Uniform CCR5 usage in a panel of HIV-1 subtype G plasma-derived functional envelope clones
A. Revilla1, E. Delgado1, Y. Vega1, L. Jiménez1, M. González-Galeano1, M. Pinilla1, A. Ocampo2, M.J. Lezaún3, R. Ojea4, G. Cilla5, R. Rodríguez6, P. Ordóñez7, S. Pérez8, R. Fernández-Rodríguez9, L. Pérez-Álvarez1, M.M. Thomson1
1Instituto de Salud Carlos III (Campus Majadahonda), Biology and Variability of HIV, Majadahonda, Spain, 2Complejo Hospitalario Xeral-Cíes, HIV Unit, Vigo, Spain, 3Txagorritxu Hospital, Microbiology, Alava, Spain, 4Complejo Hospitalario Provincial, HIV Unit, Pontevedra, Spain, 5Complejo Hospitalario Donostia, Microbiology, San Sebastián, Spain, 6Complejo Hospitalario Santa María Madre, Orense, Spain, 7Hospital Arquitecto Marcide, Microbiology, Ferrol, Spain, 8Complejo Hospitalario Universitario, Microbiology, Vigo, Spain, 9Complejo Hospitalario Cristal Piñor, Orense, Spain
Background: Subtype G represents the 4th most prevalent clade in the global HIV-1 pandemic. However, there are insufficient data on coreceptor utilization by subtype G viruses.
Objectives: To determine coreceptor usage by HIV-1 subtype G isolates using a panel of functional envelope clones obtained from plasma of HIV-1-infected individuals.
Methods: Full-length envelopes were amplified from plasma RNA by a single genome amplification RT-PCR assay and cloned into an expression vector. Envelope clones were cotransfected with an Env-deficient HIV-1 construct in 293T cells, generating pseudovirions, which were used to infect TZM-bl reporter cells, which express CCR5 and CXCR4, in addition to luciferase under HIV-1 promoter transcriptional control. Phylogenetic sequence analyses were performed with neighbour-joining trees and bootscanning. In vitro coreceptor use was determined by infection of CCR5- and CXCR4-expresing GHOST cell lines, which express green fluorescent protein (GFP) upon transcriptional activation by Tat. Four days after infection, the differential capacity of pseudovirions to infect each GHOST cell line was examined by fluorescence microscopy. X4 and R5 HIV-1 isolates were used as positive controls.
Results: Cloned envelopes from 16 individuals were highly functional for cell entry, with a signal/background luminescence ratio ≥50 in the TZM-bl assay. Phylogenetic analyses revealed that envelopes were uniformly of subtype G, 12 clustering with the Spanish-Portuguese variant, 1 with the Cuban variant, 1 with a Nigerian cluster and 1 with a Kenian virus. When tested in GHOST cells, envelopes from all 16 individuals, including 7 from AIDS patients, exhibited an R5 phenotype, which was also predicted by 4 different genotypic methods.
Conclusions: CCR5 monotropism was uniformly observed among 16 HIV-1 subtype G functional envelope clones, even at advanced stages of the infection.
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