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. 2012 Oct;86(19):10821-8.
doi: 10.1128/JVI.01428-12. Epub 2012 Aug 1.

Spatiotemporal dynamics of adenovirus membrane rupture and endosomal escape

Oana Maier  1 , Shauna A Marvin, Harald Wodrich, Edward M Campbell, Christopher M Wiethoff
Affiliations

Spatiotemporal dynamics of adenovirus membrane rupture and endosomal escape

Oana Maier et al. J Virol. 2012 Oct.

Abstract

A key step in adenovirus cell entry is viral penetration of cellular membranes to gain access to the cytoplasm and deliver the genome to the nucleus. Yet little is known about this important event in the adenoviral life cycle. Using the cytosolic protein galectin-3 (gal3) as a marker of membrane rupture with both live- and fixed-cell imaging, we demonstrate that in the majority of instances, exposure of pVI and recruitment of gal3 to ruptured membranes occur early at or near the cell surface and occur minimally in EEA-1-positive (EEA-1(+)) early endosomes or LAMP-1(+) late endosomes/lysosomes. Live-cell imaging of Ad5 egress from gal3(+) endosomes occurs most frequently from perinuclear locations. While the Ad5 capsid is observed escaping from gal3(+) endosomes, pVI appears to remain associated with the gal3(+) ruptured endosomes. Thus, Ad5 membrane rupture and endosomal escape appear to be both spatially and temporally distinct events.

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Figures

Fig 1
Fig 1
gal3 accumulates in punctate structures in Ad5-infected cells. HeLa cells were either left uninfected (A) or infected with fluorescently labeled (green) Ad5 (B), Ad2ts1 (C), or reovirus T3D (D), and at 30 min postinfection, the cells were fixed and stained for gal3 (red) or reovirus. The number of gal3 puncta per cell was counted. Bars, 5 μm. **, P < 0.001.
Fig 2
Fig 2
The number of gal3 puncta increases with increasing virus concentration. HeLa cells were infected with increasing concentrations of fluorescently labeled Ad5 (green). Cells were fixed and stained for gal3 (red) at 30 min postinfection. (A) Representative images. Bars, 5 μm. (B) The numbers of gal3 and virus puncta per cell were counted.
Fig 3
Fig 3
Ad5 membrane rupture occurring near the cell surface. (A) HeLa cells were infected with fluorescently labeled Ad5, fixed at 0 and 30 min postinfection, and stained for gal3 (red) and pVI (blue). Nuclei were stained with DAPI (gray). Bar, 5 μm. (B) U2OS cells stably expressing mCherry-gal3 were infected with Alexa 488-Ad5, and images were collected 15 min postinfection and collected at 5 Hz.
Fig 4
Fig 4
Time-resolved imaging of Alexa 488-Ad5 endosomal escape in U2OS cells expressing mCherry-gal3. Images were collected 30 min after infection at a rate of 5 Hz. (A) Frame showing cells prior to endosomal escape. (B) Traces tracking the movement of the fluorescent virions away from relatively immobile gal3+ structures. (C) Montage showing the rapid directional movements of a fluorescent virion (from white box in panel B) as monitored by MTrackJ in ImageJ.
Fig 5
Fig 5
Ad5 acquires pVI and gal3 prior to early endosomes. Fluorescently labeled Ad5 (green) was bound to HeLa cells for 1 h at 4°C, after which cells were washed and shifted to 37°C. Cells were fixed and stained for gal3 (red) and pVI (A), EEA-1 (B), or LAMP-1 (C). M, merged images. The percentage of Ad5 colocalization was determined. Error bars indicate the standard error of the mean. Bars, 5 μm.
Fig 6
Fig 6
Ad5-pVI exposure precedes gal3 recruitment. Fluorescently labeled Ad5 (green) was bound to HeLa cells for 1 h, after which the cells were washed and shifted to 37°C to allow virus internalization. (A) Cells were fixed and stained for pVI (blue) and gal3 (red) at different times post-virus internalization. Inserts represent enlarged views of gal3 puncta. (B) The numbers of gal3 and pVI puncta per cell were counted for each time point. Error bars indicate the standard error of the mean. (C) The percentages of gal3 puncta colocalizing with Ad5, pVI, or both or with gal3 alone (None) at the indicated time points were determined.
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