Adenovirus infection is dependent on regulation and accessibility of the receptor CAR

Coxsackie and Adenovirus Receptor (CAR) displays dual biological functions since it acts both as a viral receptor for the two unrelated viruses, coxsackie and adenovirus (Ad), and as a cell-adhesion molecule in mammalian cells. The number and accessibility of CAR receptors expressed at the cell surface is a major determinant for successful infection. The aim of this thesis is to evaluate how CAR and Ad infection vary depending on the physiological milieu surrounding the cell. To address the aim of the thesis, CAR-mediated Ad infection was studied under three different conditions, namely in the presence of anti-Ad neutralizing antibodies that constitute a humoral immune response to the viral capsid, in the presence of cytokines that constitute secreted pro-inflammatory mediators, and in the presence of altered intracellular signaling pathways that constitute hallmarks of cancer progression.

To examine the impact of neutralizing antibodies on CAR-mediated Ad infection, quantitative methods were developed to measure cell-associated virus and successful viral infection (gene expression). In the presence of neutralizing antibodies, Ad was hindered from interaction with CAR and infection was prevented. The impact of neutralizing antibodies was further characterized by determining the extent to which the decrease in infection resulted from hindered receptor binding versus actual viral inactivation. To examine this question, target cells were modified by introducing the expression of an Fcgamma receptor that was capable of binding and internalizing Ad-antibody complexes in a CAR-independent manner. These experiments showed that infectious virus was present in Ad-antibody complexes and that hindrance of binding to CAR likely constituted a major factor in neutralizing Ad.

To examine the impact of inflammation on Ad infection, CAR expression and Ad infection of human endothelial cells were studied in the presence and absence of the proinflammatory cytokines, tumor necrosis factor alpha (TNFalpha) and interferon gamma (IFNgamma). The data showed that these cytokines suppressed CAR protein and mRNA levels in endothelial cells and inhibited Ad infection in a time and dose-dependent manner, demon-strating that cytokine-mediated changes in cell physiology had the potential to affect CAR-dependent Ad infection by changing the availability of CAR.

Finally, to examine the impact of cancer-related intracellular signaling pathways on CAR-mediated Ad infection, several in vitro models were established to recreate progression of tumor cells from low to high-grade malignancy, a process known as epithelial to mesenchymal transition (EMT). CAR was suppressed both at the transcriptional and translational level in these models, and a novel transcriptional repressor complex involving Snail and Smads was identified. This complex mediated effective suppression of CAR as well as another cell adhesion protein E-cadherin reflecting the fact that CAR expression and Ad infection can be modulated as part of larger, long term changes in cell physiology.

In summary, these thesis studies shed new light on mechanisms involved in adenovirus interaction with host cells and on regulation and accessibility of CAR during normal and pathological conditions. As such this thesis has, in part, contributed to a better understanding of the intimate interplay between virology and cell biology.

List of scientific papers

I. Vincent T, Harvey BG, Hogan SM, Bailey CJ, Crystal RG, Leopold PL (2001). Rapid assessment of adenovirus serum neutralizing antibody titer based on quantitative, morphometric evaluation of capsid binding and intracellular trafficking: population analysis of adenovirus capsid association with cells is predictive of adenovirus infectivity. J Virol. 75(3): 1516-21
https://pubmed.ncbi.nlm.nih.gov/11152523

II. Leopold PL, Wendland RL, Vincent T, Crystal RG (2006). Neutralized adenovirus-immune complexes can mediate effective gene transfer via an Fc receptor-dependent infection pathway. J Virol. 80(20): 10237-47
https://pubmed.ncbi.nlm.nih.gov/17005701

III. Vincent T, Pettersson RF, Crystal RG, Leopold PL (2004). Cytokine-mediated downregulation of coxsackievirus-adenovirus receptor in endothelial cells. J Virol. 78(15): 8047-58
https://pubmed.ncbi.nlm.nih.gov/15254176

IV. Vincent T, Neve EPA, Kukalev A, Virtanen I, Philipson L, Leopold PL, Crystal RG, Moustakas A, Pettersson RF, Fuxe J (2007). A snail-smad transcriptional repressor complex promotes TGFbeta-mediated epithelial mesenchymal transition. [Manuscript]