CD4 occupancy triggers sequential pre-fusion conformational states of the HIV-1 envelope trimer with relevance for broadly neutralizing antibody activity

Abstract

During the entry process, the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) trimer undergoes a sequence of conformational changes triggered by both CD4 and coreceptor engagement. Resolving the conformation of these transient entry intermediates has proven challenging. Here, we fine-mapped the antigenicity of entry intermediates induced by increasing CD4 engagement of cell surface–expressed Env. Escalating CD4 triggering led to the sequential adoption of different pre-fusion conformational states of the Env trimer, up to the pre-hairpin conformation, that we assessed for antibody epitope presentation. Maximal accessibility of the coreceptor binding site was detected below Env saturation by CD4. Exposure of the fusion peptide and heptad repeat 1 (HR1) required higher CD4 occupancy. Analyzing the diverse antigenic states of the Env trimer, we obtained key insights into the transitions in epitope accessibility of broadly neutralizing antibodies (bnAbs). Several bnAbs preferentially bound CD4-triggered Env, indicating a potential capacity to neutralize both pre- and post-CD4 engagement, which needs to be explored. Assessing binding and neutralization activity of bnAbs, we confirm antibody dissociation rates as a driver of incomplete neutralization. Collectively, our findings highlight a need to resolve Env conformations that are neutralization-relevant to provide guidance for immunogen development.

A bipartite structural organization defines the SERINC family of HIV-1 restriction factors

“…This hypothesis is consistent with the genetic studies that mapped the determinants of SERINC5 sensitivity to the variable loops in gp120 20,29 , as amino acid changes in the variable loops can modulate the accessibility of MPER 30 . Moreover, the interaction with CD4, which triggers an open HIV-1 Env conformation and increases MPER accessibility 31 , rendered tier 2 and 3 viral isolates sensitive to restriction by SERINC5 21 . At present, we cannot exclude other possibilities, such as interactions with viral glycans and/or conformational movements of the spike that would enable direct SERINC5 contacts with gp120.…”

The Conserved Positive Charge in the Transmembrane Domain of HIV Gp41 Contributes to Its Intrinsic Preference for Trimerization

“…Extensive structural rearrangements of gp41 occur after CD4 engagement of the target cell and dissociation of the gp120 subunit. [20][21][22] The energetically favorable six-helix bundle formation in the extracellular domains of gp41, for example, requires significant structural rearrangements that lead to viral fusion. 23,24 In the context of full length Env, the R696A mutation did not result in profound or uniform effects on sensitivity to thermal denaturation or on infectivity decay at physiological temperature.…”

Broadly resistant HIV-1 against CD4-binding site neutralizing antibodies

“…After washing thrice with 200μl of staining buffer, the cells were stained with PE labeled antihuman IgG secondary antibody (rabbit anti-human IgG-PE, Santa Cruz) at a 1:200 dilution in 50μl of staining buffer for 45 minutes at room temperature. Following three washes with staining buffer, the cells were resuspended and analyzed on a FACSCalibur instrument (BD Biosciences, USA) using FlowJo 10 software (FlowJo, USA) [71]. Appropriate negative controls were included, such as mock-transfected 293T cells, secondary conjugated antibody labeling of Envtransduced cells as a background control for the secondary antibody, and the irrelevant antibody MERS-4V2 targeting the receptor binding domain of Middle East Respiratory Syndrome Coronavirus [72].…”