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Cellular receptors for viruses with ocular tropism
Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology. (Arnberg)
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 [en]
Adenovirus, picornavirus, receptors, antivirals
National Category
Microbiology in the medical area
Research subject
Molecular Biology; Infectious Diseases; Microbiology
Identifiers
URN: urn:nbn:se:umu:diva-42818ISBN: 978-91-7459-182-8OAI: oai:DiVA.org:umu-42818DiVA: diva2:410465
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
List of papers
1. Multivalent sialic acid conjugates inhibit adenovirus type 37 from binding to and infecting human corneal epithelial cells
Open this publication in new window or tab >>Multivalent sialic acid conjugates inhibit adenovirus type 37 from binding to and infecting human corneal epithelial cells
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2007 (English)In: Antiviral Research, ISSN 0166-3542, E-ISSN 1872-9096, Vol. 73, no 2, 92-100 p.Article in journal (Refereed) Published
Abstract [en]

Adenovirus type 37 is one of the main causative agents of epidemic keratoconjunctivitis. In a series of publications, we have reported that this virus uses sialic acid as a cellular receptor. Here we demonstrate in vitro that on a molar basis, multivalent sialic acid conjugated to human serum albumin prevents adenovirus type 37 from binding to and infecting human corneal epithelial cells 1000-fold more efficiently than monosaccharidic sialic acid. We also demonstrate that the extraordinary inhibitory effect of multivalent sialic acid is due to the ability of this compound to aggregate virions. We conclude that multivalent sialic acid may be a potential new antiviral drug, for use in the treatment of epidemic keratoconjunctivitis caused by the adenoviruses that use sialic acid as cellular receptor.

Keyword
Adenovirus, EKC, Sialic acid, Multivalent
National Category
Microbiology in the medical area
Identifiers
urn:nbn:se:umu:diva-12970 (URN)10.1016/j.antiviral.2006.08.004 (DOI)
Available from: 2007-05-11 Created: 2007-05-11 Last updated: 2011-04-18Bibliographically approved
2. Design, synthesis, and evaluation of N-acyl modified sialic acids as inhibitors of adenoviruses causing epidemic keratoconjunctivitis
Open this publication in new window or tab >>Design, synthesis, and evaluation of N-acyl modified sialic acids as inhibitors of adenoviruses causing epidemic keratoconjunctivitis
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2009 (English)In: Journal of Medicinal Chemistry, ISSN 0022-2623, E-ISSN 1520-4804, Vol. 52, no 12, 3666-3678 p.Article in journal (Refereed) Published
Abstract [en]

The adenovirus serotype Ad37 binds to and infects human corneal epithelial (HCE) cells through attachment to cellular glycoproteins carrying terminal sialic acids. By use of the crystallographic structure of the sialic acid-interacting domain of the Ad37 fiber protein in complex with sialyllactose, a set of N-acyl modified sialic acids were designed to improve binding affinity through increased hydrophobic interactions. These N-acyl modified sialic acids and their corresponding multivalent human serum albumin (HSA) conjugates were synthesized and tested in Ad37 cell binding and cell infectivity assays. Compounds bearing small substituents were as effective inhibitors as sialic acid. X-ray crystallography and overlays with the Ad37-sialyllactose complex showed that the N-acyl modified sialic acids were positioned in the same orientation as sialic acid. Their multivalent counterparts achieved a strong multivalency effect and were more effective to prevent infection than the monomers. Unfortunately, they were less active as inhibitors than multivalent sialic acid.

National Category
Microbiology in the medical area
Identifiers
urn:nbn:se:umu:diva-42373 (URN)10.1021/jm801609s (DOI)19456100 (PubMedID)
Available from: 2011-04-07 Created: 2011-04-07 Last updated: 2011-04-18Bibliographically approved
3. Sialic acid is a cellular receptor for coxsackievirus A24 variant, an emerging virus with pandemic potential
Open this publication in new window or tab >>Sialic acid is a cellular receptor for coxsackievirus A24 variant, an emerging virus with pandemic potential
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2008 (English)In: Journal of Virology, ISSN 0022-538X, E-ISSN 1098-5514, Vol. 82, no 6, 3061-3068 p.Article in journal (Refereed) Published
Abstract [en]

Binding to target cell receptors is a critical step in the virus life cycle. Coxsackievirus A24 variant (CVA24v) has pandemic potential and is a major cause of acute hemorrhagic conjunctivitis, but its cellular receptor has hitherto been unknown. Here we show that CVA24v fails to bind to and infect CHO cells defective in sialic acid expression. Binding of CVA24v to and infection of corneal epithelial cells are efficiently inhibited by treating cells with a sialic acid-cleaving enzyme or sialic acid-binding lectins and by treatment of the virus with soluble, multivalent sialic acid. Protease treatment of cells efficiently inhibited virus binding, suggesting that the receptor is a sialylated glycoprotein. Like enterovirus type 70 and influenza A virus, CVA24v can cause pandemics. Remarkably, all three viruses use the same receptor. Since several unrelated viruses with tropism for the eye use this receptor, sialic acid-based antiviral drugs that prevent virus entry may be useful for topical treatment of such infections.

Place, publisher, year, edition, pages
Baltimore: American Society for Microbiology, 2008
National Category
Microbiology in the medical area
Identifiers
urn:nbn:se:umu:diva-21102 (URN)10.1128/JVI.02470-07 (DOI)18184708 (PubMedID)
Available from: 2009-04-02 Created: 2009-04-02 Last updated: 2011-04-15Bibliographically approved
4. The GD1a glycan is a cellular receptor for adenoviruses causing epidemic keratoconjunctivitis (Letter)
Open this publication in new window or tab >>The GD1a glycan is a cellular receptor for adenoviruses causing epidemic keratoconjunctivitis (Letter)
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2011 (English)In: Nature Medicine, ISSN 1078-8956, 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.

National Category
Microbiology in the medical area
Identifiers
urn:nbn:se:umu:diva-38769 (URN)10.1038/nm.2267 (DOI)21151139 (PubMedID)
Available from: 2011-01-03 Created: 2010-12-28 Last updated: 2015-02-18Bibliographically approved

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