Single Amino Acid Changes in the Virus Capsid Permit Coxsackievirus B3 To Bind Decay-Accelerating Factor

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The coxsackievirus-adenovirus receptor (CAR) and decay-accelerating factor (DAF) have been identified as cellular receptors for coxsackievirus B3 (CVB3). The first described DAF-binding isolate was obtained during passage of the prototype strain, Nancy, on rhabdomyosarcoma (RD) cells, which express DAF but very little CAR. Here, the structure of the resulting variant, CVB3-RD, has been solved by X-ray crystallography to 2.74 Å, and a cryo-electron microscopy reconstruction of CVB3-RD complexed with DAF has been refined to 9.0 Å. This new high-resolution structure permits us to correct an error in our previous view of DAF-virus interactions, providing a new footprint of DAF that bridges two adjacent protomers. The contact sites between the virus and DAF clearly encompass CVB3-RD residues recently shown to be required for binding to DAF; these residues interact with DAF short consensus repeat 2 (SCR2), which is known to be essential for virus binding. Based on the new structure, the mode of the DAF interaction with CVB3 differs significantly from the mode reported previously for DAF binding to echoviruses. Coxsackieviruses are significant human pathogens that cause myocarditis, meningitis, and pancreatitis and have been implicated in the development of juvenile diabetes (58, 60-64). Virulence determinants have been described throughout the genome (19,20,30,51,65), including the P1 region, which encodes the structural proteins (7,10,13,25,48,49,56). The capsid surface presents a topology of structural motifs that largely dictate receptor recognition and usage, directly affecting tropism and pathogenicity.Group B coxsackieviruses (CVBs) belong to the genus Enterovirus of the family Picornaviridae. Picornaviruses are nonenveloped, positive-sense, single-stranded-RNA animal viruses with a capsid comprised of 60 protomers arranged to form an icosahedral shell ϳ300 Å in diameter with Tϭ1 (pseudo-Tϭ3) symmetry (ICTV classification) (8). In mature capsids, each protomer contains four structural proteins, VP-1, -2, -3, and -4. Structural studies have shown that capsids share common features, including a depression around the icosahedral 5-fold symmetry axes (called the "canyon") and a hydrophobic cavity located underneath the floor of the canyon (called the "pocket") (52). Biochemical and structural evidence indicates that the ligand within the pocket is a fatty acid (26, 55). For many picornaviruses, a receptor binds into the canyon and dislodges this "pocket factor," initiating conformational changes that lead to the formation of "A particles" and the subsequent uncoating of the virion (1, 12, 33, 39, 66). The major CVB receptor, the coxsackievirus-adenovirus receptor (CAR), binds within the CVB3 canyon (37) and causes the formation of A particles (16,35).A number of CVB isolates bind a second receptor, decayaccelerating factor (DAF) (CD55), a molecule that also serves as a receptor for many other enteroviruses (2,3,18,23,24,43,45). DAF, which is expressed on virtually all cell surfaces, acts to protect cells from lysis ...

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confidence: 55%

The Crystal Structure of a Coxsackievirus B3-RD Variant and a Refined 9-Angstrom Cryo-Electron Microscopy Reconstruction of the Virus Complexed with Decay-Accelerating Factor (DAF) Provide a New Footprint of DAF on the Virus Surface

Yoder

Cifuente

Pan

et al. 2012

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“…For experiments with DAF transgenic mice, we wanted to use a DAFbinding isolate that replicated efficiently in mice. We therefore tested CVB3 H3-RD, a DAF-binding derivative of the cardiovirulent isolate CVB3 H3 which differs from CVB3 H3 in only a single capsid residue (21). After inoculation with 10 5 PFU by the intraperitoneal route, CVB3 H3-RD replicated to high titers in the pancreas, liver, and heart ( Fig.…”

Section: Resultsmentioning

confidence: 99%

Expression of Human Decay-Accelerating Factor on Intestinal Epithelium of Transgenic Mice Does Not Facilitate Infection by the Enteral Route

Pan

Zhang

Odenwald

et al. 2015

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In vitro, infection of polarized human intestinal epithelial cells by coxsackievirus B3 (CVB3) depends on virus interaction with decay-accelerating factor (DAF), a receptor expressed on the apical cell surface. Although mice are highly susceptible to CVB3 infection when virus is delivered by intraperitoneal injection, infection by the enteral route is very inefficient. Murine DAF, unlike human DAF, does not bind virus, and we hypothesized that the absence of an accessible receptor on the intestinal surface is an important barrier to infection by the oral route. We generated transgenic mice that express human DAF specifically on intestinal epithelium and measured their susceptibility to infection by a DAF-binding CVB3 isolate. Human DAF permitted CVB3 to bind to the intestinal surface ex vivo and to infect polarized monolayers of small-intestinal epithelial cells derived from DAF transgenic mice. However, expression of human DAF did not facilitate infection by the enteral route either in immunocompetent animals or in animals deficient in the interferon alpha/beta receptor. These results indicate that the absence of an apical receptor on intestinal epithelium is not the major barrier to infection of mice by the oral route. IMPORTANCE CVB3 infection of human intestinal epithelial cells depends on DAF at the apical cell surface, and expression of human DAF on murine intestinal epithelial cells permits their infection in vitro. However, expression of human DAF on the intestinal surface of transgenic mice did not facilitate infection by the oral route. Although the role of intestinal DAF in human infection has not been directly examined, these results suggest that DAF is not the critical factor in mice.C oxsackieviruses belong to the genus Enterovirus of the family Picornaviridae, a group of small, nonenveloped viruses with a single-strand, positive-sense RNA genome (1). Group B coxsackieviruses (CVBs) are important causes of viral myocarditis and meningitis, and they have been proposed to be a possible triggering agent for juvenile diabetes (1).CVBs, like other enteroviruses, are transmitted by the fecaloral route and are believed to initiate infection by crossing the intestinal mucosa, which is lined by polarized epithelial cells with distinct apical and basolateral surfaces. In polarized epithelial cells, the primary CVB receptor, the coxsackievirus and adenovirus receptor (CAR) (2-4), is not expressed on the apical cell surface but is instead confined to the basolateral surface, where it functions as part of the intercellular tight junction (5); CAR is thus inaccessible to virus approaching the apical cell surface from the intestinal lumen. Some CVB isolates bind to a second receptor, decay-accelerating factor (DAF) (6-8). Unlike CAR, DAF is highly expressed on the apical surface of polarized epithelial cells, and infection of polarized cells depends on virus attachment to DAF (9). In addition to providing a docking site for virus, DAF mediates a series of intracellular signals that permit virus to move ac...

“…We next determined whether DAF was required to facilitate apical CVB infection of BeWo cells, as has been shown for other polarized cell types (9,13). First, we compared the efficiency of apical infection by a non-DAF binding CVB isolate (CVB3-Nancy) versus a DAF-binding isolate (CVB3-RD) (a single amino acid difference exists between CVB3-RD and CVB3-Nancy [49]). We found that CVB3-Nancy infected BeWo cells poorly from the apical surface (Ͻ10%) compared to CVB3-RD, indicating that DAF was necessary for efficient apical infection of these cells (Fig.…”

Section: Resultsmentioning

confidence: 99%

Lipid Raft- and Src Family Kinase-Dependent Entry of Coxsackievirus B into Human Placental Trophoblasts

Delorme-Axford

Sadovsky

Coyne

2013

bMaternal-fetal transmission of group B coxsackieviruses (CVB) during pregnancy has been associated with a number of diverse pathological outcomes, including hydrops fetalis, fetal myocarditis, meningoencephalitis, neurodevelopmental delays, congenital skin lesions, miscarriage, and/or stillbirth. Throughout pregnancy, the placenta forms a critical antimicrobial protective barrier at the maternal-fetal interface. Despite the severity of diseases accompanying fetal CVB infections, little is known regarding the strategies used by CVB to gain entry into placental trophoblasts. Here we used both a trophoblast cell line and primary human trophoblasts to demonstrate the mechanism by which CVB gains entry into polarized placental trophoblasts. Our studies revealed that the kinetics of CVB entry into placental trophoblasts are similar to those previously described for polarized intestinal epithelial cells. Likewise, CVB entry into placental trophoblasts requires decay-accelerating factor (DAF) binding and involves relocalization of the virus from the apical surface to intercellular tight junctions. In contrast, we have identified a divergent mechanism for CVB entry into polarized trophoblasts that is clathrin, caveolin-1, and dynamin II independent but requires intact lipid rafts. In addition, we found that members of the Src family of tyrosine kinases were required for CVB entry. Our studies highlight the complexity of viral entry into human placental trophoblasts and may serve as a model for mechanisms used by diverse pathogens to penetrate the placental barrier.