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      Requirements for Receptor Engagement during Infection by Adenovirus Complexed with Blood Coagulation Factor X

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          Abstract

          Human adenoviruses from multiple species bind to coagulation factor X (FX), yet the importance of this interaction in adenovirus dissemination is unknown. Upon contact with blood, vectors based on adenovirus serotype 5 (Ad5) binds to FX via the hexon protein with nanomolar affinity, leading to selective uptake of the complex into the liver and spleen. The Ad5:FX complex putatively targets heparan sulfate proteoglycans (HSPGs). The aim of this study was to elucidate the specific requirements for Ad5:FX-mediated cellular uptake in this high-affinity pathway, specifically the HSPG receptor requirements as well as the role of penton base-mediated integrin engagement in subsequent internalisation. Removal of HS sidechains by enzymatic digestion or competition with highly-sulfated heparins/heparan sulfates significantly decreased FX-mediated Ad5 cell binding in vitro and ex vivo. Removal of N-linked and, in particular, O-linked sulfate groups significantly attenuated the inhibitory capabilities of heparin, while the chemical inhibition of endogenous HSPG sulfation dose-dependently reduced FX-mediated Ad5 cellular uptake. Unlike native heparin, modified heparins lacking O- or N-linked sulfate groups were unable to inhibit Ad5 accumulation in the liver 1h after intravascular administration of adenovirus. Similar results were observed in vitro using Ad5 vectors possessing mutations ablating CAR- and/or αv integrin binding, demonstrating that attachment of the Ad5:FX complex to the cell surface involves HSPG sulfation. Interestingly, Ad5 vectors ablated for αv integrin binding showed markedly delayed cell entry, highlighting the need for an efficient post-attachment internalisation signal for optimal Ad5 uptake and transport following surface binding mediated through FX. This study therefore integrates the established model of αv integrin-dependent adenoviral infection with the high-affinity FX-mediated pathway. This has important implications for mechanisms that define organ targeting following contact of human adenoviruses with blood.

          Author Summary

          Adenoviruses can infect many cell types and cause a range of illnesses in humans, including respiratory, ocular and gastrointestinal disorders. These illnesses are rarely fatal; however, in immunocompromised individuals, especially young children, disseminated adenovirus infections can cause serious and life-threatening complications. Studies have shown that several adenoviruses including vectors based on adenovirus serotype 5 (Ad5) bind to coagulation factor X (FX) in the bloodstream. Ad5 uses the high-affinity interaction with FX to putatively bind to heparan sulfate proteoglycans (HSPGs). However, very little is known about this infection pathway. Here we demonstrate that interaction of Ad5:FX with HSPGs is solely via the HS sidechains of these ubiquitously-expressed molecules. We further show that this interaction is dependent on HS sulfation, in particular O-sulfation. Although attachment of Ad5:FX to HSPGs is independent of the coxsackievirus and adenovirus receptor (CAR) or αv integrins, efficient and rapid intracellular transport of Ad5 retains a dependence on engagement of αv integrins via the penton base protein. This is the first study to characterise the receptor requirements for cell uptake via the recently-identified, FX-mediated infection pathway, which may be of significance for the development of therapies against disseminated adenoviral disease.

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          Most cited references 71

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          Isolation of a common receptor for Coxsackie B viruses and adenoviruses 2 and 5.

          A complementary DNA clone has been isolated that encodes a coxsackievirus and adenovirus receptor (CAR). When transfected with CAR complementary DNA, nonpermissive hamster cells became susceptible to coxsackie B virus attachment and infection. Furthermore, consistent with previous studies demonstrating that adenovirus infection depends on attachment of a viral fiber to the target cell, CAR-transfected hamster cells bound adenovirus in a fiber-dependent fashion and showed a 100-fold increase in susceptibility to virus-mediated gene transfer. Identification of CAR as a receptor for these two unrelated and structurally distinct viral pathogens is important for understanding viral pathogenesis and has implications for therapeutic gene delivery with adenovirus vectors.
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            Heparan sulphate proteoglycans fine-tune mammalian physiology.

            Heparan sulphate proteoglycans reside on the plasma membrane of all animal cells studied so far and are a major component of extracellular matrices. Studies of model organisms and human diseases have demonstrated their importance in development and normal physiology. A recurrent theme is the electrostatic interaction of the heparan sulphate chains with protein ligands, which affects metabolism, transport, information transfer, support and regulation in all organ systems. The importance of these interactions is exemplified by phenotypic studies of mice and humans bearing mutations in the core proteins or the biosynthetic enzymes responsible for assembling the heparan sulphate chains.
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              Integrins alpha v beta 3 and alpha v beta 5 promote adenovirus internalization but not virus attachment.

              Adenovirus contains a heterodimeric protein complex consisting of 186 kd fiber protein that mediates high affinity virus attachment to cells and a 400 kd pentavalent subunit (penton base) that contains five Arg-Gly-Asp sequences, implying a role for integrins in adenovirus infection. We demonstrate that the vitro-nectin-binding integrins alpha v beta 3 and alpha v beta 5 promote viral infection in a novel way since antibodies against these receptors or soluble penton base block virus internalization without affecting attachment. Moreover, adenovirus binds to cultured cells lacking alpha v integrins but fail to become internalized, thus restricting infection of these cells. Transfection of alpha v(-) cells with a cDNA encoding alpha v results in the expression of integrins alpha v beta 3 and alpha v beta 5 and allows virus internalization and infection. These data indicate that adenovirus attachment and uptake into cells are separate but cooperative events that result from the interaction of distinct viral coat proteins with a receptor for attachment and alpha v integrin receptors for internalization.
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                Author and article information

                Affiliations
                [1 ]Institute of Cardiovascular and Medical Sciences, British Heart Foundation Glasgow Cardiovascular Research Centre, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom
                [2 ]Department of Molecular Cell Biology, Vrije Universiteit Medical Center (VUMC), Amsterdam, The Netherlands
                [3 ]Department of Dermatology, University of Turku and Turku University Central Hospital, Turku, Finland
                University of Michigan, United States of America
                Author notes

                Conceived and designed the experiments: ACB ALP SAN AHB. Performed the experiments: ACB ALP MRD LC. Analyzed the data: ACB ALP MRD AHB. Contributed reagents/materials/analysis tools: NvR VMK. Wrote the paper: ACB SAN AHB.

                Contributors
                Role: Editor
                Journal
                PLoS Pathog
                plos
                plospath
                PLoS Pathogens
                Public Library of Science (San Francisco, USA )
                1553-7366
                1553-7374
                October 2010
                October 2010
                7 October 2010
                : 6
                : 10
                2951380
                20949078
                10-PLPA-RA-2964R3
                10.1371/journal.ppat.1001142
                (Editor)
                Bradshaw et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
                Counts
                Pages: 17
                Categories
                Research Article
                Virology/Host Invasion and Cell Entry
                Virology/Virion Structure, Assembly, and Egress

                Infectious disease & Microbiology

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