High Temporal Resolution Imaging Reveals Endosomal Membrane Penetration and Escape of Adenoviruses in Real Time

Abstract

Summary Imaging host-pathogen interactions in real time can provide significant insight into dynamic processes and provide information about time and space of their occurences. Here we present detailed experimental instructions on how to image the membrane penetration process of the non-enveloped adenovirus in rel time. The system is based on a cell line stably expressing the lectin galectin-3 fused to a fluorophore. Membrane-lytic events during adenovirus cell entry can be monitored by the recruitment of galectin-3 to galactose-containing membrane glycoproteins on the exo-surface of ruptured membranes. The simultaneous use of fluorescently labeled adenoviral capsids allows to image the events in unmatched temporal resolution.

The Amphipathic Helix of Adenovirus Capsid Protein VI Contributes to Penton Release and Postentry Sorting

“…We previously showed that protein VI release upon entry into cells can be visualized by antibody staining, because the antibody epitope remains hidden prior to capsid disassembly (26). Likewise, we showed that cytoplasmic Gal-3, a galactose binding lectin that binds galactosyl residues exposed to the cytoplasm upon membrane damage (49), can be used to monitor AdV-induced membrane damage in living and fixed cells via Gal-3 accumulation at damaged membranes (16,23). Using these approaches, we quantified protein VI release and induced membrane damage over time for all three viruses.…”

“…The viruses were quantified as physical particles per microliter following a method described previously (40). The concentrated and dialyzed viruses were labeled with Alexa 488 dye using a small-scale proteinlabeling kit (Invitrogen) as described previously (16).…”

“…Several studies, mainly performed on human adenovirus serotype 5 (HAdV-C5), show that mature virions exit the endocytic compartment during or shortly after uptake to traverse the cytosol for efficient nuclear delivery (14)(15)(16). This process involves a cap-sid-associated membrane-lytic activity absent from PRO-P137L particles.…”

“…Following uptake into cells, protein VI is liberated and thus becomes accessible to antibody staining, providing direct evidence for entry-dependent disassembly and protein VI release (26). A recent study using cellular markers of membrane damage provided the first experimental system to show in vivo visualization of the membrane lysis step of AdV entry, demonstrating that membrane damage coincided with protein VI deployment from the AdV capsid (16,22,23). The work showed that membrane lysis and parting of the remaining capsid from the damaged membrane compartment are separated in time and subcellular space, functionally discriminating between the two events.…”

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

“…Intracellular Gal8 coordinates the destruction of disrupted late endosomes and associated bacteria by autophagy, whereas Gal3 is involved in membrane sorting by clustering glycoproteins and glycolipids, and modulation of cell-cell contacts during organogenesis [18] [19] [20]. Recently, Gal3 was reported to be recruited to endosomes disrupted by incoming human adenovirus type 5 (HAdV-C5) [13] [21]. Adenoviruses are non-enveloped human pathogens and widely used vectors in clinical gene therapy and vaccination [22] [23] [24] [25].…”