Table 1.

Single entity assays reveal cell-to-cell variability in HAdV-C infection.

Infection Step	Variability	Evidence	References
Virus binding	10–15×	Fluorescent HAdV-C2/C5 virions; epithelial cells (CAR)/alveolar macrophages (SR-A6).	[53,54,55]
Endocytosis	2–3×	Fluorescent HAdV-C5 and immunofluorescence microscopy.	[44,56,57]
Protein VI exposure	10×	Intensity of immuno-stained protein VI on endocytosed virions at 10 or 20 min pi.	[44,58]
Penetration into the cytosol	2–3×	Streptolysin-O mediated plasma membrane permeabilization and staining of cytosolic HAdV-Alexa488 species B or C by perfused anti-Alexa488 antibody. Penetration inactive HAdV-C2_TS1-Alexa488 served as negative control for variability. Data are in agreement wth thin section electron microscopy resolving single virions in endosomes.	[44,47,48,59,60]
Nuclear targeting	low	Fluorescent HAdV-C2/C5 virions.	[49,61,62]
Uncoating of virion DNA	low	Fluorescent HAdV-C2/C5 virions and clickable virion DNA.	[49,63]
Nuclear import	3×/15×	Confocal microscopy of DNA-associated protein VII & clickable viral DNA. Note: the protein VII-based immuno-staining does not detect mis-delivered viral DNA in the cytosol, unlike click-staining.	[31,44,49]
E1A, E1B-55K early transcription	10–15×	scRNA-FISH, in combination with localization of incoming viral DNA by click chemistry.	[31]
Major late transcription	10–15×	scRNA-FISH in human lung epithelial cells.	[31]
DNA replication	Variable onset	Click chemistry and sc DNA-FISH.	[31]
Assembly	?	Co-assembly model of virions from components suggests that there is a large excess of unassembled over virion-incorporated capsomers.	[64,65]
Proteolytic maturation	10% light, 90% heavy particles	HAdV-C5 particles isolated from producer cells by CsCl density gradient centrifugation assays. Two bands are typically visible on the gradient: “light” particles with unprocessed structural proteins and infectious “heavy” particles with proteolytically processed structural proteins.	[66,67,68]
Egress	73% lytic; 27% nonlytic	Single well, single plaque assays by live cell imaging.	[42,69]
Lysis/Persistence		Simultaneous single-cell in situ analyses of HAdV-C5 gene expression, suppression of the E1A promoter by IFN, and activation by Ire1α/XBP1 axis of the unfolded protein response pathway.	[70,71,72]