A Cluster of Basic Amino Acids in the Factor X Serine Protease Mediates Surface Attachment of Adenovirus/FX Complexes

Duffy, Margaret R.; Bradshaw, Angela C.; Parker, Alan L.; McVey, John H.; Baker, Andrew H.

doi:10.1128/jvi.05382-11

Cited by 34 publications

(29 citation statements)

References 15 publications

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“…To exclude the possibility that adenovirus uses CAR to enter hepatocytes in the absence of heparan sulfate, we investigated the ability of the vector AdY477A, in which CAR binding is ablated, to transduce Ext1 HEP A substantial amount of experimental evidence indicates that Ad.FX can bind directly to heparin and to HS (6,23,24,43). Therefore, not surprisingly, heparin injected intravenously into mice immediately prior to Ad5 injection blocks liver uptake (6).…”

Section: Discussionmentioning

confidence: 99%

“…In contrast, Ad5 binding to HSPGs requires the presence of blood coagulation factor X (FX), which binds to the Ad5 hexon when the virus comes in contact with blood (7,(21)(22)(23). The interaction of Ad.FX and HS is mediated by electrostatic interactions between the heparin binding exosite of the FX serine protease domain and the sulfate groups of HS (6,(23)(24)(25). FX is required for Ad5 transduction in vivo in wild-type mice.…”

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confidence: 99%

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Hepatocyte Heparan Sulfate Is Required for Adeno-Associated Virus 2 but Dispensable for Adenovirus 5 Liver Transduction In Vivo

Zaiss

Foley

Lawrence

et al. 2016

J Virol

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Adeno-associated virus 2 (AAV2) and adenovirus 5 (Ad5) are promising gene therapy vectors. Both display liver tropism and are currently thought to enter hepatocytes in vivo through cell surface heparan sulfate proteoglycans (HSPGs). To test directly this hypothesis, we created mice that lack Ext1, an enzyme required for heparan sulfate biosynthesis, in hepatocytes. A better understanding of how viral vectors enter cells in vivo is critical to improve their therapeutic use. Adeno-associated virus 2 (AAV2) and adenovirus 5 (Ad5) vectors have shown promise in clinical trials for treatment of a wide variety of diseases (1, 2). Both vectors, when injected intravenously into mice, exhibit transgene expression in liver (3-5). Heparan sulfate proteoglycans (HSPGs) are the primary receptors currently thought to facilitate AAV2 and Ad5 entry into hepatocytes (6-8).HSPGs are present both on the cell surface and in the extracellular matrix (9, 10). They consist of a protein core posttranslationally modified to contain heparan sulfate (HS) chains (11). HS biosynthesis occurs by polymerization of alternating glucuronic acid and N-acetylglucosamine residues (12-14), catalyzed by an enzyme complex composed of EXT1 and EXT2 (15). EXT1 and EXT2 are essential molecules required for HS synthesis; cells lacking either molecule do not synthesize HS (16,49).AAV2 binds directly to cell surface HSPGs via an HS-binding motif on the virus capsid (3,17,18). AAV capsid modifications that alter the cluster of positive amino acids that constitute the HS binding motif abrogate liver transduction (3,19,20), suggesting that the ability of the capsid to bind to HS is critical for AAV2 liver transduction in vivo. In contrast, Ad5 binding to HSPGs requires the presence of blood coagulation factor X (FX), which binds to the Ad5 hexon when the virus comes in contact with blood (7,(21)(22)(23). The interaction of Ad.FX and HS is mediated by electrostatic interactions between the heparin binding exosite of the FX serine protease domain and the sulfate groups of HS (6,(23)(24)(25). FX is required for Ad5 transduction in vivo in wild-type mice. In the absence of FX, or when viruses with mutant hexon proteins unable to bind FX are used, Ad5 liver transduction is essentially completely abrogated (7, 21-23, 26, 27).

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Hepatocyte Heparan Sulfate Is Required for Adeno-Associated Virus 2 but Dispensable for Adenovirus 5 Liver Transduction In Vivo

Zaiss

Foley

Lawrence

et al. 2016

J Virol

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“…The mechanism is based on the fact that they bind to blood coagulation factor X upon injection into the blood stream. Factor X (bound to the vector particles) mediates a bridging to heparan surface proteoglycans (HSPGs) on the hepatocyte surface, and this triggers the uptake of viral particles by hepatocytes (Waddington et al 2008;Alba et al 2009;Bradshaw et al 2010;Duffy et al 2011). The bioluminescence recording of freely moving mice can only be performed on the dorsal side of animals, since the cage bottom is covered with nontransparent litter.…”

Section: Visualization Of Circadian Liver Gene Expression By Whole-bomentioning

confidence: 99%

Real-time recording of circadian liver gene expression in freely moving mice reveals the phase-setting behavior of hepatocyte clocks

et al. 2013

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The mammalian circadian timing system consists of a master pacemaker in the suprachiasmatic nucleus (SCN) in the hypothalamus, which is thought to set the phase of slave oscillators in virtually all body cells. However, due to the lack of appropriate in vivo recording technologies, it has been difficult to study how the SCN synchronizes oscillators in peripheral tissues. Here we describe the real-time recording of bioluminescence emitted by hepatocytes expressing circadian luciferase reporter genes in freely moving mice. The technology employs a device dubbed RT-Biolumicorder, which consists of a cylindrical cage with reflecting conical walls that channel photons toward a photomultiplier tube. The monitoring of circadian liver gene expression revealed that hepatocyte oscillators of SCN-lesioned mice synchronized more rapidly to feeding cycles than hepatocyte clocks of intact mice. Hence, the SCN uses signaling pathways that counteract those of feeding rhythms when their phase is in conflict with its own phase.

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“…In the FX SP domain, seven basic amino acids (R273, K276, R306, R347, K351, K420, and R424) have been shown to mediate surface attachment of HAdv-FX complexes to HSPGs on hepatocytes (32). Previous analyses identified that K420 and R424 are the most critical residues for heparin binding to FXa, and together with the other charged amino acids, they form the so-called heparin-binding exosite on the surface of the FX SP domain (33).…”

Section: Fvii Binds Hadv5 Hexon In An Altered Orientation Compared Tomentioning

confidence: 99%

Coagulation Factor Binding Orientation and Dimerization May Influence Infectivity of Adenovirus-Coagulation Factor Complexes

Irons

Flatt

Doronin

et al. 2013

J Virol

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d Adenoviruses (Ads) are promising vectors for therapeutic interventions in humans. When injected into the bloodstream, Ad vectors can bind several vitamin K-dependent blood coagulation factors, which contributes to virus sequestration in the liver by facilitating transduction of hepatocytes. Although both coagulation factors FVII and FX bind the hexon protein of human Ad serotype 5 (HAdv5) with a very high affinity, only FX appears to play a role in mediating Ad-hepatocyte transduction in vivo. To understand the discrepancy between efficacy of FVII binding to hexon and its apparently poor capacity for supporting virus cell entry, we analyzed the HAdv5-FVII complex by using high-resolution cryo-electron microscopy (cryo-EM) followed by molecular dynamic flexible fitting (MDFF) simulations. The results indicate that although hexon amino acids T423, E424, and T425, identified earlier as critical for FX binding, are also involved in mediating binding of FVII, the FVII GLA domain sits within the surface-exposed hexon trimer depression in a different orientation from that found for FX. Furthermore, we found that when bound to hexon, two proximal FVII molecules interact via their serine protease (SP) domains and bury potential heparan sulfate proteoglycan (HSPG) receptor binding residues within the dimer interface. In contrast, earlier cryo-EM studies of the Ad-FX interaction showed no evidence of dimer formation. Dimerization of FVII bound to Ad may be a contributing mechanistic factor for the differential infectivity of Ad-FX and Ad-FVII complexes, despite high-affinity binding of both these coagulation factors to the virus. V iral vectors based on adenoviruses (Ads) specific to human and animal species have been adapted widely for gene transfer applications both in vitro and in vivo. Extensive in vitro analyses have revealed a model of Ad cell infection whereby the initial virus attachment to a plasma membrane-localized receptor via the fiber protein is followed by virus penton interaction with integrins that mediate virion internalization into the cell (1, 2). To date, a number of cellular proteins that serve as functional high-affinity virus attachment receptors have been identified. For the most common vectors, based on species C human adenovirus serotype 5 (HAdv5), as well as for HAdv of species A, D, E, and F, it was found that a tight junction protein designated coxsackie and adenovirus receptor (CAR) can serve as a high-affinity attachment receptor (3-5). The species B Ads may utilize CD46 and/or DSG2 proteins to gain entry into host cells (6-8), while several species D Ads may utilize CD46, sialic acid, or GD1a glycan to enter the cell (9, 10). It was also shown that HAdv can bind a variety of integrin classes that interact with a penton RGD amino acid motif to trigger internalization of cell-bound virus particles into the cell (2).Although this canonical pathway of HAdv cell entry operates efficiently in vitro and explains the topology and functional interdependence between viral capsid proteins (11, 12), ...

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A Cluster of Basic Amino Acids in the Factor X Serine Protease Mediates Surface Attachment of Adenovirus/FX Complexes

Cited by 34 publications

References 15 publications

Hepatocyte Heparan Sulfate Is Required for Adeno-Associated Virus 2 but Dispensable for Adenovirus 5 Liver Transduction In Vivo

Hepatocyte Heparan Sulfate Is Required for Adeno-Associated Virus 2 but Dispensable for Adenovirus 5 Liver Transduction In Vivo

Real-time recording of circadian liver gene expression in freely moving mice reveals the phase-setting behavior of hepatocyte clocks

Coagulation Factor Binding Orientation and Dimerization May Influence Infectivity of Adenovirus-Coagulation Factor Complexes

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