MAGI-1 PDZ2 Domain Blockade Averts Adenovirus Infection via Enhanced Proteolysis of the Apical Coxsackievirus and Adenovirus Receptor.

Adenoviruses (AdVs) are etiological agents of gastrointestinal, heart, eye, and respiratory tract infections that can be lethal for immunosuppressed people. Many AdVs use the coxsackievirus and adenovirus receptor (CAR) as a primary receptor. The CAR isoform resulting from alternative splicing that includes the eighth exon, CAREx8, localizes to the apical surface of polarized epithelial cells and is responsible for the initiation of AdV infection. We have shown that the membrane level of CAREx8 is tightly regulated by two MAGI-1 PDZ domains, PDZ2 and PDZ4, resulting in increased or decreased AdV transduction, respectively. We hypothesized that targeting the interactions between the MAGI-1 PDZ2 domain and CAREx8 would decrease the apical CAREx8 expression level and prevent AdV infection. Decoy peptides that target MAGI-1 PDZ2 were synthesized (TAT-E6 and TAT-NET1). PDZ2 binding peptides decreased CAREx8 expression and reduced AdV transduction. CAREx8 degradation was triggered by the activation of the regulated intramembrane proteolysis (RIP) pathway through a disintegrin and metalloproteinase (ADAM17) and γ-secretase. Further analysis revealed that ADAM17 interacts directly with the MAGI-1 PDZ3 domain, and blocking the PDZ2 domain enhanced the accessibility of ADAM17 to the substrate (CAREx8). Finally, we validated the efficacy of TAT-PDZ2 peptides in protecting the epithelia from AdV transduction in vivo using a novel transgenic animal model. Our data suggest that TAT-PDZ2 binding peptides are novel anti-AdV molecules that act by enhanced RIP of CAREx8 and decreased AdV entry. This strategy has additional translational potential for targeting other viral receptors that have PDZ binding domains, such as the angiotensin-converting enzyme 2 receptor. IMPORTANCE Adenovirus is a common threat in immunosuppressed populations and military recruits. There are no currently approved treatments/prophylactic agents that protect from most AdV infections. Here, we developed peptide-based small molecules that can suppress AdV infection of polarized epithelia by targeting the AdV receptor, coxsackievirus and adenovirus receptor (CAREx8). The newly discovered peptides target a specific PDZ domain of the CAREx8-interacting protein MAGI-1 and decrease AdV transduction in multiple polarized epithelial models. Peptide-induced CAREx8 degradation is triggered by extracellular domain (ECD) shedding through ADAM17 followed by γ-secretase-mediated nuclear translocation of the C-terminal domain. The enhanced shedding of the CAREx8 ECD further protected the epithelium from AdV infection. Taken together, these novel molecules protect the epithelium from AdV infection. This approach may be applicable to the development of novel antiviral molecules against other viruses that use a receptor with a PDZ binding domain.

Generation and evaluation of a chimeric antibody against coxsackievirus and adenovirus receptor for cancer therapy.

Coxsackievirus and adenovirus receptor (CAR) is a single-pass transmembrane protein that is associated with adenoviral infection. CAR is involved in the formation of epithelial tight junctions and promotes tumor growth in some cancers. Previously, we developed mouse monoclonal antibodies against human CAR and found that one, mu6G10A, significantly inhibited tumor growth in xenografts of human cancer cells. Herein, we generated and characterized a mouse-human chimeric anti-CAR antibody (ch6G10A) from mu6G10A. ch6G10A had binding activity, inducing antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity, and in vivo anti-tumor activity against CAR-expressing prostate cancer DU-145 cells. In addition, cancer tissue array analysis confirmed that CAR is highly expressed in neuroendocrine lung cancers including small cell lung cancer, and treatment with ch6G10A effectively inhibited in vivo subcutaneous tumor growth of NCI-H69 small cell lung cancer cells in nude mice. Moreover, treatment with mu6G10A effectively inhibited both in vivo orthotopic tumor growth and distant metastatic formation in mouse xenograft models of a highly metastatic subline of human small cell lung cancer DMS273 cells. These results suggest that targeting therapy to CAR with a therapeutic antibody might be effective against several cancer types including small cell lung cancer.

A novel monoclonal antibody targeting coxsackie virus and adenovirus receptor inhibits tumor growth in vivo.

To create a new anti-tumor antibody, we conducted signal sequence trap by retrovirus-meditated expression method and identified coxsackie virus and adenovirus receptor (CXADR) as an appropriate target. We developed monoclonal antibodies against human CXADR and found that one antibody (6G10A) significantly inhibited the growth of subcutaneous as well as orthotopic xenografts of human prostate cancer cells in vivo. Furthermore, 6G10A also inhibited other cancer xenografts expressing CXADR, such as pancreatic and colorectal cancer cells. Knockdown and overexpression of CXADR confirmed the dependence of its anti-tumor activity on CXADR expression. Our studies of its action demonstrated that 6G10A exerted its anti-tumor activity primarily through both antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity. Moreover, 6G10A reacted with human tumor tissues, such as prostate, lung, and brain, each of which express CXADR. Although we need further evaluation of its reactivity and safety in human tissues, our results show that a novel anti-CXADR antibody may be a feasible candidate for cancer immunotherapy.

Effect of lovastatin on coxsackievirus B3 infection in human endothelial cells.

OBJECTIVE: The coxsackie and adenovirus receptor (CAR) mediates the entry of coxsackievirus B (CVB) and adenovirus into host cells and is, therefore, a key determinant for the molecular pathogenesis of viral diseases such as myocarditis. The aim was to investigate the influence of HMG-CoA reductase inhibitor lovastatin on CAR expression in endothelial cells. METHODS: Human umbilical vein endothelial cells (HUVECs) were exposed to different concentrations of lovastatin (0.05-5 µmol/l) for up to 48 h. Alterations in CAR expression were examined by quantitative real-time PCR (qRT-PCR) and flow cytometry. In addition, after treatment with 1 µmol/l lovastatin for 48 h, HUVECs were infected for 8 h with CVB3 and virus replication was detected by qRT-PCR using viral-specific TaqMan probes. RESULTS: We found that lovastatin decreases CAR mRNA expression by up to 80% (p < 0.01) and CAR protein expression by up to 19% (p < 0.01), in a concentration-dependent manner. Moreover, virus replication of CVB3 was significantly inhibited after lovastatin treatment (p < 0.05). The signaling mechanism of CAR down-regulation by lovastatin depends on the Rac1/Cdc42 pathway. CONCLUSION: This study shows for the first time that lovastatin reduces the expression of CAR and subsequently the replication of CVB3 in HUVECs.