2 research outputs found
Stimulated Emission Depletion Nanoscopy Reveals Time-Course of Human Immunodeficiency Virus Proteolytic Maturation
Concomitant with
human immunodeficiency virus type 1 (HIV-1) budding
from a host cell, cleavage of the structural Gag polyproteins by the
viral protease (PR) triggers complete remodeling of virion architecture.
This maturation process is essential for virus infectivity. Electron
tomography provided structures of immature and mature HIV-1 with a
diameter of 120–140 nm, but information about the sequence
and dynamics of structural rearrangements is lacking. Here, we employed
super-resolution STED (stimulated emission depletion) fluorescence
nanoscopy of HIV-1 carrying labeled Gag to visualize the virion architecture.
The incomplete Gag lattice of immature virions was clearly distinguishable
from the condensed distribution of mature protein subunits. Synchronized
activation of PR within purified particles by photocleavage of a caged
PR inhibitor enabled time-resolved <i>in situ</i> observation
of the induction of proteolysis and maturation by super-resolution
microscopy. This study shows the rearrangement of subviral structures
in a super-resolution light microscope over time, outwitting phototoxicity
and fluorophore bleaching through synchronization of a biological
process by an optical switch
Super-resolution Microscopy of Clickable Amino Acids Reveals the Effects of Fluorescent Protein Tagging on Protein Assemblies
The advent of super-resolution microscopy (nanoscopy) has set high standards for fluorescence tagging. Fluorescent proteins (FPs) are convenient tags in conventional imaging, but their use in nanoscopy has been questioned due to their relatively large size and propensity to form multimers. Here, we compared the nanoscale organization of proteins with or without FP tags by introducing the unnatural amino acid propargyl-l-lysine (PRK) in 26 proteins known to form multimolecular arrangements and into their FP-tagged variants. We revealed the proteins by coupling synthetic fluorophores to PRK <i>via</i> click chemistry and visualized them using ground-state depletion microscopy followed by individual molecule return, as well as stimulated emission depletion microscopy. The arrangements formed by the FP-tagged and nontagged proteins were similar. Mild, but statistically significant differences were observed for only six proteins (23% of all proteins tested). This suggests that FP-based nanoscopy is generally reliable. Unnatural amino acids should be a reliable alternative for the few proteins that are sensitive to FP tagging