A dynamic three-step mechanism drives the HIV-1 pre-fusion reaction

Abstract

Little is known about the intermolecular dynamics and stoichiometry of the interactions of the human immunodeficiency virus type 1 (HIV-1) envelope (Env) protein with its receptors and co-receptors on the host cell surface. Here we analyze time-resolved HIV-1 Env interactions with T-cell surface glycoprotein CD4 (CD4) and C-C chemokine receptor type 5 (CCR5) or C-X-C chemokine receptor type 4 (CXCR4) on the surface of cells, by combining multicolor super-resolution localization microscopy (direct stochastic optical reconstruction microscopy) with fluorescence fluctuation spectroscopy imaging. Utilizing the primary isolate JR-FL and laboratory HXB2 strains, we reveal the time-resolved stoichiometry of CD4 and CCR5 or CXCR4 in the pre-fusion complex with HIV-1 Env. The HIV-1 Env pre-fusion dynamics for both R5- and X4-tropic strains consists of a three-step mechanism, which seems to differ in stoichiometry. Analyses with the monoclonal HIV-1-neutralizing antibody b12 indicate that the mechanism of inhibition differs between JR-FL and HXB2 Env. The molecular insights obtained here identify assemblies of HIV-1 Env with receptors and co-receptors as potential novel targets for inhibitor design.

Fix your membrane receptor imaging: Actin cytoskeleton and CD4 membrane organization disruption by chemical fixation

“…vation through its ability to enhance T-cell receptor (TCR)mediated signaling by binding to the antigen-presenting major histocompatibility complex II (MHCII) (12). Besides its importance in immune signaling, CD4 is also the primary cellular receptor for human immunodeficiency viruses (HIV) (12,13). The importance of super-resolution in the study of membrane receptor organization and function cannot be overstated.…”

“…The importance of super-resolution in the study of membrane receptor organization and function cannot be overstated. A recent example is the characterization of the spatiotemporal dynamics and stoichiometry of the interactions between CD4 (and co-receptors) and HIV-1 in the context of viral entry, impossible to achieve without molecular imaging approaches (13).…”

Quantitative FRET-FLIM-BlaM to Assess the Extent of HIV-1 Fusion in Live Cells

“…In that respect, our group has been utilizing advanced imaging approaches in virology for the last decade and we are convinced that these new methodologies will be very useful to assess retrovirus fusion and infection with higher accuracy at the single cell level. To be able to quantify the relative number of internalized viral particles per cell in a highly physiological environment is the first step to couple these assays with other approaches, such as single cell RNAseq [16] or, more importantly, single molecule microscopy [17]. The methodology presented here provides more detailed information about HIV-1 fusogenicity and can be applied in a wide range of studies, i.e., single cell susceptibility to HIV-1 fusion in the context of metabolic studies, or in the presence/absence of specific cellular surface receptors to study their involvement in HIV-1 fusion.…”

Fluorescence Microscopy of the HIV-1 Envelope

“…Thus, they can greatly affect the behaviour of Env by fixing specific structures. Introducing artificial antigen peptides in Env, such as the FLAG-tag [63], which are recognised by independent antibodies, can overcome this limitation. Anti-Env broadly neutralising antibodies themselves are also the subject of intensive research [64].…”