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The GD1a glycan is a cellular receptor for adenoviruses causing epidemic keratoconjunctivitis (Letter)
Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology.
Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology.
University of Tübingen.
Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology.
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2011 (English)In: Nature Medicine, ISSN 1078-8956, E-ISSN 1546-170X, Vol. 17, no 1, 105-109 p.Article in journal (Refereed) Published
Abstract [en]

Adenovirus type 37 (Ad37) is a leading cause of epidemic keratoconjunctivitis (EKC), a severe and highly contagious ocular disease. Whereas most other adenoviruses infect cells by engaging CD46 or the coxsackie and adenovirus receptor (CAR), Ad37 binds previously unknown sialic acid-containing cell surface molecules. By glycan array screening, we show here that the receptor-recognizing knob domain of the Ad37 fiber protein specifically binds a branched hexasaccharide that is present in the GD1a ganglioside and that features two terminal sialic acids. Soluble GD1a glycan and GD1a-binding antibodies efficiently prevented Ad37 virions from binding and infecting corneal cells. Unexpectedly, the receptor is constituted by one or more glycoproteins containing the GD1a glycan motif rather than the ganglioside itself, as shown by binding, infection and flow cytometry experiments. Molecular modeling, nuclear magnetic resonance and X-ray crystallography reveal that the two terminal sialic acids dock into two of three previously established sialic acid-binding sites in the trimeric Ad37 knob. Surface plasmon resonance analysis shows that the knob-GD1a glycan interaction has high affinity. Our findings therefore form a basis for the design and development of sialic acid-containing antiviral drugs for topical treatment of EKC.

Place, publisher, year, edition, pages
2011. Vol. 17, no 1, 105-109 p.
National Category
Microbiology in the medical area
Identifiers
URN: urn:nbn:se:umu:diva-38769DOI: 10.1038/nm.2267PubMedID: 21151139OAI: oai:DiVA.org:umu-38769DiVA: diva2:382648
Available from: 2011-01-03 Created: 2010-12-28 Last updated: 2017-12-11Bibliographically approved
In thesis
1. Cellular receptors for viruses with ocular tropism
Open this publication in new window or tab >>Cellular receptors for viruses with ocular tropism
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Several viruses from different virus families are known to cause ocular infections, e.g. members of the Adenoviridae, Picornaviridae and the Herpesviridae families. These infections are spread by contact and in the case of adenoviruses (Ads) and picornaviruses they are also highly contagious. The ocular infections caused by Ads and picornaviruses are called epidemic keratoconjunctivitis (EKC) and acute hemorrhagic conjunctivitis (AHC), respectively. Historically, EKC is caused mainly by three types of Ads from species D: Ad8, Ad19 and Ad37. The infection is characterized by keratitis and conjunctivitis but also involves pain, edema, lacrimation and blurred vision. AHC is caused mainly by two types of picornaviruses: coxsackievirus A24v (CVA24v) and enterovirus 70 (EV70), and is characterized by pain, redness, excessive tearing, swelling and subconjunctival hemorrhages. In addition, blurred vision, keratitis, malaise, myalgia, fever, headache and upper respiratory tract symptoms can also be experienced. Both infections are problematic in many parts of the world, affecting millions of people every year. Despite the great need, the only treatment available today is supportive treatment; no antiviral drugs are available to combat these common viral infections.

Ad37 has previously been reported to use sialic acid (SA) as its cellular receptor. Since there is no antiviral treatment available against EKC we wanted to evaluate the inhibitory effect of SA-based antiviral compounds on Ad37 binding to and infection of ocular cells. We found that multivalent compounds consisting of SA linked to a globular carrier molecule, in this case human serum albumin, efficiently blocked Ad37 binding and infection at low concentrations. Further attempts were then made to improve the effect by chemically modifying SA monosaccharides. However, no enhanced inhibitory effect was accomplished and the conclusion was that the best inhibitors are based on unmodified SA. We next hypothesized that development of efficient SA-based binding inhibitors may require detailed knowledge about the structure of the SA-containing receptor. Using a battery of biological and biochemical experiments, including glycan array, binding and infection assays, X-ray crystallography and surface plasmon resonance (SPR); we identified a specific glycan involved in the binding and infection of Ad37. This glycan turned out to be a branched, di-SA-containing motif corresponding to the glycan motif of the ganglioside GD1a. However, the ganglioside itself did not function as a cellular receptor, as shown by a number of binding and infection assays. Instead, the receptor consisted of one or more glycoproteins that contain the GD1a glycan motif. This glycan docked with both its SAs into the trimeric Ad37 knob resulting in a very strong interaction as compared to most other protein-glycan interactions. Hopefully, this finding will aid development of more potent inhibitors of Ad37 binding and infection.

The receptor for CVA24v, one of the main causative agents of AHC, has been unknown until now. We showed that this ocular virus, like Ad37, is also able to use SA as a receptor on corneal cells but not on conjunctival cells. This suggested that CVA24v may use two different receptors. As for Ad37, the receptor used by CVA24v on corneal cells also appears to be one or more sialic acid-containing glycoproteins. We believe that these findings may be a starting point for design and development of candidate drugs for topical treatment of AHC.

Place, publisher, year, edition, pages
Umeå: Umeå university, 2011. 123 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1414
Keyword
Adenovirus, picornavirus, receptors, antivirals
National Category
Microbiology in the medical area
Research subject
Molecular Biology; Infectious Diseases; Microbiology
Identifiers
urn:nbn:se:umu:diva-42818 (URN)978-91-7459-182-8 (ISBN)
Public defence
2011-05-13, Major Groove, by 6L, Norrlands universitetssjukhus, Umeå, 13:00 (English)
Opponent
Supervisors
Available from: 2011-04-15 Created: 2011-04-13 Last updated: 2011-04-20Bibliographically approved
2. Early host cell interactions and antivirals against ocular adenoviruses
Open this publication in new window or tab >>Early host cell interactions and antivirals against ocular adenoviruses
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Alternative title[sv]
Tidiga värd cells interaktioner och antiviraler mot okulära adenovirus
Abstract [en]

Viruses are common causative agents of ocular infection among humans. Epidemic keratoconjuntivitis (EKC) is a severe and contagious ocular disease with reported outbreaks worldwide. It is estimated that this disease affects 20-40 million individuals every year, which leads to huge socioeconomic costs for the affected countries. EKC is characterized by keratitis and conjunctivitis but is also associated with pain, edema, lacrimation, and decreased vision that can prolong for months after the infection and in rare cases years. This disease is caused by human adenoviruses (HAdVs), which belong to the family of Adenoviridae. Currently, there is no available treatment against EKC.

EKC is mainly caused by HAdV-8, HAdV-19, HAdV-37, HAdV-53, HAdV-54, and HAdV-56, which belong to species D HAdVs. HAdV-8, HAdV-19 and HAdV-37 have previously been shown to use sialic acid (SA)-containing glycans as cellular receptors to bind to and infect human corneal epithelial (HCE) cells. To characterize the receptor in more detail, we performed a glycan array, which included SA-containing glycans. A branched hexasaccharide terminating with SA in each arm was identified as a candidate receptor. This glycan corresponds to the glycan motif found on a ganglioside, GD1a. By performing a series of biological and biochemical experiments we confirmed the function of the GD1a glycan as a cellular receptor for EKC-causing HAdVs. However, the glycan used as a receptor was linked to plasma membrane protein(s) through O-glycosidic bonds, rather than to a lipid (as in the ganglioside). X-ray crystallography analysis showed that the two terminal SA:s interacted with two of the three previously identified SA-binding sites on the knob domain of the HAdV-37 capsid protein known as the fiber.

Based on the structural features of the GD1a:HAdV-37 knob interaction, we assumed that a three-armed molecule with each arm terminating with SA would be an efficient inhibitor. Such molecules were designed, synthesized and found to efficiently prevent HAdV-37 binding to and infection of corneal cells. These results indicate that trisialic acids-containing compounds may be used for treatment of EKC.

After binding to its primary receptor, most HAdVs have been shown to interact with αVβ3 and αVβ5 integrins to enter human cells. This interaction occurs through the RGD (arginine-alanine-aspartic acid) motif in the capsid protein known as the penton base. However, it was not clear if corneal epithelial cells express αVβ3 and αVβ5 integrins. Thus, to better understand additional early steps of infection by EKC-causing HAdVs, we performed binding and infection competition experiments using human corneal epithelial cells and siRNA, integrin specific antibodies, peptides and RGD-containing ligands indicating that α3, αV, β1 affected HAdV-37 infection of but not binding to HCE cells. We could also see that HAdV-37 co-localize with α3 and αV at after entry into HCE cells. In situ histochemistry confirmed that the expression of α3 and αV in human corneal tissue. Overall, our results suggest that αV and α3 integrins are important for HAdV-37 infection of corneal cells.

Altogether, these results provide further insight into the biology of HAdVs and open up for development of novel antiviral drugs.

Place, publisher, year, edition, pages
Umeå: Umeå University, 2015. 90 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1697
Keyword
Adenovirus, Virus host interactions, Antivirals, Sialic acid, Integrins, Epidemic keratoconjuntivitis
National Category
Microbiology in the medical area
Identifiers
urn:nbn:se:umu:diva-99907 (URN)978-91-7601-211-6 (ISBN)
Public defence
2015-03-13, Hörsal Betula, 6M, Norrlands universitetssjukhus, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2015-02-20 Created: 2015-02-16 Last updated: 2015-02-18Bibliographically approved

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