A Quantitative Live-Cell Superresolution Imaging Framework for Measuring the Mobility of Single Molecules at Sites of Virus Assembly

Abstract

The insurgence of superresolution microscopy into the fields of virology and microbiology has begun to enable the mapping of molecular assemblies critical for host–pathogen interfaces that organize on a scale below the resolution limit of the light microscope. It is, however, challenging to completely understand the molecular interactions between host and pathogen from strictly time-invariant observations. Herein, we describe a method using simultaneous dual-color superresolution microscopy to gain both structural and dynamic information about HIV-1 assembly. Specifically, we demonstrate the reconstruction of single virus assembly sites using live-cell photo-activated localization microscopy (PALM) while concurrently assessing the sub-viral mobility of the HIV-1 envelope glycoprotein during interaction with the viral lattice. We propose that our method is broadly applicable to elucidating pathogen and host protein–protein interactions through quantification of the dynamics of these proteins at the nanoscale.

Single-Molecule FRET Imaging of Virus Spike–Host Interactions

“…Imaging macromolecules at the single-molecule/single-particle level has advanced our understanding of both static and dynamic aspects in virus–host interactions, merited by avoiding the averaging-out effect from traditional ensemble-level measurements. Those imaging techniques, such as single-particle cryoEM/cryoET, single-particle optical tracking, and super-resolution fluorescence microscopy exerted significant roles in addressing fundamental questions with regards to structures, dynamics, and functions of virus molecules underlying virus–host interactions [ 38 , 39 , 40 , 41 , 42 , 43 , 44 ].…”

The envelope cytoplasmic tail regulates HIV-1 assembly and spread

“…To explain the defect in spreading infection of W757A virus, and the inability of Env to polarise to virus assembly sites at the VS, single particle tracking (SPT) was performed on Env trimers proximal and distal to super-resolved sites of virus assembly to interrogate the mobility of wildtype EnvCT and W757A mutants (Groves et al, 2020). This approach has previously elucidated the dynamics of Env mobility at the cell surface, revealing that during nascent virus assembly Env becomes trapped or corralled in the Gag lattice to facilitate incorporation into virions (Pezeshkian et al, 2019;Buttler et al, 2018) (Fig.…”

“…Human IgG (926-32232, Licor). The BG18-QD625 antibody fab fragment probe was produced recombinantly for single particle tracking as previously described (Groves et al, 2020).…”

“…Al., (Groves et al, 2020). Briefly, CEM-A T cells in complete RPMI medium (10% fetal bovine serum; mM L-glutamine; 1% penicillin/streptomycin, and 1%…”

“…hypoxanthine/thymidine; Corning, USA) were seeded on 25 mm No. 1.0 glass coverslips (Warner Instruments; Hamden, CT, USA) and infected with VSV-G pseudotyped virus packaged with the pSV-NL4-3 HIV-1 reference genome, encoding the previously described CA-Skylan-S nanobody probe, with deletions in the gag-p6 late-domain, pol, vif, vpr, and nef genes (Groves et al, 2020). Forty-eight hours post-infection, cells were stained with BG18-QD625 probe and coverslips were transferred to specimen holders and mounted on a custombuilt ring-TIRF microscope with a live-cell chamber maintaining 37° C and 5% CO2.…”