Functional Effects of Coxsackievirus and Adenovirus Receptor Glycosylation on Homophilic Adhesion and Adenoviral Infection▿

The coxsackievirus and adenovirus receptor (CAR) is both a viral receptor and homophilic adhesion protein. The extracellular portion of CAR consists of two immunoglobulin (Ig)-like domains, each with a consensus sequence for N-glycosylation. We used chemical, genetic, and biochemical studies to show that both sites are glycosylated and contribute to the function of CAR. Although the glycosylation of CAR does not alter cell surface levels or junctional localization, it affects both adhesion and adenovirus infection in unique ways. CAR-mediated adhesion appears to require at least one site of glycosylation since cells expressing CAR without glycosylation do not cluster with each other. In contrast, glycosylation of the Ig-like domain proximal to the membrane is key to the cooperative behavior of adenovirus binding and infection. Contrary to the hypothesis that cooperativity improves viral infection, our data show that although glycosylation of the D2 domain is required for adenovirus cooperative binding, it has a negative consequence upon infection. This is the first report dissecting the adhesion and receptor activities of CAR, revealing that factors other than the binding interface play a significant role in the function of CAR. These data have important implications for both cancers with altered glycosylation states and cancer treatments using oncolytic adenovirus

Adenovirus Co-Opts Neutrophilic Inflammation to Enhance Transduction of Epithelial Cells

Human adenoviruses (HAdV) cause a variety of infections in human hosts, from self-limited upper respiratory tract infections in otherwise healthy people to fulminant pneumonia and death in immunocompromised patients. Many HAdV enter polarized epithelial cells by using the primary receptor, the Coxsackievirus and adenovirus receptor (CAR). Recently published data demonstrate that a potent neutrophil (PMN) chemoattractant, interleukin-8 (IL-8), stimulates airway epithelial cells to increase expression of the apical isoform of CAR (CAREx8), which results in increased epithelial HAdV type 5 (HAdV5) infection. However, the mechanism for PMN-enhanced epithelial HAdV5 transduction remains unclear. In this manuscript, the molecular mechanisms behind PMN mediated enhancement of epithelial HAdV5 transduction are characterized using an MDCK cell line that stably expresses human CAREx8 under a doxycycline inducible promoter (MDCK-CAREx8 cells). Contrary to our hypothesis, PMN exposure does not enhance HAdV5 entry by increasing CAREx8 expression nor through activation of non-specific epithelial endocytic pathways. Instead, PMN serine proteases are responsible for PMN-mediated enhancement of HAdV5 transduction in MDCK-CAREx8 cells. This is evidenced by reduced transduction upon inhibition of PMN serine proteases and increased transduction upon exposure to exogenous human neutrophil elastase (HNE). Furthermore, HNE exposure activates epithelial autophagic flux, which, even when triggered through other mechanisms, results in a similar enhancement of epithelial HAdV5 transduction. Inhibition of F-actin with cytochalasin D partially attenuates PMN mediated enhancement of HAdV transduction. Taken together, these findings suggest that HAdV5 can leverage innate immune responses to establish infections

Isoform-specific regulation and localization of the coxsackie and adenovirus receptor in human airway epithelia.

Adenovirus is an important respiratory pathogen. Adenovirus fiber from most serotypes co-opts the Coxsackie-Adenovirus Receptor (CAR) to bind and enter cells. However, CAR is a cell adhesion molecule localized on the basolateral membrane of polarized epithelia. Separation from the lumen of the airways by tight junctions renders airway epithelia resistant to inhaled adenovirus infection. Although a role for CAR in viral spread and egress has been established, the mechanism of initial respiratory infection remains controversial. CAR exists in several protein isoforms including two transmembrane isoforms that differ only at the carboxy-terminus (CAR(Ex7) and CAR(Ex8)). We found low-level expression of the CAR(Ex8) isoform in well-differentiated human airway epithelia. Surprisingly, in contrast to CAR(Ex7), CAR(Ex8) localizes to the apical membrane of epithelia where it augments adenovirus infection. Interestingly, despite sharing a similar class of PDZ-binding domain with CAR(Ex7), CAR(Ex8) differentially interacts with PICK1, PSD-95, and MAGI-1b. MAGI-1b appears to stoichiometrically regulate the degradation of CAR(Ex8) providing a potential mechanism for the apical localization of CAR(Ex8) in airway epithelial. In summary, apical localization of CAR(Ex8) may be responsible for initiation of respiratory adenoviral infections and this localization appears to be regulated by interactions with PDZ-domain containing proteins

Sidestream Smoke Exposure Increases the Susceptibility of Airway Epithelia to Adenoviral Infection

<div><h3>Background</h3><p>Although significant epidemiological evidence indicates that cigarette smoke exposure increases the incidence and severity of viral infection, the molecular mechanisms behind the increased susceptibility of the respiratory tract to viral pathogens are unclear. Adenoviruses are non-enveloped DNA viruses and important causative agents of acute respiratory disease. The Coxsackievirus and adenovirus receptor (CAR) is the primary receptor for many adenoviruses. We hypothesized that cigarette smoke exposure increases epithelial susceptibility to adenovirus infection by increasing the abundance of apical CAR.</p> <h3>Methodology and Findings</h3><p>Cultured human airway epithelial cells (CaLu-3) were used as a model to investigate the effect of sidestream cigarette smoke (SSS), mainstream cigarette smoke (MSS), or control air exposure on the susceptibility of polarized respiratory epithelia to adenoviral infection. Using a Cultex air-liquid interface exposure system, we have discovered novel differences in epithelial susceptibility between SSS and MSS exposures. SSS exposure upregulates an eight-exon isoform of CAR and increases adenoviral entry from the apical surface whilst MSS exposure is similar to control air exposure. Additionally, the level of cellular glycogen synthase kinase 3β (GSK3β) is downregulated by SSS exposure and treatment with a specific GSK3β inhibitor recapitulates the effects of SSS exposure on CAR expression and viral infection.</p> <h3>Conclusions</h3><p>This is the first time that SSS exposure has been shown to directly enhance the susceptibility of a polarized epithelium to infection by a common respiratory viral pathogen. This work provides a novel understanding of the impact of SSS on the burden of respiratory viral infections and may lead to new strategies to alter viral infections. Moreover, since GSK3β inhibitors are under intense clinical investigation as therapeutics for a diverse range of diseases, studies such as these might provide insight to extend the use of clinically relevant therapeutics and increase the understanding of potential side effects.</p> </div

GSK3β is downregulated in polarized CaLu-3 cells 18 h post-GSK3β inhibitor (SB415286) treatment.

<p>A) Total mRNA expression of GSK3β in control (white) or SB415286 (black) treated CaLu-3 epithelia (four biological replicates per condition measured in duplicate in each qPCR assay; mean values from three independent experiments relative to control<u>+</u>SE of the mean). B) Representative Western blot analysis of GSK3β, GSK3β-pS9, and β-actin protein levels. Quantification of C) GSK3β or D) GSK3β-pS9 protein levels, relative to β-actin (mean values from three independent experiments expressed as a percentage of control<u>+</u>SE of the mean). *p<0.05.</p

CAR expression is increased in polarized CaLu-3 cells 18 h post-sidestream cigarette smoke (SSS) exposure relative to mainstream cigarette smoke (MSS), or filtered air (SSFA or MSFA) exposure.

<p>A: Total CAR mRNA (three to four biological replicates per condition measured in duplicate in each qPCR assay; three independent experiments) and B: CAR^Ex8 mRNA (three to four biological replicates per condition measured in duplicate in each qPCR assay; three independent experiments) quantification using quantitative RT-PCR. Mean values of three independent experiments relative to control<u>+</u>SE of the mean. C: Total CAR and D: CAR^Ex8 protein and corresponding β-actin expression by Western blot (representative) and Multi-Guage image analysis (mean values from three independent experiments expressed as a percentage of control<u>+</u>SE of the mean). *p<0.05.</p

CAR expression is upregulated in polarized CaLu-3 cells 18 h post apical treatment with 45 µM of GSK3β inhibitor (SB415286).

<p>A: Total CAR and B: CAR^Ex8 mRNA levels by quantitative RT-PCR (three biological replicates per condition measured in duplicate in each qPCR assay; mean values from three independent experiments relative to control<u>+</u>SE of the mean) and C: total CAR and D: CAR^Ex8 and corresponding β-actin protein expression by Western blot (representative) and quantification using Multi-Guage densitometric analysis (mean values from three independent experiments expressed as a percentage of control<u>+</u>SE of the mean). Apical biotinylation of polarized CaLu-3 cells 18 h post-treatment with 45 µM SB415286 shows increased protein levels of E: total CAR and F: CAR^Ex8 specifically (representative blot shown from three independent experiments). *p<0.05.</p

GSK3β is downregulated 18 h post- sidestream cigarette smoke (SSS) exposure in comparison to air (SSFA or MSFA) or mainstream cigarette smoke (MSS) exposure.

<p>A: Analysis of total GSK3β mRNA levels by quantitative RT-PCR (four to six biological replicates per condition measured in duplicate in each qPCR assay; three independent experiments; mean values from three independent experiments relative to control<u>+</u>SE of the mean). B: GSK3β and C: GSK3β-pS9 protein levels, representative Western blot and densitometric analysis, relative to β-actin (mean values from three independent experiments (duplicate gels per experiment) expressed as a percentage of control<u>+</u>SE of the mean). *p<0.05.</p