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Related Experiment Videos

A sensitive, quantitative assay for human immunodeficiency virus type 1 integration.

Una O'Doherty1, William J Swiggard, Deepa Jeyakumar

  • 1Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6060, USA. unao@mail.med.upenn.edu

Journal of Virology
|October 9, 2002
PubMed
Summary
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A new nested PCR assay accurately quantifies rare human immunodeficiency virus type 1 (HIV-1) integration events in resting T cells. This sensitive method aids in understanding latent HIV-1 reservoirs and may help monitor novel eradication therapies.

Area of Science:

  • Virology
  • Molecular Biology
  • Immunology

Background:

  • Latent reservoirs of human immunodeficiency virus type 1 (HIV-1) provirus are established in resting T cells, particularly in patients on antiretroviral therapy.
  • Understanding the mechanisms of HIV-1 integration is crucial for dissecting reservoir formation and persistence.

Purpose of the Study:

  • To develop and validate a sensitive, quantitative assay for measuring HIV-1 integration events in T cells.
  • To provide a tool for studying the establishment and persistence of latent HIV-1 reservoirs.

Main Methods:

  • A fluorescence-monitored, nested PCR assay was developed.
  • The assay involves a preamplification step using primers for genomic Alu elements and HIV-1 gag sequences, followed by kinetic PCR quantifying HIV-1 long terminal repeat sequences.

Related Experiment Videos

  • A T-cell-based integration standard was created to reflect the randomness of HIV-1 integration.

    • Main Results:

    • The assay is 10 to 100 times more sensitive than previous quantitative Alu PCR-based integration assays.
    • The method specifically detects integration events, showing no detection of proviruses with integrase inhibitors or integrase-deficient viruses.
    • The assay successfully quantifies rare integration events in resting T cells.

    Conclusions:

    • This novel nested PCR assay provides a sensitive and specific method for quantifying HIV-1 integration.
    • The assay offers new insights into the accumulation and persistence of latent HIV-1 reservoirs.
    • This method may be clinically useful for monitoring the eradication of latent HIV-1 by novel therapies.