Illuminating the virus life cycle with single-molecule FRET imaging

Abstract

Single-molecule Förster resonance energy transfer (smFRET) imaging has emerged as a powerful tool to probe conformational dynamics of viral proteins, identify novel structural intermediates that are hiding in averaging population-based measurements, permit access to the energetics of transitions and as such to the precise molecular mechanisms of viral replication. One strength of smFRET is the capability of characterizing biological molecules in their fully hydrated/native state, which are not necessarily available to other structural methods. Elegant experimental design for physiologically relevant conditions, such as intact virions, has permitted the detection of previously unknown conformational states of viral glycoproteins, revealed asymmetric intermediates, and allowed access to the real-time imaging of conformational changes during viral fusion. As more laboratories are applying smFRET, our understanding of the molecular mechanisms and the dynamic nature of viral proteins throughout the virus life cycle are predicted to improve and assist the development of novel antiviral therapies and vaccine design.

Real-Time Conformational Dynamics of SARS-CoV-2 Spikes on Virus Particles

“…A unique strength of single-molecule imaging is its capacity to directly reveal both the structural and kinetic features that define biological function (Lu et al, 2019b;Roy et al, 2008). To extract such information for the SARS-CoV-2 S protein, we employed hidden Markov modeling (HMM) (McKinney et al, 2006) to idealize individual smFRET traces.…”

An intact amber-free HIV-1 system for in-virus protein bioorthogonal click labeling that delineates envelope conformational dynamics

“…Attaching donor/acceptor FRET-paired fluorophores to two sites of the host molecule within their Förster distance of a few nanometers is a prerequisite for applying smFRET 36 . We have previously used enzymatic labeling for smFRET imaging of HIV-1 Env 32,[37][38][39] , in which customized Cy3/Cy5 FRET-paired dyes are placed into Env gp120 variable loops V1 and V4, respectively, by enzymes that recognize introduced short peptides (Q3 and A1) and transfer dye-conjugated substrates to specific residues within these peptides 40,41 . This approach results in the insertion of 6 and 12 amino acids into V1 and V4, respectively, of gp120.…”

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 ].…”

“…One unique strength of smFRET is to reveal the timing, order, and frequency of conformational states and state-to-state transitions of S in situ on the surface of viral particles ( Figure 3 D) [ 40 , 41 , 47 ]. Kinetic analyses deployed in smFRET include the transition density for state-to-state transition (plotted as initial state vs. final state), the hidden Markov modeling for idealizing molecular motions, and dwelling time for estimating transitional rates.…”