Fig. 4

Changes in RC before and after LVC. Infectivity and protein structural stability of EL9 mutants. a RC estimated as RLU (relative units of luciferase) production (y axis) of the co-cultured viruses obtained before and after LVC (Oct-14 and Apr-15, blue bars) tested in TZM-bl cells. b Infectivity of the pseudoviruses generated with Gp160 Env expression plasmids derived from A (April-06), B1 (c4- May-12 and c7 March- 04) and B2 variants. Four B2 clones from samples previous to LVC (c4, c3, c6 and c8) and 6 B2 clones from Oct-14 (c16, c11, c15, c8, c20, c14) are included. Time points used for pseudoviruses generation was included in brackets. A pNL4-3 pseudovirus is also included as a reference. All these experiments were performed in duplicate c We compared infectivity expressed as RLU (relative units of luciferase) for WT, ESC (L592R mutation) and ESC + COM (L592R and K588R mutations) Env-EL9 sequences. Pseudoviruses were constructed from B2 c-11 with L592R mutation by site-directed mutagenesis. The infectivity values were normalized according to the RC of the pNL4-3 control virus (value = 1). Bars represent standard deviation from two experiments with three replicates per experiment d DOPE of the protein structure of the WT (black), WT with the escape mutation L592R (ESC, white) and WT with both escape and compensatory mutations (ESC + COM, dark gray). Note that in terms of protein stability, the order is the following: WT (more stable) > L592R with K588R > L592R (less stable). e Illustration of the best-fitting 3D structural models (with zoom in the mutation positions) for the WT (the orange and blue positions correspond to those that will suffer the escape and compensatory mutations, respectively), ESC (the red position corresponds to that with the escape mutation) and ESC + COM (the red and pink positions correspond to those with the escape mutation and compensatory mutations, respectively) states