Single Virus Tracing: Visualization of the Infection Pathway of a Virus into a Living Cell

Abstract

Approach, contact, retreat; approach, contact, penetrate. With a number of fleeting subsecond contacts, the single virus moves across the cellular membrane (paths 1, 2 in the graphic) before it is suddenly engulfed (path 3). Although only a few viruses are successful, once inside they enter the nucleus in a matter of minutes (path 4), much faster than previously thought. Single virus tracing, the application of real‐time single‐molecule techniques to this biomedical problem, enables us to view the live scenes of viral infection with high spatial and temporal resolution and under ideal physiological conditions in a living cell. In this way, the “movie script” of a viral infection is obtained.

HIV-1–cellular interactions analyzed by single virus tracing

“…Measurements were performed on a wide-field fluorescence microscope (NIKON Te300) with single-molecule sensitivity (Bräuchle et al 2002;Seisenberger et al 2001). A schematic of the setup is shown in Fig.…”

“…Single virus tracing (SVT) uses ultra-sensitive wide-field fluorescence microscopy to track individual virus particles in real time with high spatial resolution and single molecule sensitivity (Bräuchle et al 2002;Seisenberger et al 2001). As the fluorescence is emitted by a single, sub-diffraction limited particle, the position of the particle can be determined to an accuracy of \40 nm from the peak position of the observed fluorescent spot.…”

“…With this technique, the individual steps of the entry process and the dynamics of these processes can be elucidated. SVT has been utilized to investigate the entry and trafficking of several viruses including adeno-associated virus (Bräuchle et al 2002;Seisenberger et al 2001), adenovirus (Suomalainen et al 1999), simian virus 40 (Damm et al 2005), murine polyoma virus (Ewers et al 2005), HIV (McDonald et al 2002), influenza virus (Lakadamyali et al 2003;Rust et al 2004), rabies virus (Finke et al 2004) and murine leukemia virus (Lehmann et al 2005). From the trajectories of individual viruses, the interactions between these particles and different cellular components can be directly observed.…”

Nonergodicity, fluctuations, and criticality in heterogeneous diffusion processes

“…Similar situations are encountered in cell membranes [8,9]. The experimental evidence for subdiffusion in the crowded cytoplasm of living cells ranges from the motion of small labeled proteins [10,11] over mRNA molecules and chromosomal loci [12,13], lipid and insulin granules [14,15], virus particles [16,17], as well as chromosomal telomeres [18] and Cajal bodies [19] inside the nucleus. Subdiffusion was observed for membrane resident proteins experimentally [9,20] and for membrane lipid molecules in computer simulations [21].…”

Pulsed Interleaved Excitation

“…Ultrasensitive fluorescence methods allow one to investigate the interactions and dynamics of biomolecules with high accuracy even on the level of single fluorophores. Such ultrasensitive methods include fluorescence correlation spectroscopy (FCS) (1,2) and fluorescence cross-correlation spectroscopy (FCCS) (3,4), burst analysis (5), single-molecule studies (6), singlepair fluorescence resonance energy transfer (spFRET) experiments (7)(8)(9)(10)(11), and single virus tracing (12,13).…”