A Spotlight on Viruses—Application of Click Chemistry to Visualize Virus-Cell Interactions

Abstract

The replication of a virus within its host cell involves numerous interactions between viral and cellular factors, which have to be tightly controlled in space and time. The intricate interplay between viral exploitation of cellular pathways and the intrinsic host defense mechanisms is difficult to unravel by traditional bulk approaches. In recent years, novel fluorescence microscopy techniques and single virus tracking have transformed the investigation of dynamic virus-host interactions. A prerequisite for the application of these imaging-based methods is the attachment of a fluorescent label to the structure of interest. However, their small size, limited coding capacity and multifunctional proteins render viruses particularly challenging targets for fluorescent labeling approaches. Click chemistry in conjunction with genetic code expansion provides virologists with a novel toolbox for site-specific, minimally invasive labeling of virion components, whose potential has just recently begun to be exploited. Here, we summarize recent achievements, current developments and future challenges for the labeling of viral nucleic acids, proteins, glycoproteins or lipids using click chemistry in order to study dynamic processes in virus-cell interactions.

Imaging the adenovirus infection cycle

“…The following section provides a brief overview of the most frequently used FM approaches and labeling strategies. For more detailed reviews, the reader is referred to .…”

HIV-1 uncoating by release of viral cDNA from capsid-like structures in the nucleus of infected cells

“…Viral cDNA can be visualized in fixed cells using fluorescence in-situ hybridization (FISH) (Marini et al, 2015) or its derivatives using branched probes (Chin et al, 2015), but the harsh assay conditions destroy the native cellular environment and impair immunofluorescence analysis. Incorporation of the modified nucleoside 5-ethynyl-2’-deoxyuridine (EdU) allowed the detection of actively transcribing HIV-1 complexes by visualizing de novo synthesized viral DNA via click chemistry (Peng et al, 2015; Stultz et al, 2017), but this approach is also limited to fixed cells and cellular extraction precludes high resolution analysis (Müller et al, 2019). To overcome these limitations, we adapted a live cell compatible genetically encoded system (ANCHOR) that allows single molecule gene labeling (Germier et al, 2017; Saad et al, 2014), and has previously been applied for visualization of viral DNA (Blanco-Rodriguez et al, 2020; Komatsu et al, 2018; Mariamé et al, 2018).…”

Covalent labeling of nucleic acids

“…During a significant part of their replication cycles, viruses do not exist in the form of morphologically distinct virus particles, but either as subviral nucleoprotein complexes, as genetic information only, or as newly-synthesized virion components. 24 Detecting viral nucleic acids is therefore a central objective for scientists who want to image virus-cell interactions. After metabolic labeling using various ethynyl-containing nucleosides at 1-2.5 mM (and fluorescent labeling via CuAAC), DNA trafficking could be visualized at single-molecule resolution within mammalian cells using super-resolution microscopy.…”

“…21,22 Some studies also point out applications of nucleic acid labeling in molecular imaging of tumors and infection. 23,24 The existing and potential medical applications are in turn a driving force for biotechnology.…”