Hepatocyte Heparan Sulfate Is Required for Adeno-Associated Virus 2 but Dispensable for Adenovirus 5 Liver Transduction In Vivo.
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. Ext1(HEP) mutant mice exhibit an 8-fold reduction of heparan sulfate in primary hepatocytes and a 5-fold reduction of heparan sulfate in whole liver tissue. Conditional hepatocyte Ext1 gene deletion greatly reduced AAV2 liver transduction following intravenous injection. Ad5 transduction requires blood coagulation factor X (FX); FX binds to the Ad5 capsid hexon protein and bridges the virus to HSPGs on the cell surface. Ad5.FX transduction was abrogated in primary hepatocytes from Ext1(HEP) mice. However, in contrast to the case with AAV2, Ad5 transduction was not significantly reduced in the livers of Ext1(HEP) mice. FX remained essential for Ad5 transduction in vivo in Ext1(HEP) mice. We conclude that while AAV2 requires HSPGs for entry into mouse hepatocytes, HSPGs are dispensable for Ad5 hepatocyte transduction in vivo. This study reopens the question of how adenovirus enters cells in vivo.


OBJECTIVE
Our understanding of how viruses enter cells, and how they can be used as therapeutic vectors to manage disease, begins with identification of the cell surface receptors to which viruses bind and which mediate viral entry. Both adeno-associated virus 2 and adenovirus 5 are currently thought to enter hepatocytes in vivo through heparan sulfate proteoglycans (HSPGs). However, direct evidence for these conclusions is lacking. Experiments presented herein, in which hepatic heparan sulfate synthesis was genetically abolished, demonstrated that HSPGs are not likely to function as hepatocyte Ad5 receptors in vivo. The data also demonstrate that HSPGs are required for hepatocyte transduction by AAV2. These results reopen the question of the identity of the Ad5 receptor in vivo and emphasize the necessity of demonstrating the nature of the receptor by genetic means, both for understanding Ad5 entry into cells in vivo and for optimization of Ad5 vectors as therapeutic agents.
Department of Biological Chemistry, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California, USA Department of Medical and Molecular Pharmacology, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California, USA.
Anne K
AK
Zaiss
Department of Cellular and Molecular Medicine, University of California San Diego, La Jolla, California, USA.
Erin M
EM
Foley
Department of Cellular and Molecular Medicine, University of California San Diego, La Jolla, California, USA.
Roger
R
Lawrence
Department of Biological Chemistry, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California, USA Department of Medical and Molecular Pharmacology, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California, USA.
Lina S
LS
Schneider
Department of Biological Chemistry, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California, USA Department of Medical and Molecular Pharmacology, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California, USA.
Hamidreza
H
Hoveida
Department of Cellular and Molecular Medicine, University of California San Diego, La Jolla, California, USA.
Patrick
P
Secrest
Department of Biological Chemistry, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California, USA Department of Medical and Molecular Pharmacology, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California, USA.
Arthur B
AB
Catapang
Sanford-Burnham Medical Research Institute, La Jolla, California, USA.
Yu
Y
Yamaguchi
Translational Research Laboratory, IDIBELL-Institut Catala d'Oncologia, Barcelona, Spain.
Ramon
R
Alemany
Department of Pediatrics and Medicine, Emory University, Atlanta, Georgia, USA.
Dmitry M
DM
Shayakhmetov
Department of Cellular and Molecular Medicine, University of California San Diego, La Jolla, California, USA.
Jeffrey D
JD
Esko
Department of Biological Chemistry, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California, USA Department of Medical and Molecular Pharmacology, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California, USA hherschman@mednet.ucla.edu.
Harvey R
HR
Herschman
D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D002478:Cells, Cultured; D000229:Dependovirus / Q000235:genetics*; D005260:Female; D005822:Genetic Vectors; D006497:Heparan Sulfate / Q000378:metabolism*; D022781:Hepatocytes / Q000737:chemistry / Q000821:virology*; D008099:Liver / Q000737:chemistry / Q000821:virology*; D008297:Male; D051379:Mice; D011991:Receptors, Virus / Q000378:metabolism*; D014161:Transduction, Genetic
D006497:Heparan Sulfate; D011991:Receptors, Virus