HIV-1 coreceptor usage determination in clinical isolates using clonal and population-based genotypic and phenotypic assays.
Orally bioavailable CXCR4 and CCR5 coreceptor antagonists are being developed for the treatment of HIV-1 infection. A new tropism-testing platform, which offers various options depending on the needs, was established. Each option has specific characteristics in terms of sensitivity, information, throughput and cost. The platform consists of four assays, all based on a one-step RT-PCR of the main part of the HIV envelope glycoprotein gp120 (called 'NH(2)-V4'). Population-based sequencing of gp120's V3 loop is generally cheap and easy to run, and was chosen as the first test in the platform's cascade. Given its drawbacks such as limited sensitivity, additional tests were developed. A sensitive assay using NH(2)-V4 gp120 clonal sequencing and tropism prediction enabled us to demonstrate the quasispecies diversity present in 13 patient samples. For phenotyping, an eGFP-containing HIV backbone deleted for NH(2)-V4 was constructed and used for clonal and population tropism determination. As expected, clonal NH(2)-V4 gp120 phenotyping demonstrated significant correlation between prediction algorithms and phenotype-based classification. The absence of the N-linked glycosylation motif in V3 was associated with CXCR4 usage. Finally, population NH(2)-V4 gp120 phenotypic tropism determination appeared to be a promising tool for the detection of minority species present in the amplified envelope fragments.
Van Baelen
Kurt
K
Virco BVBA, Generaal De Wittelaan L11 B4, 2800 Mechelen, Belgium. kvbaele1@vrcbe.jnj.com
Vandenbroucke
Ina
I
Rondelez
Evelien
E
Van Eygen
Veerle
V
Vermeiren
Hans
H
Stuyver
Lieven J
LJ
D000465:Algorithms; D000595:Amino Acid Sequence; D065100:CCR5 Receptor Antagonists; D005838:Genotype; D015699:HIV Envelope Protein gp120 / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D015658:HIV Infections / Q000821:virology; D015497:HIV-1 / Q000145:classification / Q000302:isolation & purification / Q000502:physiology*; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010641:Phenotype; D010802:Phylogeny; D019713:Receptors, CCR5 / Q000378:metabolism*; D019718:Receptors, CXCR4 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D014773:Virology / Q000379:methods
D065100:CCR5 Receptor Antagonists; D015699:HIV Envelope Protein gp120; C059762:HIV envelope protein gp120 (305-321); D010446:Peptide Fragments; D019713:Receptors, CCR5; D019718:Receptors, CXCR4