (rate constants 2-4 x 10(4) M-1 s-1), causing fluorescence quenching and reduced tryptophan accessibility.
    • The intermediate form lacks polymerase activity but binds primer/template with high affinity.
    • The final isomerization is a slow first-order reaction, 12-fold faster for HIV-2 (1.2 h-1) than HIV-1 RT (0.1 h-1), and is the rate-limiting step.
    • Structural analysis suggests the p51 thumb subdomain and p66 palm subdomain interactions are involved in maturation.

    Conclusions:

    • The maturation of HIV RT is a complex, multi-step process involving subunit association and a slow isomerization.
    • The intermediate heterodimer is structurally distinct and functionally different from the mature enzyme.
    • Differences in isomerization rates between HIV-1 and HIV-2 RT may relate to specific subdomain interactions, potentially impacting antiviral drug development.