Single-particle virology

Abstract

The development of advanced experimental methodologies, such as optical tweezers, scanning-probe and super-resolved optical microscopies, has led to the evolution of single-molecule biophysics, a field of science that allows direct access to the mechanistic detail of biomolecular structure and function. The extension of single-molecule methods to the investigation of particles such as viruses permits unprecedented insights into the behavior of supramolecular assemblies. Here we address the scope of viral exploration at the level of individual particles. In an era of increased awareness towards virology, single-particle approaches are expected to facilitate the in-depth understanding, and hence combating, of viral diseases.

Keywords: Atomic force microscopy; Optical tweezers; Single-molecule mechanics; Super-resolution microscopy; Viral genome packaging; Viral genome release.

Fig. 1

Schematics of measuring the mechanical function of the φ29 bacteriophage portal motor (Smith et al. 2001). The force generated by the DNA-packaging portal motor is transmitted, via the dsDNA molecule, to an optically trapped latex bead attached to the free DNA end, allowing for the measurement of the force. This particular packaging motor stalls at a force of about 60 pN

Fig. 2

Imaging virions with atomic force microscopy (AFM) in non-contact mode, by using photothermal excitation to resonate the cantilever. a Height-contrast AFM image of a T7 bacteriophage particle attached to poly-l-lysine-coated mica. Based on the surface topography, this icosahedral virion is facing towards the buffer solution (phosphate-buffered saline, PBS) with its 3-fold symmetry axis. Individual capsomeres can be discerned in the image. b Topographical height profile plot along the axis of the particle image (indicated by the dotted straight line). c Height-contrast AFM image of a T7 bacteriophage particle pointing towards the buffer solution with its tail. To immobilize the tail fibers, the sample was chemically fixed with 2.5% glutaraldehyde and imaged in PBS. d Topographical height profile plot along the axis of a tail fiber (indicated by the dotted freehand line)

Fig. 3

Imaging virions with AFM in jumping mode (fast force mapping). a Height-contrast AFM image of a T7 bacteriophage particle attached to poly-l-lysine-coated mica. b Example of a force versus distance curve that lies behind every pixel of the FFM image

Fig. 4

Nanoindentation of a viral capsid. a Schematics of the experiment. The capsid and AFM cantilever tip are indicated with a realistic relative scale. Mechanical information can be collected, in the form of force versus distance data, during both the indentation and retraction phases of the experiment. b Force versus distance plot obtained on a T7 phage particle (Vörös et al. 2017). Force sawteeth during indentation and retraction point at buckling and unbuckling events, respectively, which result in stepwise, 6-nm reversible structural changes in the capsid. The slope of the indentation trace may be used to calculate the stiffness of the capsid

Fig. 5

Super-resolution microscopy techniques employed in single-particle virus research. a Principles of stochastic methods (STORM and PALM). b Principles of the deterministic approach in STED. c Principle of the shaping of the point spread function (PSF). d Example of influenzavirus A (IAV) infection in a human dendritic cell. Blue color corresponds to Alcian blue in the cellular environment, gray to the DAPI-stained nucleus, and green to IAV nucleoprotein labeled with FITC-conjugated antibody. Adapted from (Baharom et al. 2017)

Fig. 6

a Schematics of investigating the genomic DNA release from individual phage particles by using a microfluidic device, total internal reflection fluorescence (TIRF) microscopy, and DNA-intercalating fluorophores. b Time-resolved images of the release of dsDNA from a single λ-phage particle. DNA ejection was triggered by adding LamB (maltoporin), an E. coli outer-membrane protein, and the DNA molecule was visualized by rapid staining with the fluorescent dye SUBR Gold present in the buffer solution of the microfluidic chamber. Time delay between consecutive DNA images is 0.25 s. Upper and lower image series were recorded in the presence of 10 mM NaCl and 10 MgSO₄, respectively. Adapted from (Grayson et al. 2007)