A Direct and Versatile Assay Measuring Membrane Penetration of Adenovirus in Single Cells

Abstract

Endocytosis is the most prevalent entry port for viruses into cells, but viruses must escape from the lumen of endosomes to ensure that viral genomes reach a site for replication and progeny formation. Endosomal escape also helps viruses bypass endolysosomal degradation and presentation to certain Toll-like intrinsic immunity receptors. The mechanisms for cytosolic delivery of nonenveloped viruses or nucleocapsids from enveloped viruses are poorly understood, in part because no quantitative assays are readily available which directly measure the penetration of viruses into the cytosol. Following uptake by clathrin-mediated endocytosis or macropinocytosis, the nonenveloped adenoviruses penetrate from endosomes to the cytosol, and they traffic with cellular motors on microtubules to the nucleus for replication. In this report, we present a novel single-cell imaging assay which quantitatively measures individual cytosolic viruses and distinguishes them from endosomal viruses or viruses at the plasma membrane. Using this assay, we showed that the penetration of human adenoviruses of the species C and B occurs rapidly after virus uptake. Efficient penetration does not require acidic pH in endosomes. This assay is versatile and can be adapted to other adenoviruses and members of other nonenveloped and enveloped virus families.

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

“…HAdV-C5 enters epithelial cells through CAR and integrin receptors, and HAdV-B3 through CD46 and desmoglein-2 receptors [3] [47] [48] [49]. Both viruses penetrate HeLa cells independent of endosomal acidification with an efficiency higher than 70% (HAdV-C5) or about 40% (HAdV-B3) within 30 min of cold-synchronized infection [30]. Uninfected cells had diffuse cytoplasmic red fluorescence and variable levels of nuclear red fluorescence, in contrast to the prominent cytoplasmic mCherry-Gal3 foci in both types of infected cells, indicating that membrane rupture was independent of the nature of entry receptors ( (Fig.…”

“…HAdV-C2 and C5 enter epithelial cells by receptor-mediated endocytosis and a stepwise uncoating program initiated at the plasma membrane by the differential movements of two receptors, integrin and coxsackievirus adenovirus receptor (CAR) [26] [27] [28]. 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].…”

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

“…Virion motility in TC7 cells was determined by spinning disc confocal microscopy in the time window of 30-90 mpi. Cytosolic virions are the predominant entity in this time window, as indicated by previous studies showing that the half-time for virion penetration from endosomes is 15 min and that virions predominantly dock at the NPC after 30 min of infection Gastaldelli et al, 2008;Greber et al, 1997Greber et al, , 1993Greber and Way, 2006;Imelli et al, 2009;Meier et al, 2002;Suomalainen et al, 2013Suomalainen et al, , 2001Trotman et al, 2001). Movies with a length of 200 s were recorded at an image acquisition rate of 25 frames per second (Hz).…”

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

“…La détection de l'ADNv, couplée au marquage d'une protéine de la capside par immunofluorescence, permet de préciser les événements survenant lors des étapes précoces de l'infection par l'AdV. Notamment, en couplant cette technique de marquage de l'ADNv avec une méthode précédemment mise au point pour quantifier les particules d'AdV échappées des endosomes [8], l'équipe a mis en évidence un phéno-mène précédemment insoupçonné : la présence de l'ADNv nu, non protégé par la capside, dans le cytoplasme. Ce phénomène n'est pas observable après traitement des cellules par la leptomycine B, un inhibiteur des mécanismes d'export via les pores nucléaires qui empêche le désassemblage complet de la capside de l'AdV.…”