Range of CD4-Bound Conformations of HIV-1 gp120, as Defined Using Conditional CD4-Induced Antibodies

Abstract

The HIV envelope binds cellular CD4 and undergoes a range of conformational changes that lead to membrane fusion and delivery of the viral nucleocapsid into the cellular cytoplasm. This binding to CD4 reveals cryptic and highly conserved epitopes, the molecular nature of which is still not fully understood. The atomic structures of CD4 complexed with gp120 core molecules (a form of gp120 in which the V1, V2, and V3 loops and N and C termini have been truncated) have indicated that a hallmark feature of the CD4-bound conformation is the bridging sheet minidomain. Variations in the orientation of the bridging sheet hairpins have been revealed when CD4-liganded gp120 was compared to CD4-unliganded trimeric envelope structures. Hence, there appears to be a number of conformational transitions possible in HIV-1 monomeric gp120 that are affected by CD4 binding. The spectrum of CD4-bound conformations has been interrogated in this study by using a well-characterized panel of conditional, CD4-induced (CD4i) monoclonal antibodies (MAbs) that bind HIV-1 gp120 and its mutations under various conditions. Two distinct CD4i epitopes of the outer domain were studied: the first comprises the bridging sheet, while the second contains elements of the V2 loop. Furthermore, we show that the unliganded extended monomeric core of gp120 (core e ) assumes an intermediate CD4i conformation in solution that further undergoes detectable rearrangements upon association with CD4. These discoveries impact both accepted paradigms concerning gp120 structure and the field of HIV immunogen design. IMPORTANCE Elucidation of the conformational transitions that the HIV-1 envelope protein undergoes during the course of entry into CD4؉ cells is fundamental to our understanding of HIV biology. The binding of CD4 triggers a range of gp120 structural rearrangements that could present targets for future drug design and development of preventive vaccines. Here we have systematically interrogated and scrutinized these conformational transitions using a panel of antibody probes that share a specific preference for the CD4i conformations. These have been employed to study a collection of gp120 mutations and truncations. Through these analyses, we propose 4 distinct sequential steps in CD4i transitions of gp120 conformations, each defined by antibody specificities and structural requirements of the HIV envelope monomer. As a result, we not only provide new insights into this dynamic process but also define probes to further investigate HIV infection. V iral tropism is mediated by the specific binding of the viral spike protein to its corresponding cell surface receptor. Evolution has driven human immunodeficiency virus (HIV) to elaborate on this canonical paradigm, introducing a series of orchestrated sequential events involving two receptors: CD4 as a primary receptor (1-3) and a chemokine receptor (CXCR4 or CCR5) as a subsequent coreceptor (4-10). However, many critical details of the molecular mechanisms by which CD4 triggers a number of conformati...

Unique binding modes for the broad neutralizing activity of single-chain variable fragments (scFv) targeting CD4-induced epitopes

“…Then, the chemokine receptors bind to the bridging sheet region on gp120 and the entry step follows the membrane fusion through gp41 [ 6 – 9 ]. The bridging sheet consists of a four-stranded β-sheet structure composed of two double-strand β-sheet structures, hairpin 1 (H1 containing β2 and β3 in the stem of the V1/V2 loops) and hairpin 2 (H2 containing β20 and β21 in the C4 region) [ 5 , 10 ]. These β-sheets are highly conserved among HIV-1 strains because the structure and amino acid sequences are critical for the interaction with the N-terminus of CCR5, suggesting it is the main component of the co-receptor binding site [ 11 , 12 ].…”

“…The subtle difference in their reacting epitopes may account for the difference in neutralization potency among anti-CD4i scFvs. Analysis using a series of Env mutants revealed that individual anti-CD4i mAbs had various dependencies for the H1, V1/V2 loops and V3 stem regions [ 10 , 18 ]. In addition, the binding of CD4 significantly affected the dependency on these regions [ 10 , 18 ].…”

Insights into the molecular mechanism underlying CD4-dependency and neutralization sensitivity of HIV-1: a comparative molecular dynamics study on gp120s from isolates with different phenotypes

“…Because V4 and V5 are connected to b20 (one of bridging-sheet elements) and a5 (layer 3) through rigid b19 and b24, respectively, it is very likely that dynamical behaviours of these two surface loops may mediate/modulate conformational dynamics of the b20-b21 hairpin and layer 3, respectively. The stronger mobility of V4 and V5 in R2-gp120 likely makes a larger contribution to increasing uctuations of b20-b21 and a5, respectively, which have been shown important for triggering the formation of bridging sheet 16,58,59 and for communication between inner and outer domains, 15,16 respectively.…”

“…6E) will enlarge the cavity located between the inner and outer domains, allowing b20-b21 hairpin to move in the same direction as layers 1 and 2. Previous studies 37,40,58 have suggested that the opening of inter-domain cavity followed by the rearrangement of b20-b21 hairpin is a crucial initial step for triggering gp120's conformational transition from the unliganded to the CD4bound state since the reorientation of b20-b21 results in the formation of the mature CD4-Phe43-binding pocket and the disruption of hydrogen bonds between b3 and b21, which is a prerequisite for further repositioning of V1/V2 and the formation of the mature bridge sheet. In the motional mode along the eigenvector 3 ( Fig.…”

“…6F), b21 and the four b-stands (bA to bD) within the V1/V2 region move collectively outwards and downwards, thus likely allowing for a full extension and reorientation of these substructures which, although not observed in our simulations due to limited simulation time, were considered as crucial events in gp120 conformational transition. 58 Of interest is that the homology model showing a full-extension downward orientation of V1/V2 relative to gp120 core has been constructed by Langley et al, 37 who suggest that the CD4-independent gp120 has a higher probability to adopt this intermediate state than CD4-dependent gp120 when unliganded by CD4. In addition, the bending and twisting motions of the V1/V2 region along the eigenvectors 1 and 2, respectively, may help to disrupt its adhesion with the V3 loop, which is fully exposed in the CD4-bound state of gp120 17,63 and, upon exposure, participates in the formation of the mature coreceptorbinding site or of the CD4i epitope.…”