*2.1. Virus*

HIV-1 pNL4-3p10-17 and p81Ap10-17 molecular constructs were obtained from B. Chesebro, (National Institute of Allergy and Infectious Diseases, Hamilton, Montana 59840, USA) and contain the whole HIV-1 genome [45]. The HIV-1 p81A p10-17 clone was generated by replacing the pNL4-3p10-17 a 659 bp long sequence of env. This nucleotide sequence belongs to the R5-tropic HIV-1 Ba-L and includes the V1, V2, V3 variable domains, whereas NL4-3 is a CXCR4-tropic strain, 81A replicates in cells of the MDM lineage. These plasmids were transfected in 293T through the FuGENE 6TM (Roche), a lipidic, not liposomial reagent.

HIV-1 clinical isolates #17 (X4), #6 (R5) and #10 (R5/X4) were obtained from patients enrolled from the Katholieke Universiteit Leuven (Rega institute, Leuven, Belgium) and expanded in peripheral blood mononuclear cells (PBMC).

The laboratory-adapted HIV-1 X4 strain IIIB was expanded in H9 cells and obtained from supernatants at day 8 post infection. The laboratory-adapted HIV-1 adapted R5-tropic strain Ba-L was expanded in MDM [46,47].

All the strains were purified from supernatants of the respective cultures after centrifugation at 20,000 rpm for 2 h, filtered through 0.45 μm filter, DNase I treated, and concentrated with a Centricon Plus-20 membrane with a 100,000 molecular weight cut-o ff (Millipore Corporation, Bedford, Mass.) to remove contaminating cytokines and growth factors which might interfere with signal transduction analysis. Concentrated virus was stored in aliquots at −70 ◦C until use. Stock virus titers were determined with a colorimetric reverse transcriptase activity assay (Roche Molecular Biochemicals, Indianapolis, USA).
