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Updated: May 28, 2026

Amplification, Next-generation Sequencing, and Genomic DNA Mapping of Retroviral Integration Sites
09:31

Amplification, Next-generation Sequencing, and Genomic DNA Mapping of Retroviral Integration Sites

Published on: March 22, 2016

The HIV-1 integrase monomer induces a specific interaction with LTR DNA for concerted integration

Krishan K Pandey1, Sibes Bera, Duane P Grandgenett

  • 1Institute for Molecular Virology, Saint Louis University Health Sciences Center, Saint Louis, Missouri 63104, United States.

Biochemistry
|October 14, 2011
PubMed

Insights

The human immunodeficiency virus type 1 (HIV) integrase (IN) monomer efficiently catalyzes concerted integration of U5 long terminal repeat (LTR) DNA substrates. Optimal integration occurs at a dimer:DNA ratio of 1, with activity decreasing as substrate length increases.

Area of Science:

  • Molecular Biology
  • Virology
  • Structural Biology

Background:

  • The assembly mechanism of the human immunodeficiency virus type 1 (HIV) synaptic complex (SC) for concerted integration remains unclear.
  • Previous studies indicate HIV SC and prototype foamy virus (PFV) intasomes utilize a tetramer of integrase (IN) for integration catalysis.

Purpose of the Study:

  • To investigate the role of HIV integrase (IN) in the concerted integration process.
  • To determine the optimal conditions and substrate requirements for HIV IN-mediated integration.
  • To explore the binding interactions between HIV IN and U5 long terminal repeat (LTR) oligonucleotide (ODN) substrates.

Main Methods:

  • Purification of HIV IN under specific buffer conditions (EDTA, MgSO4).
  • Assays for concerted integration of U5 LTR ODN substrates into circular target DNA.
  • Determination of optimal molar ratios of IN to U5 DNA.
  • Analysis of integration activity with varying ODN lengths and fidelity measurements.
  • DNaseI protection assays to determine IN binding footprints on modified DNA.

Main Results:

  • HIV IN purified under specific conditions was predominantly monomeric.
  • Efficient concerted integration was observed with micromolar concentrations of 3'-OH recessed and blunt-ended U5 LTR ODNs (19-42 bp).
  • An optimal IN dimer:DNA end molar ratio of 1 was identified for concerted integration.
  • Integration activity decreased with increasing ODN length (18/20 bp to 40/42 bp).
  • Average fidelity for 5 bp host site duplication was 56% with recessed and blunt-ended substrates.
  • DNaseI protection assays indicated viral sequences beyond 21 bp from the terminus were not essential for IN binding.

Conclusions:

  • HIV IN binds differentially to shorter (18/20 bp) versus longer (40/42 bp) ODN substrates for concerted integration.
  • The HIV IN monomer is a potential candidate for crystallization studies of IN-DNA complexes, potentially including strand transfer inhibitors.

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