The Coxsackievirus and Adenovirus Receptor Has a Short Half-Life in Epithelial Cells.

The coxsackievirus and adenovirus receptor (CAR) is an essential cellular protein that is involved in cell adhesion, cell signaling, and viral infection. The 8-exon encoded isoform (CAREx8) resides at the apical surface of polarized epithelia, where it is accessible as a receptor for adenovirus entering the airway lumen. Given its pivotal role in viral infection, it is a target for antiviral strategies. To understand the regulation of CAREx8 and determine the feasibility of receptor downregulation, the half-life of total and apical localized CAREx8 was determined and correlated with adenovirus transduction. Total and apical CAREx8 has a relatively short half-life of approximately 2 h. The half-life of apical CAREx8 correlates well with adenovirus transduction. These results suggest that antiviral strategies that aim to degrade the primary receptor for apical adenovirus infection will be effective within a relatively short time frame after application.

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.

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.

Production of isoform-specific knockdown/knockout Madin-Darby canine kidney epithelial cells using CRISPR/Cas9.

CRISPR-Cas9 gene editing has made it possible to specifically edit genes in a myriad of target cells. Here, a method for isoform-specific editing and clonal selection in Madin-Darby canine kidney (MDCK) epithelial cells is described in detail. This approach was used to address a long-standing question in virology of how adenovirus enters polarized epithelia from the apical surface. Our method relies on selecting two sgRNA sequences, cloning them into a suitable fluorescently labeled Cas9 vector system, and subsequently transfecting our MDCK epithelium and selecting isoform-specific Coxsackievirus and adenovirus receptor knockout clones. Utilization of this method is readily applicable to many other genetic targets in epithelial cells.•Simultaneous utilization of an sgRNA upstream and an sgRNA downstream of a target sequence allows for deletion of the intervening sequence, including whole exons.•Sorting of cells positive for fluorescent marker gene expression enhances the identification of partial and biallelic gene knockout.•PCR screening allows relatively fast and efficient determination of isoform-specific deletion.

Isoform specific editing of the coxsackievirus and adenovirus receptor.

The Coxsackievirus and adenovirus receptor (CAR) is both a viral receptor and cell adhesion protein. CAR has two transmembrane isoforms that localize distinctly in polarized epithelial cells. Whereas the seven exon-encoded isoform (CAREx7) exhibits basolateral localization, the eight exon-encoded isoform (CAREx8) can localize to the apical epithelial surface where it can mediate luminal adenovirus infection. To further understand the distinct biological functions of these two isoforms, CRISPR/Cas9 genomic editing was used to specifically delete the eighth exon of the CXADR gene in a Madine Darby Canine Kidney (MDCK) cell line with a stably integrated lentiviral doxycycline-inducible CAREx8 cDNA. The gene-edited clone demonstrated a significant reduction in adenovirus susceptibility when both partially and fully polarized, and doxycycline-induction of CAREx8 restored sensitivity to adenovirus. These data reinforce the importance of CAREx8 in apical adenovirus infection and provide a new model cell line to probe isoform specific biological functions of CAR.

Poliovirus Receptor: More than a simple viral receptor.

The human poliovirus receptor (PVR) is a cell surface protein with a multitude of functions in human biology. PVR was initially identified as the receptor for the human poliovirus and recent discoveries have given a greater insight into both its morphology and its function. Alternative splicing of the PVR gene results in a total of 4 alternatively spliced isoforms. Two of these isoforms lack a complete transmembrane domain and are considered soluble and block viral infection; the remaining two transmembrane isoforms differ only at their extreme C-terminal domains resulting in differential localization in epithelia and polarity of viral infection. In addition to its role as a receptor for the human poliovirus, several native biological functions have also been uncovered. PVR is an important cell adhesion protein and is involved in the transendothelial migration of leukocytes. Through its interactions with CD226 and TIGIT, transmembrane proteins found on leukocytes, PVR is a key regulator of the cell-mediated immune response. As PVR is differentially regulated in a broad spectrum of cancers, it has a strong potential for clinical use as a biomarker. PVR is also a possible target for novel cancer therapies. Utilizing its natural tropism for PVR, a genetically modified form of the live attenuated poliovirus vaccine is currently being tested for its ability to locate and destroy certain tumors. These recent studies emphasize the importance of PVR in human biology and demonstrate its utility beyond being a viral receptor protein.

The PDZ3 domain of the cellular scaffolding protein MAGI-1 interacts with the Coxsackievirus and adenovirus receptor (CAR).

The Coxsackievirus and adenovirus receptor (CAR) is an essential cellular protein that is involved in cell-cell adhesion, protein trafficking, and viral infection. The major isoform of CAR is selectively sorted to the basolateral membrane of polarized epithelial cells where it co-localizes with the cellular scaffolding protein membrane-associated guanylate kinase with inverted domain structure-1 (MAGI-1). Previously, we demonstrated CAR interacts with MAGI-1 through a PDZ-domain dependent interaction. Here, we show that the PDZ3 domain of MAGI-1 is exclusively responsible for the high affinity interaction between the seven exon isoform of CAR and MAGI-1 using yeast-two-hybrid analysis and confirming this interaction biochemically and in cellular lysates by in vitro pull down assay and co-immunoprecipitation. The high affinity interaction between the PDZ3 domain and CAR C-terminus was measured by fluorescence resonance energy transfer. Further, we investigated the biological relevance of this high affinity interaction between CAR and the PDZ3 domain of MAGI-1 and found that it does not alter CAR-mediated adenovirus infection. By contrast, interruption of this high affinity interaction altered the localization of MAGI-1 indicating that CAR is able to traffic MAGI-1 to cell junctions. These data deepen the molecular understanding of the interaction between CAR and MAGI-1 and indicate that although CAR plays a role in trafficking PDZ-based scaffolding proteins to cellular junctions, association with a high affinity intracellular binding partner does not significantly alter adenovirus binding and entry via CAR.