A novel denaturing heteroduplex tracking assay for genotypic prediction of HIV-1 tropism

Binshan Shi, Barbara Weiser, Linda M. Styer, Kimdar Kemal, Cheryl Brunner, Kathryn Anastos, Harold Burger

Research output: Contribution to journalArticle

5 Scopus citations

Abstract

Human immunodeficiency virus type 1 (HIV-1) is characterized by sequence variability. The third variable region (V3) of the HIV-1 envelope glycoprotein gp120 plays a key role in determination of viral coreceptor usage (tropism) and pathogenesis. This report describes a novel denaturing heteroduplex tracking assay (HTA) to analyze the genetic variation of HIV-1 V3 DNA. It improved upon previous non-denaturing HTA approaches to distinguish HIV-1 CCR5 and CXCR4 tropic viruses in mixed populations. The modifications included the use of a single-stranded fluorescent probe based on the consensus V3 sequence of HIV-1 CCR5 tropic viruses, Locked Nucleic Acid (LNA) " clamps" at both ends of heteroduplex DNA, and denaturing gel electrophoresis using Mutation Detection Enhancement (MDE®) as matrix. The analysis demonstrated that the LNA " clamps" increased its melting temperature (Tm) and the thermal stability of heteroduplex DNA. The partially denaturing gel used a defined concentration of formamide, and significantly induced mobility shifts of heteroduplex DNA that was dependent on the number and patterns of DNA mismatches and insertions/deletions. This new technique successfully detected tropisms of 53 HIV-1 V3 clones of known tropism, and was able to separate and detect multiple V3 DNA variants encoding tropisms for CCR5 or CXCR4 in a mixture. The assay had the sensitivity to detect 0.5% minority species. This method may be useful as a research tool for analysis of viral quasispecies and for genotypic prediction of HIV-1 tropism in clinical specimens.

Original languageEnglish (US)
Pages (from-to)108-117
Number of pages10
JournalJournal of Virological Methods
Volume185
Issue number1
DOIs
StatePublished - Oct 2012
Externally publishedYes

Keywords

  • Denaturing heteroduplex tracking assay
  • HIV-1 gp120 V3
  • Locked Nucleic Acid clamps
  • Viral tropism

ASJC Scopus subject areas

  • Virology

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