A novel denaturing heteroduplex tracking assay for genotypic prediction of HIV-1 tropism.
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 (T(m)) 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.
Division of Infectious Diseases, Wadsworth Center, New York State Department of Health, Albany, NY, USA. binshan.shi@acphs.edu
Binshan
B
Shi
Barbara
B
Weiser
Linda M
LM
Styer
Kimdar
K
Kemal
Cheryl
C
Brunner
Kathryn
K
Anastos
Harold
H
Burger
D016679:Genome, Viral; D005838:Genotype; D015497:HIV-1 / Q000235:genetics* / Q000502:physiology*; D020180:Heteroduplex Analysis / Q000379:methods*; D006801:Humans; D009691:Nucleic Acid Denaturation; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes; D012680:Sensitivity and Specificity; D044366:Transition Temperature; D056189:Viral Tropism; D014773:Virology / Q000379:methods*
D015345:Oligonucleotide Probes