Tracking Viral Genomes in Host Cells at Single-Molecule Resolution

Abstract

Viral DNA trafficking in cells has large impacts on physiology and disease development. Current methods lack the resolution and accuracy to visualize and quantify viral DNA trafficking at single-molecule resolution. We developed a noninvasive protocol for accurate quantification of viral DNA-genome (vDNA) trafficking in single cells. Ethynyl-modified nucleosides were used to metabolically label newly synthesized adenovirus, herpes virus, and vaccinia virus vDNA, without affecting infectivity. Superresolution microscopy and copper(I)-catalyzed azide-alkyne cycloaddition (click) reactions allowed visualization of infection at single vDNA resolution within mammalian cells. Analysis of adenovirus infection revealed that a large pool of capsid-free vDNA accumulated in the cytosol upon virus uncoating, indicating that nuclear import of incoming vDNA is a bottleneck. The method described here is applicable for the entire replication cycle of DNA viruses and offers opportunities to localize cellular and viral effector machineries on newly replicated viral DNA, or innate immune sensors on cytoplasmic viral DNA.

The nuclear export factor CRM1 controls juxta-nuclear microtubule-dependent virus transport

“…However, depending on the cell type, adenovirus is either arrested (stuck) at the MTOC or dispersed in the distal cytoplasm. The latter phenotype is observed for example in HeLa cells, which are known to have a distinct microtubule distribution (Li et al, 2012;Strunze et al, 2005;Wang et al, 2013). LMB precludes the uncoating and the delivery of viral DNA into the nucleus and blocks infection.…”

“…The effective resolution here was estimated from the average full width at half maximum (FWHM) of the signal from single adenovirus particles (n=236). The procedure was identical to that described previously (Wang et al, 2013).…”

“…The incoming virion sheds accessory proteins in a stepwise manner during entry (Greber et al, 1993;Nakano et al, 2000). The cytosolic particles are leaky containers comprising the hexon protein and viral DNA, but lacking the pentons and some stabilizing proteins (Luisoni et al, 2015;Puntener et al, 2011;Wang et al, 2013). They are different from intact virions, which are regular icosahedral T=25 (where T is the triangular number, a standard way to describe the subunit number and organization of the facets of an icosahedron) particles of ∼90 nm in diameter (Liu et al, 2010;Reddy et al, 2010).…”

Endosomophagy clears disrupted early endosomes but not virus particles during virus entry into cells     

“…Virus penetration into the cytosol occurs from early endosomes in a pHindependent manner [29] [30]. It requires the membrane-lytic viral protein VI [31] [32] [33], lysosomal secretion, the sphingolipid ceramide [34] [35], and gates the pathway for viral DNA genome separation from the capsid and nuclear delivery of the viral genome [36] [37]. Here, we describe a novel observation, the recruitment of Gal3, ubiquitin and the poly-ubiquitin binding protein p62/SQSTM1 to ruptured early endosomes, followed by clearance of disrupted membranes without clearance of virus particles from the in-fected cell.…”

Suivi à la trace du parcours intracellulaire de l’adénovirus au cours des étapes précoces du cycle infectieux

“…La présence d'ADNv dans le l'efficacité de la réplication virale. Dans le cas de l'AdV, seuls deux analogues, l'EdC et l'EdA, permettent de marquer de façon efficace l'ADNv sans compromettre sa capacité à se répliquer [7]. Cette spé-cificité dépend du virus utilisé puisque d'autres virus, comme l'HSV-1 ou le virus de la vaccine, requièrent d'autres analogues nucléotidiques.…”