Improved detection of CXCR4-using HIV by V3 genotyping: application of population-based and "deep" sequencing to plasma RNA and proviral DNA

J Acquir Immune Defic Syndr. 2010 Aug;54(5):506-10. doi: 10.1097/QAI.0b013e3181d0558f.


Background: Tropism testing should rule out CXCR4-using HIV before treatment with CCR5 antagonists. Currently, the recombinant phenotypic Trofile assay (Monogram) is most widely utilized; however, genotypic tests may represent alternative methods.

Methods: Independent triplicate amplifications of the HIV gp120 V3 region were made from either plasma HIV RNA or proviral DNA. These underwent standard, population-based sequencing with an ABI3730 (RNA n = 63; DNA n = 40), or "deep" sequencing with a Roche/454 Genome Sequencer-FLX (RNA n = 12; DNA n = 12). Position-specific scoring matrices (PSSMX4/R5) (-6.96 cutoff) and geno2pheno[coreceptor] (5% false-positive rate) inferred tropism from V3 sequence. These methods were then independently validated with a separate, blinded dataset (n = 278) of screening samples from the maraviroc MOTIVATE trials.

Results: Standard sequencing of HIV RNA with PSSM yielded 69% sensitivity and 91% specificity, relative to Trofile. The validation dataset gave 75% sensitivity and 83% specificity. Proviral DNA plus PSSM gave 77% sensitivity and 71% specificity. "Deep" sequencing of HIV RNA detected >2% inferred-CXCR4-using virus in 8/8 samples called non-R5 by Trofile, and <2% in 4/4 samples called R5.

Conclusions: Triplicate analyses of V3 standard sequence data detect greater proportions of CXCR4-using samples than previously achieved. Sequencing proviral DNA and "deep" V3 sequencing may also be useful tools for assessing tropism.

MeSH terms

  • DNA, Viral / genetics
  • HIV Envelope Protein gp120 / genetics*
  • HIV Infections / virology*
  • HIV-1 / classification*
  • HIV-1 / genetics
  • HIV-1 / isolation & purification
  • HIV-1 / physiology*
  • Humans
  • Plasma / virology*
  • Proviruses / genetics
  • RNA, Viral / genetics
  • Receptors, CXCR4 / analysis
  • Receptors, HIV / analysis*
  • Sequence Analysis, DNA
  • Viral Tropism*


  • DNA, Viral
  • HIV Envelope Protein gp120
  • RNA, Viral
  • Receptors, CXCR4
  • Receptors, HIV