The Highly Conserved Layer-3 Component of the HIV-1 gp120 Inner Domain Is Critical for CD4-Required Conformational Transitions

Abstract

The trimeric envelope glycoprotein (Env) of human immunodeficiency virus type 1 (HIV-1) mediates virus entry into host cells. CD4 engagement with the gp120 exterior envelope glycoprotein subunit represents the first step during HIV-1 entry. CD4-induced conformational changes in the gp120 inner domain involve three potentially flexible topological layers (layers 1, 2, and 3). Structural rearrangements between layer 1 and layer 2 have been shown to facilitate the transition of the envelope glycoprotein trimer from the unliganded to the CD4-bound state and to stabilize gp120-CD4 interaction. However, our understanding of CD4-induced conformational changes in the gp120 inner domain remains incomplete. Here, we report that a highly conserved element of the gp120 inner domain, layer 3, plays a pivot-like role in these allosteric changes. In the unliganded state, layer 3 modulates the association of gp120 with the Env trimer, probably by influencing the relationship of the gp120 inner and outer domains. Importantly, layer 3 governs the efficiency of the initial gp120 interaction with CD4, a function that can also be fulfilled by filling the Phe43 cavity. This work defines the functional importance of layer 3 and completes a picture detailing the role of the gp120 inner domain in CD4-induced conformational transitions in the HIV-1 Env trimer.

Molecular architecture of the uncleaved HIV-1 envelope glycoprotein trimer

“…S4). These observations are consistent with previous studies suggesting that layered movement occurs within the gp120 inner domain upon CD4 binding (17,25,28).…”

“…The full-length unliganded gp120 subunit shown in our cryo-EM map exhibits a conformation different from that observed in the CD4-bound gp120 core (16). The improved resolution of the current map allows positioning of the secondary structure elements in gp120 and a detailed assessment of the conformational changes collectively associated with Env cleavage and CD4 binding (25)(26)(27)(28).…”

Conformational Evaluation of HIV-1 Trimeric Envelope Glycoproteins Using a Cell-based ELISA Assay

“…Figure 1 schematically represents the general procedure and the exposure of CD4i epitopes following treatment with sCD4 or by coexpression of cellular CD4 18 . In Figure 2, we used sCD4 to induce Env conformational changes that expose CD4i mAbs 17b and 48d epitopes which overlap the coreceptor binding site 24,29 , whereas the outer-domain recognizing mAb 2G12 is not affected by this treatment as expected 18,24 .…”

“…Using the general procedure described above, we adapted the protocol to assay the impact of soluble CD4 (sCD4) and coexpressed cellular CD4 on the exposure of CD4i epitopes on either wild-type (wt) or mutated Env, as described previously 18,24,25,28 . Figure 1 schematically represents the general procedure and the exposure of CD4i epitopes following treatment with sCD4 or by coexpression of cellular CD4 18 .…”

“…Here we describe in detail a method that allows for fast and easy determination of the conformation of cellularly-expressed Env trimers 18,[24][25][26] . Following transient transfection of Env in a human adherent cell line the binding of Env-specific antibodies is detected using a simple chemiluminescence reaction.…”