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Measurement of In Vitro Integration Activity of HIV-1 Preintegration Complexes
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DNA ultra-sensitive quantification, a technology for studying HIV unintegrated linear DNA.

Hélène Marie Roux1, Suzanne Figueiredo1, Lucas Sareoua1

  • 1Université Paris Cité, Institut Cochin, INSERM U1016, CNRS, UMR8104, 75014 Paris, France.

Cell Reports Methods
|May 9, 2023
PubMed
Summary

Unintegrated linear HIV DNA (ULDs) can cause pre-integrative latency. A new ultra-sensitive method, DUSQ, accurately quantifies ULDs in patient samples, enabling tracking of HIV latency.

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Area of Science:

  • Virology
  • Molecular Biology
  • Immunology

Background:

  • Unintegrated HIV DNA constitutes a significant portion of viral DNA in patients.
  • Linear forms (unintegrated linear DNAs [ULDs]) are key substrates for viral integration and replication.
  • Detecting ULDs is challenging due to limitations in current techniques' sensitivity and specificity.

Purpose of the Study:

  • To develop a highly sensitive and specific method for quantifying unintegrated linear HIV DNA (ULDs).
  • To investigate the stability and half-life of ULDs in different cellular states.
  • To demonstrate the utility of the developed method for tracking pre-integrative HIV latency in patient samples.

Main Methods:

  • Development of DNA ultra-sensitive quantification (DUSQ) technology.
  • Combination of linker-mediated PCR and next-generation sequencing (NGS) with molecular barcodes.
  • Analysis of ULDs in cells with varying activity levels and in patient-derived samples.

Main Results:

  • The DUSQ method offers ultra-sensitive, specific, and high-throughput quantification of ULDs.
  • ULDs exhibit a half-life of up to 11 days in resting CD4+ T cells.
  • Successful quantification of ULDs in HIV-1 infected patient samples, validating DUSQ's in vivo application.

Conclusions:

  • DUSQ is a powerful tool for quantifying rare DNA molecules like ULDs.
  • The findings provide a novel approach to track pre-integrative HIV latency.
  • DUSQ technology holds potential for broader applications in detecting other rare DNA targets.