腺相关病毒2在体内进行肝脏转导时需要肝细胞硫酸乙酰肝素，但腺病毒5则不需要。
Hepatocyte Heparan Sulfate Is Required for Adeno-Associated Virus 2 but Dispensable for Adenovirus 5 Liver Transduction In Vivo.
作者信息
Zaiss Anne K, Foley Erin M, Lawrence Roger, Schneider Lina S, Hoveida Hamidreza, Secrest Patrick, Catapang Arthur B, Yamaguchi Yu, Alemany Ramon, Shayakhmetov Dmitry M, Esko Jeffrey D, Herschman Harvey R
机构信息
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.
Department of Cellular and Molecular Medicine, University of California San Diego, La Jolla, California, USA.
出版信息
J Virol. 2015 Oct 21;90(1):412-20. doi: 10.1128/JVI.01939-15. Print 2016 Jan 1.
UNLABELLED
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.
IMPORTANCE
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.
未标记
腺相关病毒2（AAV2）和腺病毒5（Ad5）是很有前景的基因治疗载体。两者均表现出肝脏嗜性，目前认为它们在体内通过细胞表面硫酸乙酰肝素蛋白聚糖（HSPG）进入肝细胞。为了直接验证这一假设，我们构建了肝细胞中缺乏硫酸乙酰肝素生物合成所需酶Ext1的小鼠。Ext1（HEP）突变小鼠原代肝细胞中的硫酸乙酰肝素减少了8倍，全肝组织中的硫酸乙酰肝素减少了5倍。条件性肝细胞Ext1基因缺失大大降低了静脉注射后AAV2在肝脏中的转导。Ad5转导需要凝血因子X（FX）；FX与Ad5衣壳六邻体蛋白结合，并将病毒桥接到细胞表面的HSPG。来自Ext1（HEP）小鼠的原代肝细胞中Ad5.FX转导被废除。然而，与AAV2的情况不同，Ext1（HEP）小鼠肝脏中的Ad5转导没有显著降低。FX在Ext1（HEP）小鼠体内对Ad5转导仍然至关重要。我们得出结论，虽然AAV2进入小鼠肝细胞需要HSPG，但HSPG在体内对Ad5肝细胞转导是可有可无的。这项研究重新开启了腺病毒如何在体内进入细胞的问题。
重要性
我们对病毒如何进入细胞以及如何将它们用作治疗疾病的载体的理解，始于识别病毒结合并介导病毒进入的细胞表面受体。目前认为腺相关病毒2和腺病毒5在体内均通过硫酸乙酰肝素蛋白聚糖（HSPG）进入肝细胞。然而，缺乏这些结论的直接证据。本文所展示的实验，其中通过基因手段消除了肝脏硫酸乙酰肝素的合成，表明HSPG在体内不太可能作为肝细胞Ad5受体发挥作用。数据还表明，AAV2对肝细胞转导需要HSPG。这些结果重新开启了体内Ad5受体身份的问题，并强调了通过基因手段证明受体性质的必要性，这对于理解Ad5在体内进入细胞以及优化Ad5载体作为治疗剂都很重要。
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