NOTE:The reference numbering from the original has been maintained in this citation list.

Adenovirus binds its receptor (CAR), enters cells, and replicates. It must then escape to the environment to infect a new host. We found that following infection, human airway epithelia first released adenovirus to the basolateral surface. Virus then traveled between epithelial cells to emerge on the apical surface. Adenovirus fiber protein, which is produced during viral replication, facilitated apical escape. Fiber binds CAR, which sits on the basolateral membrane where it maintains tight junction integrity. When fiber bound CAR, it disrupted junctional integrity, allowing virus to filter between the cells and emerge apically. Thus, adenovirus exploits its receptor for two important but distinct steps in its life cycle: entry into host cells and escape across epithelial barriers to the environment.

Although recombinant adenoviruses are attractive vectors for gene transfer to airway epithelia, they have proven to be relatively inefficient. To investigate the mechanisms of adenovirus-mediated gene transfer to airway epithelia, we examined the role of adenovirus fiber and penton base, the two proteins involved in attachment to and entry of virus into the cell. We used human airway epithelia grown under conditions that allow differentiation and development of a ciliated apical surface that closely resembles the in vivo condition. We found that addition of fiber protein inhibited virus binding and vector-mediated gene transfer to immature airway epithelia, as well as to primary cultures of rat hepatocytes and HeLa cells. However, fiber protein had no effect on vector binding and gene transfer to ciliated airway epithelia. We obtained similar results with addition of penton base protein: the protein inhibited gene transfer to immature epithelia, whereas there was no effect with ciliated epithelia. Moreover, infection was not attenuated with an adenovirus containing a mutation in penton base that prevents the interaction with cell surface integrins. These data suggest that the receptors required for efficient infection by adenovirus are either not present or not available on the apical surface of ciliated human airway epithelia. The results explain the reason for inefficient gene transfer and suggest approaches for improvement. ( J. Clin. Invest. 1997. 100: 1144-1149.)

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Adenovirus DNA is tightly bound to the nuclear matrix throughout the course of infection. Analysis of adenovirus DNA from infected HeLa cell nuclei after extraction with lithium diiodosalicylate and digestion with restriction enzymes demonstrated that the sites of tightest attachment occur in the terminal fragments of the linear viral chromosome. Analysis of viruses mutated in the precursor terminal protein coding sequence demonstrated that the terminal protein, which is covalently attached to the 5' end of each DNA strand, mediates the tight binding. Virions containing chromosomes with mutant terminal proteins were unpackaged and viral DNA accumulated in the nucleus at a normal rate and competed for the limiting component during transcription complex formation, but their early genes were transcribed at reduced efficiency by both RNA polymerases II and III. The transcriptional defects were not complemented by coinfection with a wild-type virus. We propose that the adenovirus chromosome may exist as a single chromatin domain during infection and that binding of DNA to the nuclear matrix may play a critical role in adenovirus transcription.