Surface mapping of Env protein from HIV by chemical modifications

The heterotrimeric HIV Env glycoproteins gp120 and gp41 form a specialized type I viral membrane fusion complex that mediates viral entry. Aimed at novel vaccine design, the Tat and the wt or the V2-deleted trimeric Env (V2-Env) were chosen to develop a combined vaccine inducing immune responses against early and late HIV products to block different steps of HIV replication in infected cells. A surprising observation was that env and Tat form a tight 1:1 complex. Several information are available on the atomic structure of Env: X-ray structure of SIV gp120 core and of HIV gp120 core bound to CD4 and an overall picture of the trimer obtained by cryo-electron tomography. To better understand the true atomic structure of trimeric env, and characterize the binding site for Tat, we investigated the cysteine and lysine residues reactivity of trimeric env compared to monomeric gp120, and the effect of Tat binding on the accessibility of the reactive residues. gp120 SF160V2, trimeric gp140 SF160V2 and Tat were from ISS. Disulfide reactivity was measured by reaction with TCEP in the presence of 5 mM SBDF. Lysine reactivity was measured at different times in the presence of TNBS. Labelled proteins were freed from reagents excess, denaturated, carboxymethylated and deglycosylated. Finally the proteins were digested with chymotripsin, labelled peptides separated by HPLC, peaks collected and identified by MS. Both in gp120 and gp140 six disulfide bridges were labelled, both in the presence and in the absence of Tat. Instead 19 of 31 lysine were labelled in gp120, and 22 of 39 on gp140. One lysine, labelled in gp120, is absent in gp140. The Tat presence suppress a single lysine labelling in both gp120 and gp140, and increases the labelling extent of a lysine in gp140 only. Labelled residues were identified on the 3-D structure of gp120 and on the “in silico” model of the complex gp120-Tat.