2013
DOI: 10.1128/jvi.01833-13
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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 … Show more

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Cited by 67 publications
(88 citation statements)
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“…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.…”
Section: Resultsmentioning
confidence: 99%
See 1 more Smart Citation
“…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.…”
Section: Resultsmentioning
confidence: 99%
“…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].…”
Section: Introductionmentioning
confidence: 99%
“…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).…”
Section: Inhibition Of Crm1 Increases Microtubule-dependent Motility mentioning
confidence: 99%
“…La dĂ©tection de l'ADNv, couplĂ©e au marquage d'une protĂ©ine de la capside par immunofluorescence, permet de prĂ©ciser les Ă©vĂ©nements survenant lors des Ă©tapes prĂ©coces de l'infection par l'AdV. Notamment, en couplant cette technique de marquage de l'ADNv avec une mĂ©thode prĂ©cĂ©demment mise au point pour quantifier les particules d'AdV Ă©chappĂ©es des endosomes [8], l'Ă©quipe a mis en Ă©vidence un phĂ©no-mĂšne prĂ©cĂ©demment insoupçonnĂ© : la prĂ©sence de l'ADNv nu, non protĂ©gĂ© par la capside, dans le cytoplasme. Ce phĂ©nomĂšne n'est pas observable aprĂšs traitement des cellules par la leptomycine B, un inhibiteur des mĂ©canismes d'export via les pores nuclĂ©aires qui empĂȘche le dĂ©sassemblage complet de la capside de l'AdV.…”
Section: Resultsunclassified