Construction of a panel of functional HIV-1 envelope clones of subtype F
E. Delgado1, A. Revilla1, P. Nebreda1, M. Ríos2, G. Cilla3, L. María Jesús4, A. Mariño5, A. Rakhmanova6, S. Pérez-Castro7, S. Prieto Menchero8, S. Herráez9, M. González-Galeano1, M. Sánchez1, M. Pinilla1, L. Pérez-Álvarez1, M.M. Thomson1
1Instituto de Salud Carlos III, Biology and Variability of HIV, Majadahonda, Spain, 2Centro Nacional de Referencia de VIH de Chile, Santiago, Chile, 3Hospital Donostia, Guipúzcoa, Spain, 4Hospital Txagorritxu, Álava, Spain, 5Hospital Arquitecto Marcide, El Ferrol, Spain, 6Botkin Infectious Diseases Hospital, St. Petersbourg, Russian Federation, 7Hospital do Mexoeiro, Complejo Hospitalario Universitario de Vigo, Vigo, Spain, 8Hospital de Fuenlabrada, Fuenlabrada, Spain, 9Hospìtal de Basurto, Basurto, Spain
Background: HIV-1 subtype F circulates in Central Africa, Romania and Brazil. BF recombinants, with envelopes mostly of subtype F, are prevalent in Argentina and Uruguay, and most BF circulating recombinant forms have originated in South America. To date, only 2 weakly functional envelope clones of subtype F and one BF recombinant have been reported.
Objective: To obtain a panel of functional HIV-1 envelope clones of subtype F derived from plasma of HIV-1 infected patients.
Methods: Full-length HIV-1 envelopes, amplified by RT-PCR from plasma RNA using a single genome amplification assay, were cloned into an expression vector. Envelope clones were cotransfected with an Env-deficient HIV-1 plasmid in 293T cells, generating pseudovirions, which were used to test envelope functionality through infection of TZM-bl reporter cells, expressing CD4, CCR5, CXCR4, and luciferase under HIV-1 promoter control. Clones with a signal/background luminiscence ratio >50 were considered highly functional. Phylogenetic sequence analyses were performed with neighbour-joining trees using MEGA and with bootscanning using SimPlot. Coreceptor usage was predicted analysing V3 sequences with Geno2pheno, web PSSM, the 11/25 rule and the net charge.
Results: Highly functional envelope clones were derived from eleven HIV-1 infected patients of diverse geographic origins: Africa (DRC, Equatorial Guinea, and Nigeria), Europe (Spain, Portugal, Romania and Russia) and South America (Chile and Paraguay). Phylogenetic analyses revealed that 7 envelopes were F1, 2 F2, 1 CRF12_BF, 1 CRF44_BF and 1 A1/F2 recombinant. Predicted coreceptor usage was CCR5 for the eleven envelopes, including 3 from AIDS patients.
Conclusions: We report the first panel of highly functional HIV-1 envelope clones of subtype F and recombinant forms derived from subtype F, of diverse geographic origins (Africa, South America and Europe). Functional envelope clones may be useful for neutralization studies in vaccine research, as well as for analyses of efficacy of entry inhibitor drugs and coreceptor usage.
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