A sensitive phenotypic assay for the determination of human immunodeficiency virus type 1 tropism.
OBJECTIVE
To develop a sensitive phenotypic assay based on recombinant viruses (RVs) for characterizing HIV-1 tropism.


METHODS
Viral tropism was assessed in 159 plasma samples. The env gene was amplified and ligated into pNL-lacZ/env-Ren, which carries a luciferase reporter gene. Resulting constructs were transfected into HEK293T cells to generate RVs. To assess co-receptor tropism, U87.CD4.CXCR4/CCR5 cells were infected and luciferase activity was measured.


RESULTS
RVs containing env from different HIV-1 subtypes were replication competent. Minor variants were detectable in 1% of the viral population. Tropism was determined in 65% of samples with a viral load of <1000 copies/mL. The phenotypic assay described here was validated with the Trofile™ and Trofile™ES assays. Considering the Trofile™ assay as a reference, the sensitivity for R5 and R5X4/X4 detection was 90% and 77%, and the specificity was 77% and 90%, respectively. Our assay was 86% concordant with Trofile™ (90% for R5 and 77% for R5X4/X4). When our system was compared with Trofile™ES, the concordance was 89% (86% for R5 and 92% for R5X4/X4), the sensitivity for R5 was 86% and for R5X4/X4 was 92%, and the specificity for R5 was 92% and for R5X4/X4 was 86%. The phenotypic results were compared with those obtained using the following V3 genotypic prediction tools: position-specific scoring matrix; geno2pheno[coreceptor]; C4.5; C4.5 using positions 8 and 12; PART; support vector machines; and the charge rule.


CONCLUSIONS
We describe a system to assess co-receptor tropism based on the generation of chimeric replication-competent viruses with high sensitivity in the detection of minor populations. A good correlation of our results with Trofile™ assays was found.
AIDS Immunopathology Unit, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Majadahonda, Madrid, Spain.
Nuria
N
González
Mayte
M
Pérez-Olmeda
Elena
E
Mateos
Almudena
A
Cascajero
Amparo
A
Alvarez
Sanne
S
Spijkers
Javier
J
García-Pérez
Sonsoles
S
Sánchez-Palomino
Ezequiel
E
Ruiz-Mateos
Manuel
M
Leal
José
J
Alcami
D045744:Cell Line, Tumor; D017930:Genes, Reporter; D057809:HEK293 Cells; D015497:HIV-1 / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D006801:Humans; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D010641:Phenotype; D019713:Receptors, CCR5 / Q000378:metabolism*; D019718:Receptors, CXCR4 / Q000378:metabolism*; D011995:Recombination, Genetic; D012680:Sensitivity and Specificity; D056189:Viral Tropism / Q000502:physiology*; D054299:env Gene Products, Human Immunodeficiency Virus / Q000235:genetics / Q000378:metabolism
D019713:Receptors, CCR5; D019718:Receptors, CXCR4; D054299:env Gene Products, Human Immunodeficiency Virus; D008156:Luciferases