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Adenovirus-host interactions: implications for tropism and therapy
Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Human adenoviruses (HAdVs) are common viruses often associated withgastrointestinal, ocular and respiratory infections. They can infect a widevariety of cells, both dividing and non-dividing. HAdVs attach to and infecttarget cells through interactions with cellular receptors. It has also beenshown that HAdVs can use soluble host components in body fluids forindirect binding to target cells, a feature that enables the usage of new typesof receptors resulting in a more efficient HAdV infection. We thereforeevaluated the influence of soluble components from four different bodyfluids on HAdV infection of epithelial cells, representing the respiratory andocular tropism of most HAdVs. We found that plasma, saliva, and tear fluidpromote binding and infection of HAdV-5 (species C) and that plasmapromotes infection of HAdV-31 (species A). Further binding and infectionexperiments identified coagulation factor IX (FIX) and X (FX) as thecomponents of plasma responsible for increase of HAdV-5 infection whileFIX alone mediates increase of HAdV-31 infection. We found that as little as1% of the physiological concentration of these factors is required to facilitatemaximum binding.

The effect of coagulation factors on HAdV infection was thereafterextended to include all species A HAdVs: HAdV-12, -18 and -31. Species AHAdVs normally cause infections involving the airways and/or the intestine.These infections are often mild but species A HAdVs in general, and HAdV-31 in particular, have been shown to cause severe and life-threateninginfections in immunocompromised patients. We show here that FIXefficiently increase HAdV-18 and -31 (but not HAdV-12) binding andinfection of human epithelial cells, representing the respiratory andgastrointestinal tropism. FIX was shown to interact with the hexon proteinof HAdV-31 and surface plasmon resonance analysis revealed that theHAdV-31:FIX interaction is slightly stronger than that of the HAdV-5:FIX/FX interactions, but more interestingly, the half-lives of theseinteractions are profoundly different. By performing binding and infectionexperiments using cells expressing specific glycosaminoglycans (GAGs) and ivGAG-cleaving enzymes we found that the HAdV-31:FIX and HAdV-5:FIX/FX complexes bind to heparan sulfate-containing GAGs on targetcells, but we could also see a difference in GAG dependence and specificitybetween these complexes.We conclude that the use of coagulation factors might be of moreimportance than previously recognized and that this may affect not only theliver tropism seen when administering adenovirus vectors into thecirculation but also regulate primary infections by wild-type viruses of theirnatural target cells. We also believe that our findings may contribute tobetter design of HAdV-based vectors for gene and cancer therapy and thatthe interaction between the HAdV-31 hexon and FIX may serve as a targetfor antiviral treatment.

HAdV vectors are mainly based on HAdV-5 and several problems haverecently become evident when using these vectors. Major challenges withHAdV-5 based vectors include pre-existing neutralizing antibodies, pooraccess to the receptor CAR (coxsackie and adenovirus receptor), and offtarget effects to the liver due to interactions with coagulation factors. Theneed for new HAdV vectors devoid of these problems is evident.HAdV-52 is one of only three HAdVs that are equipped with two differentfiber proteins, one long and one short. We show here, by means of bindingand infection experiments, that HAdV-52 can use CAR as a cellular receptor,but that most of the binding is dependent on sialic acid-containingglycoproteins. Flow cytometry, ELISA and surface plasmon resonanceanalyses revealed that the terminal knob domain of the long fiber (52LFK)binds to CAR, and the knob domain of the short fiber (52SFK) binds tosialylated glycoproteins. X-ray crystallographic analysis of 52SFK in complexwith sialic acid revealed a new sialic acid binding site compared to otherknown adenovirus:glycan interactions. Moreover, glycan array analysisidentified α2,8-linked oligosialic acid, mimicking the naturally occurringpolysialic acid (PSia), as a potential sialic acid-containing glycan receptor for52SFK. ELISA and surface plasmon resonance confirmed the ability of52SFK to interact with PSia. Flow cytometry analysis also showed a fivefold vincrease in binding of 52SFK to PSia-expressing cells compared to controlcells. X-ray crystallographic analysis of 52SFK in complex with oligo-PSiarevealed engagement at the non-reducing end of oligo-PSia to the canonicalsialic acid-binding site, but also suggested the presence of a 'steering rim'consisting of positively charged amino acids contributing to the contact bylong-range electrostatic interactions.

PSia is nearly absent on cells in healthy adults but can be expressed inhigh amounts on several types of cancers including: glioma, neuroblastomaand lung cancer. We show here that the short fiber of HAdV-52 bindsspecifically to PSia. Taking into account that HAdV-52 has a supposedly lowseroprevalence and is incapable of interacting with coagulation factors webelieve that HAdV-52 based vectors can be useful for treatment of cancertypes with elevated PSia expression.

Place, publisher, year, edition, pages
Umeå: Umeå universitet , 2016. , 64 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1798
Keyword [en]
Adenoviridae, Amino Acids, Antiviral Agents, Epithelial Cells, Factor IX
National Category
Microbiology in the medical area
Identifiers
URN: urn:nbn:se:umu:diva-119850ISBN: 978-91-7601-453-0 (print)OAI: oai:DiVA.org:umu-119850DiVA: diva2:924985
Public defence
2016-05-27, Biomedicinhuset, sal E04, Norrlands universitetssjukhus, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2016-05-04 Created: 2016-04-29 Last updated: 2016-05-26Bibliographically approved
List of papers
1. Coagulation factors IX and X enhance binding and infection of adenovirus types 5 and 31 in human epithelial cells
Open this publication in new window or tab >>Coagulation factors IX and X enhance binding and infection of adenovirus types 5 and 31 in human epithelial cells
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2009 (English)In: Journal of Virology, ISSN 0022-538X, E-ISSN 1098-5514, Vol. 83, no 8, 3816-3825 p.Article in journal (Refereed) Published
Abstract [en]

Most adenoviruses bind directly to the coxsackie and adenovirus receptor (CAR) on target cells in vitro, but recent research has shown that adenoviruses can also use soluble components in body fluids for indirect binding to target cells. These mechanisms have been identified upon addressing the questions of how to de- and retarget adenovirus-based vectors for human gene and cancer therapy, but the newly identified mechanisms also suggest that the role of body fluids and their components may also be of importance for natural, primary infections. Here we demonstrate that plasma, saliva, and tear fluid promote binding and infection of adenovirus type 5 (Ad5) in respiratory and ocular epithelial cells, which corresponds to the natural tropism of most adenoviruses, and that plasma promotes infection by Ad31. By using a set of binding and infection experiments, we also found that Ad5 and Ad31 require coagulation factors IX (FIX) or X (FX) or just FIX, respectively, for efficient binding and infection. The concentrations of these factors that were required for maximum binding were 1/100th of the physiological concentrations. Preincubation of virions with heparin or pretreatment of cells with heparinase I indicated that the role of cell surface heparan sulfate during FIX- and FX-mediated adenovirus binding and infection is mechanistically serotype specific. We conclude that the use of coagulation factors by adenoviruses may be of importance not only for the liver tropism seen when administering adenovirus vectors to the circulation but also during primary infections by wild-type viruses of their natural target cell types.

National Category
Microbiology in the medical area
Identifiers
urn:nbn:se:umu:diva-21099 (URN)10.1128/JVI.02562-08 (DOI)19158249 (PubMedID)
Available from: 2009-04-02 Created: 2009-04-02 Last updated: 2017-12-13Bibliographically approved
2. Coagulation factor IX mediates serotype-specific binding of species A adenoviruses to host cells
Open this publication in new window or tab >>Coagulation factor IX mediates serotype-specific binding of species A adenoviruses to host cells
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2011 (English)In: Journal of Virology, ISSN 0022-538X, E-ISSN 1098-5514, Vol. 85, no 24, 13420-13431 p.Article in journal (Refereed) Published
Abstract [en]

Human species A adenoviruses (HAdVs) comprise three serotypes: HAdV-12, -18, and -31. These viruses are common pathogens and cause systemic infections that usually involve the airways and/or intestine. In immunocompromised individuals, species A adenoviruses in general, and HAdV-31 in particular, cause life-threatening infections. By combining binding and infection experiments, we demonstrate that coagulation factor IX (FIX) efficiently enhances binding and infection by HAdV-18 and HAdV-31, but not by HAdV-12, in epithelial cells originating from the airways or intestine. This is markedly different from the mechanism for HAdV-5 and other human adenoviruses, which utilize coagulation factor X (FX) for infection of host cells. Surface plasmon resonance experiments revealed that the affinity of the HAdV-31 hexon-FIX interaction is higher than that of the HAdV-5 hexon-FX interaction and that the half-lives of these interactions are profoundly different. Moreover, both HAdV-31-FIX and HAdV-5-FX complexes bind to heparan sulfate-containing glycosaminoglycans (GAGs) on target cells, but binding studies utilizing cells expressing specific GAGs and GAG-cleaving enzymes revealed differences in GAG dependence and specificity between these two complexes. These findings add to our understanding of the intricate infection pathways used by human adenoviruses, and they may contribute to better design of HAdV-based vectors for gene and cancer therapy. Furthermore, the interaction between the HAdV-31 hexon and FIX may also serve as a target for antiviral treatment.

Place, publisher, year, edition, pages
Washington: The American Society For Microbiology, 2011
National Category
Microbiology in the medical area
Identifiers
urn:nbn:se:umu:diva-50925 (URN)10.1128/JVI.06088-11 (DOI)000297642000058 ()
Available from: 2012-01-20 Created: 2012-01-02 Last updated: 2017-12-08Bibliographically approved
3. Human Adenovirus 52 Uses Sialic Acid-containing Glycoproteins and the Coxsackie and Adenovirus Receptor for Binding to Target Cells
Open this publication in new window or tab >>Human Adenovirus 52 Uses Sialic Acid-containing Glycoproteins and the Coxsackie and Adenovirus Receptor for Binding to Target Cells
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2015 (English)In: PLoS Pathogens, ISSN 1553-7366, E-ISSN 1553-7374, Vol. 11, no 2, e1004657Article in journal (Refereed) Published
Abstract [en]

Most adenoviruses attach to host cells by means of the protruding fiber protein that binds to host cells via the coxsackievirus and adenovirus receptor (CAR) protein. Human adenovirus type 52 (HAdV-52) is one of only three gastroenteritis-causing HAdVs that are equipped with two different fiber proteins, one long and one short. Here we show, by means of virion-cell binding and infection experiments, that HAdV-52 can also attach to host cells via CAR, but most of the binding depends on sialylated glycoproteins. Glycan microarray, flow cytometry, surface plasmon resonance and ELISA analyses reveal that the terminal knob domain of the long fiber (52LFK) binds to CAR, and the knob domain of the short fiber (52SFK) binds to sialylated glycoproteins. X-ray crystallographic analysis of 52SFK in complex with 2-O-methylated sialic acid combined with functional studies of knob mutants revealed a new sialic acid binding site compared to other, known adenovirus: glycan interactions. Our findings shed light on adenovirus biology and may help to improve targeting of adenovirus-based vectors for gene therapy.

National Category
Microbiology in the medical area
Identifiers
urn:nbn:se:umu:diva-103565 (URN)10.1371/journal.ppat.1004657 (DOI)000352083400038 ()25674795 (PubMedID)
Available from: 2015-05-25 Created: 2015-05-21 Last updated: 2017-12-04Bibliographically approved
4. Human adenovirus 52 short fiber binds to polysialic acid
Open this publication in new window or tab >>Human adenovirus 52 short fiber binds to polysialic acid
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(English)Manuscript (preprint) (Other academic)
National Category
Microbiology
Identifiers
urn:nbn:se:umu:diva-119848 (URN)
Available from: 2016-04-29 Created: 2016-04-29 Last updated: 2016-11-22

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