Fig. 5.

The effect of the I423A change on CD4 dependence and Env conformation. a Recombinant viruses carrying the wild-type (WT) or I423A HIV-1_JR-FLEnv were incubated with CD4-negative, CCR5-expressing Cf2Th cells in the presence of the CD4-mimetic compound DMJ-II-121 or sCD4 at the indicated concentrations. The percentage of maximal infection for each virus variant is reported here. The maximal infection levels after DMJ-II-121 addition were 297,656 and 70,626 relative luciferase units for viruses with the WT and I423A Envs, respectively. Error bars represent s.e.m. b Infection of cells with different levels of CD4. Left, parallel titration of wild-type (WT) or I423A HIV-1_JR-FL on affinofile cells that were induced to express low or high levels of CD4. Right, the effect of different levels of CD4 expression, induced by doxycycline treatment of affinofile cells, on the infectivity of WT or I423A HIV-1_JR-FL. Maximal infection levels were 675,978 and 613,708 relative luciferase units for viruses with the WT and I423A Envs, respectively. c Single-molecule fluorescence resonance energy transfer (smFRET) probes were placed in the gp120 V1 and V4 loops of WT or I423A HIV-1_JR-FL Envs. The FRET trajectories were compiled into population FRET histograms and fit to the Gaussian distributions associated with each conformational state, according to a hidden Markov model¹⁸. The percentage of the population that occupies each state as well as the number of molecules analyzed is shown and represents the average of two independent experiments