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Solution structure of the coxsackievirus and adenovirus receptor domain 2.

ABSTRACT: The coxsackievirus and adenovirus receptor (CAR) mediates entry of coxsackievirus and adenovirus. CAR possesses an extracellular region that is comprised of 2 immunoglobulin domains termed CAR-D1 and CAR-D2. In the present work, the solution structure of CAR-D2, consisting of residues 142-235 of human CAR, has been determined by NMR spectroscopy. CAR-D2 is shown to be a beta-sandwich motif comprised of two beta-sheets, which are stabilized by two disulfide bonds. The first beta-sheet is comprised of beta-strands A, B, and E, and the second beta-sheet is comprised of beta-strands C, F, and G. A relatively hydrophobic helix is found between beta-strands C and E, which replaces beta-strand D of the classical c-type immunoglobulin fold.

SUBMITTER: Jiang S

PROVIDER: S-EPMC2203326 | biostudies-literature | 2007 Mar

REPOSITORIES: biostudies-literature

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Solution structure of the coxsackievirus and adenovirus receptor domain 2.

Jiang Shaokai S Caffrey Michael M

Protein science : a publication of the Protein Society 20070301 3

The coxsackievirus and adenovirus receptor (CAR) mediates entry of coxsackievirus and adenovirus. CAR possesses an extracellular region that is comprised of 2 immunoglobulin domains termed CAR-D1 and CAR-D2. In the present work, the solution structure of CAR-D2, consisting of residues 142-235 of human CAR, has been determined by NMR spectroscopy. CAR-D2 is shown to be a beta-sandwich motif comprised of two beta-sheets, which are stabilized by two disulfide bonds. The first beta-sheet is comprise ...[more]

PMID: 17322536

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Project description:The coxsackievirus and adenovirus receptor (CAR) is a member of the immunoglobulin superfamily (IgSF) and functions as a receptor for coxsackie B viruses (CVBs). The extracellular portion of CAR comprises two glycosylated immunoglobulin-like domains, D1 and D2. CAR-D1 binds to the virus and is essential for virus infection; however, it is not known whether D2 is also important for infection, and the role of glycosylation has not been explored. To understand the function of these structural components in CAR-mediated CVB3 infection, we generated a panel of human (h) CAR deletion and substitution mutants and analyzed their functionality as CVB receptors, examining both virus binding and replication. Lack of glycosylation of the CAR-D1 or -D2 domains did not adversely affect CVB3 binding or infection, indicating that the glycosylation of CAR is not required for its receptor functions. Deletion of the D2 domain reduced CVB3 binding, with a proportionate reduction in the efficiency of virus infection. Replacement of D2 with the homologous D2 domain from chicken CAR, or with the heterologous type C2 immunoglobulin-like domain from IgSF11, another IgSF member, fully restored receptor function; however, replacement of CAR-D2 with domains from CD155 or CD80 restored function only in part. These data indicate that glycosylation of the extracellular domain of hCAR plays no role in CVB3 receptor function and that CAR-D2 is not specifically required. The D2 domain may function largely as a spacer permitting virus access to D1; however, the data may also suggest that D2 affects virus binding by influencing the conformation of D1.An important step in virus infection is the initial interaction of the virus with its cellular receptor. Although the role in infection of the extracellular CAR-D1, cytoplasmic, and transmembrane domains have been analyzed extensively, nothing is known about the function of CAR-D2 and the extracellular glycosylation of CAR. Our data indicate that glycosylation of the extracellular CAR domain has only minor importance for the function of CAR as CVB3 receptor and that the D2 domain is not essential per se but contributes to receptor function by promoting the exposure of the D1 domain on the cell surface. These results contribute to our understanding of the coxsackievirus-receptor interactions.

Project description: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.

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Solution structure of the coxsackievirus and adenovirus receptor domain 2.

Publications

Solution structure of the coxsackievirus and adenovirus receptor domain 2.

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