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Early host cell interactions and antivirals against ocular adenoviruses
Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology. (Niklas Arnberg)ORCID iD: 0000-0002-4440-3181
2015 (English)Doctoral thesis, comprehensive summary (Other academic)Alternative title
Tidiga värd cells interaktioner och antiviraler mot okulära adenovirus (Swedish)
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 [en]
Adenovirus, Virus host interactions, Antivirals, Sialic acid, Integrins, Epidemic keratoconjuntivitis
National Category
Microbiology in the medical area
Identifiers
URN: urn:nbn:se:umu:diva-99907ISBN: 978-91-7601-211-6 (print)OAI: oai:DiVA.org:umu-99907DiVA: diva2:788590
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
List of papers
1. 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, 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.

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: 2017-12-11Bibliographically approved
2. A Potent Trivalent Sialic Acid Inhibitor of Adenovirus Type 37 Infection of Human Corneal Cells
Open this publication in new window or tab >>A Potent Trivalent Sialic Acid Inhibitor of Adenovirus Type 37 Infection of Human Corneal Cells
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2011 (English)In: Angewandte Chemie International Edition, ISSN 1433-7851, E-ISSN 1521-3773, Vol. 50, no 29, 6519-6521 p.Article in journal (Refereed) Published
Place, publisher, year, edition, pages
Wiley, 2011
Keyword
adenoviruses, antiviral agents, crystal-structure elucidation, sialic acids, surface plasmon resonance
National Category
Chemical Sciences
Identifiers
urn:nbn:se:umu:diva-45043 (URN)10.1002/anie.201101559 (DOI)21648036 (PubMedID)
Available from: 2011-06-22 Created: 2011-06-20 Last updated: 2017-12-11Bibliographically approved
3. Triazole linker-based trivalent sialic acid inhibitors of adenovirus type 37 infection of human corneal epithelial cells
Open this publication in new window or tab >>Triazole linker-based trivalent sialic acid inhibitors of adenovirus type 37 infection of human corneal epithelial cells
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2015 (English)In: Organic and biomolecular chemistry, ISSN 1477-0520, E-ISSN 1477-0539, Vol. 13, no 35, 9194-9205 p.Article in journal (Refereed) Published
Abstract [en]

Adenovirus type 37 (Ad37) is one of the principal agents responsible for epidemic keratoconjunctivitis (EKC), a severe ocular infection that remains without any available treatment. Recently, a trivalent sialic acid derivative (ME0322, Angew. Chem. Int. Ed., 2011, 50, 6519) was shown to function as a highly potent inhibitor of Ad37, efficiently preventing the attachment of the virion to the host cells and subsequent infection. Here, new trivalent sialic acid derivatives were designed, synthesized and their inhibitory properties against Ad37 infection of the human corneal epithelial cells were investigated. In comparison to ME0322, the best compound (17a) was found to be over three orders of magnitude more potent in a cell-attachment assay (IC50 = 1.4 nM) and about 140 times more potent in a cell-infection assay (IC50 = 2.9nM). X-ray crystallographic analysis demonstrated a trivalent binding mode of all compounds to the Ad37 fiber knob. For the most potent compound ophthalmic toxicity in rabbits was investigated and it was concluded that repeated eye administration did not cause any adverse effects.

National Category
Microbiology in the medical area
Identifiers
urn:nbn:se:umu:diva-100014 (URN)10.1039/C5OB01025J (DOI)000360115100007 ()
Available from: 2015-02-18 Created: 2015-02-18 Last updated: 2017-12-04Bibliographically approved
4. Involvement of corneal integrins during infection of human adenovirus type 37
Open this publication in new window or tab >>Involvement of corneal integrins during infection of human adenovirus type 37
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(English)Manuscript (preprint) (Other academic)
National Category
Microbiology in the medical area
Identifiers
urn:nbn:se:umu:diva-100015 (URN)
Available from: 2015-02-18 Created: 2015-02-18 Last updated: 2015-02-18Bibliographically approved

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