HIV-1 Tropism Determination Using a Phenotypic Env Recombinant Viral Assay Highlights Overestimation of CXCR4-Usage by Genotypic Prediction Algorithms for CRRF01_AE and CRF02_AG
Figure 1
Viral RNA was extracted from patient plasma RT-PCR amplified. Env amplicons spanning the Env ectodomain were further amplified through an inner PCR. Five independent PCRs were pooled to minimize PCR-selection. Recombinant viruses were produced by co-transfecting HEK293T cells with Afe I-linearized, luciferase-tagged, Env-deleted, viral backbone and patient-derived PCR amplicon. Normalized amounts of recombinant viruses were used to infect U87.CD4.CCR5 or U87.CD4.CXCR4 indicator cells. Infection was monitored by quantifying luminescence in the cell lysates. Depending on the outcome of the infection, viruses were classified as either CCR5 tropic, CXCR4 tropic or dual/mixed. The same patient-derived PCR amplicon used for viral production was sequenced and tropism inferred by Geno2Pheno[coreceptor] and webPSSM algorithms. The phenotypic and genotypic results were compared. Abbreviations: Env EC: Env ectodomain; gp41-TM-CT: gp41 Transmembrane+cytoplasmic tail.
