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Coagulation factors IX and X enhance binding and infection of adenovirus types 5 and 31 in human epithelial cells
Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology.
Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology.
Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology.
Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology.
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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.

Place, publisher, year, edition, pages
2009. Vol. 83, no 8, 3816-3825 p.
National Category
Microbiology in the medical area
Identifiers
URN: urn:nbn:se:umu:diva-21099DOI: 10.1128/JVI.02562-08PubMedID: 19158249OAI: oai:DiVA.org:umu-21099DiVA: diva2:210597
Available from: 2009-04-02 Created: 2009-04-02 Last updated: 2017-12-13Bibliographically approved
In thesis
1. Mechanisms involved in adenovirus binding to and infection of host cells
Open this publication in new window or tab >>Mechanisms involved in adenovirus binding to and infection of host cells
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The adenovirus (Ad) family consists of 52 different human types, which are divided into seven species (A-G). Human Ads cause disease in the respiratory tract, lymphoid tissue, intestine, urinary tract, and/or in the eye. Most, but not all Ads have been demonstrated to use the coxsackie-adenovirus receptor (CAR) as an efficient receptor in vitro, but CAR has been questioned as an in vivo-receptor for various reasons. Thus, there are reasons to believe that Ads use other mechanisms for binding to target cells. In an attempt to investigate the impact of tear fluid during in vitro infection of ocular Ads (i.e. Ad37), using corneal cells, we found that human tear fluid promoted infection of an Ad with pronounced respiratory tropism (i.e. Ad5) used here as a control, but surprisingly not of Ad37. Furthermore using a virus overlay protein blotting assay we found that Ad5 bound to several tear fluid proteins. One of these, human lactoferrin (hLf) which is a component that belongs to the innate immune system in various body fluids, was alone able to promote both binding and infection of all species C Ads (Ad1, Ad2, Ad5, Ad6) in epithelial cells. hLf was also found to promote gene delivery (GFP) from an Ad5-based vector. Further we have identified lactoferricin (Lfcin), the N-terminal part of hLf, as to be responsible for this effect. We also show that plasma, saliva, and tear fluid promote infection of Ad5 in respiratory and ocular epithelial cells, and that plasma promotes infection of Ad31. The component in plasma that is responsible for this effect is likely to be coagulation factor IX (FIX) and X (FX), since both these factors were able to promote binding and infection of Ad5 and/or Ad31 in epithelial cells. Finally, we show that the excess of fiber production from initial Ad infection and the release of fibers before the particle itself is released caused masking of the tropism-specific receptors in both infected and non-infected surrounding cells. This means that the overproduction of fibers affects the ability of Ad to spread within tissues.

We conclude that soluble components in body fluids, such as hLf, FIX, and FX have the ability to mediate binding and infection of selected human Ads (species C and Ad31) in epithelial cells that represent the tropism of these Ads. We suggest that these components may serve as bridges between the virion and the cell surface. This is contributes to the knowledge about Ad lifecycle, and might help to improve the de-/retargeting of gene therapy based on Ad vectors.

Place, publisher, year, edition, pages
Umeå: Umeå university, 2009. 71 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1301
Keyword
Adenovirus, binding, infection, lactoferrin, coagulation factor IX and X, fiber
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:umu:diva-27646 (URN)978-91-7264-876-0 (ISBN)
Public defence
2009-12-04, Betula, Byggnad 6M, NUS, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2009-11-16 Created: 2009-11-12 Last updated: 2011-04-07Bibliographically approved
2. Soluble components in body fluids as mediators of adenovirus infections
Open this publication in new window or tab >>Soluble components in body fluids as mediators of adenovirus infections
2013 (English)Licentiate thesis, comprehensive summary (Other academic)
Place, publisher, year, edition, pages
Umeå: Umeå universitet, 2013. 43 p.
National Category
Microbiology in the medical area
Research subject
Medical Virology
Identifiers
urn:nbn:se:umu:diva-70173 (URN)978-91-7459-661-8 (ISBN)
Presentation
2013-05-17, A103, NUS, Umeå, 13:00 (English)
Opponent
Supervisors
Available from: 2013-08-05 Created: 2013-05-06 Last updated: 2013-08-05Bibliographically approved
3. Adenovirus-host interactions: implications for tropism and therapy
Open this publication in new window or tab >>Adenovirus-host interactions: implications for tropism and therapy
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
Adenoviridae, Amino Acids, Antiviral Agents, Epithelial Cells, Factor IX
National Category
Microbiology in the medical area
Identifiers
urn:nbn:se:umu:diva-119850 (URN)978-91-7601-453-0 (ISBN)
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

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