Characterization of A Stable HIV-1 B/C Recombinant, Soluble, and Trimeric Envelope Glycoprotein (Env) Highly Resistant to CD4-Induced Conformational Changes

Kumar R, et al., 292(38):15849-15858, J Biol Chem., 2017

The group reports on an HIV-1 B/C recombinant, native-like trimeric Env protein that is highly resistant to CD4-induced conformational changes but displays epitopes recognized by a diverse array of bnAbs. Such features make this B/C recombinant trimeric Env a useful addition to the pool of other recently identified native-like HIV-1 Env trimers suitable for use as antigenic bait for bnAb isolation, structural studies, and use as potential immunogens. The engagement of the HIV-1 gp120 glycoprotein to the host CD4 protein triggers conformational changes in gp120 that allow its binding to co-receptors and is necessary for virus entry to establish infection. In the present study, his6-tagged protein sCD4 (D1-D2) was immobilized on Ni-NTA sensors and its binding to the varying concentrations (500, 250, 125, 62.5, 32.25, 16, and 8 nM) of either BG505 or LT5.J4b12C SOSIP.664 was measured using Octet Red96 instrument. To achieve double referencing, blank Ni-NTA sensors were also dipped into the same SOSIP samples to subtract background (nonspecific) binding of SOSIP Envs to the Ni-NTA biosensor. Kinetic analysis was conducted with samples diluted in phosphate-buffered saline, pH 7.2, containing 0.01% (w/v) bovine serum albumin and 0.002% (v/v) Tween 20. For data analysis a 1:1 binding model was applied to fit the association and dissociation curves. Kinetics measurements on BLI showed that LT5.J4b12C SOSIP.664 binds to sCD4 with slower on-rate and faster off-rate (resulting in app. 3-fold lower affinity) compared with a binding of BG505 SOSIP.664 under the same experimental conditions. These data indicate that increased resistance of LT5.J4b12C SOSIP.664 to CD4-induced conformational changes is associated with less favorable binding kinetics to CD4.

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