Adeno-associated Virus (AAV) Serotypes Have Distinctive Interactions with Domains of the Cellular AAV Receptor
Adeno-associated Virus (AAV) Serotypes Have Distinctive Interactions with Domains of the Cellular AAV Receptor
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DOI：
10.1128/jvi.0391-17
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发表时间：
2017-09-01
影响因子：
5.4
通讯作者：
Carette, Jan E.
中科院分区：
文献类型：
--
作者：
Pillay, Sirika;Zou, Wei;Carette, Jan E.
腺相关病毒（AAV）的进入是通过其与特定表面聚糖和蛋白质受体的相互作用来确定的。腺相关病毒受体（AAVR）（也称为KIAA 0319 L）是转导来源于多种AAV血清型（包括进化上遥远的血清型AAV 2和AAV 5）的载体所需的必需细胞受体。在这里，我们进一步生物化学表征AAV-AAVR相互作用，并定义AAVR胞外域内促进这种相互作用的结构域。通过使用病毒覆盖测定，先前显示人细胞膜制备物中的主要AAV 2结合蛋白对应于分子量为150 kDa的糖蛋白。通过建立纯化程序，通过二维电泳进行进一步的蛋白质分离，并利用质谱，我们现在表明，这种糖蛋白是相同的AAVR。虽然我们发现AAVR是一种N-连接的糖基化蛋白，但这种糖基化不是AAV 2结合或功能转导的严格要求。使用基因互补与缺失构建体和病毒覆盖试验与个别域的组合，我们发现，AAV 2功能上主要与第二个Ig样多囊肾病（PKD）重复结构域（PKD 2）存在于AAVR的胞外域相互作用。相反，AAV 5主要通过AAVR的第一个、最远膜的PKD结构域（PKD 1）相互作用以促进转导。此外，其他AAV血清型，包括AAV 1和-8，需要PKD 1和PKD 2的组合以实现最佳转导。这些结果表明，尽管他们的共享依赖AAVR作为一个关键的入口受体，不同的AAV血清型已经发展出独特的相互作用与相同receptor.IMPORTANCE在过去的十年中，AAV载体已成为领先的基因传递工具的治疗应用和生物医学研究。然而，AAV生命周期的基本方面，包括AAV如何与宿主细胞因子相互作用以促进感染，仅部分了解。特别地，AAV受体对AAV载体转导效率和向性有显著贡献。最近鉴定的AAV受体（AAVR）是多种血清型的关键宿主受体，包括研究最多的血清型AAV 2。AAVR直接与AAV 2颗粒结合，并且限制病毒转导的速率。更详细地定义AAV-AAVR界面对于理解AAV如何与其细胞受体接合以及受体如何促进进入过程是重要的。在这里，我们进一步定义了AAV-AAVR相互作用，遗传和生化，并表明，不同的AAV血清型与Ig样PKD结构域的AAVR有离散的相互作用。这些发现揭示了这些细小病毒内AAVR接合的意想不到的分歧。
Adeno-associated virus (AAV) entry is determined by its interactions with specific surface glycans and a proteinaceous receptor(s). Adeno-associated virus receptor (AAVR) (also named KIAA0319L) is an essential cellular receptor required for the transduction of vectors derived from multiple AAV serotypes, including the evolutionarily distant serotypes AAV2 and AAV5. Here, we further biochemically characterize the AAV-AAVR interaction and define the domains within the ectodomain of AAVR that facilitate this interaction. By using a virus overlay assay, it was previously shown that the major AAV2 binding protein in membrane preparations of human cells corresponds to a glycoprotein with a molecular mass of 150 kDa. By establishing a purification procedure, performing further protein separation by two-dimensional electrophoresis, and utilizing mass spectrometry, we now show that this glycoprotein is identical to AAVR. While we find that AAVR is an N-linked glycosylated protein, this glycosylation is not a strict requirement for AAV2 binding or functional transduction. Using a combination of genetic complementation with deletion constructs and virus overlay assays with individual domains, we find that AAV2 functionally interacts predominantly with the second Ig-like polycystic kidney disease (PKD) repeat domain (PKD2) present in the ectodomain of AAVR. In contrast, AAV5 interacts primarily through the first, most membrane-distal, PKD domain (PKD1) of AAVR to promote transduction. Furthermore, other AAV serotypes, including AAV1 and -8, require a combination of PKD1 and PKD2 for optimal transduction. These results suggest that despite their shared dependence on AAVR as a critical entry receptor, different AAV serotypes have evolved distinctive interactions with the same receptor.IMPORTANCE Over the past decade, AAV vectors have emerged as leading gene delivery tools for therapeutic applications and biomedical research. However, fundamental aspects of the AAV life cycle, including how AAV interacts with host cellular factors to facilitate infection, are only partly understood. In particular, AAV receptors contribute significantly to AAV vector transduction efficiency and tropism. The recently identified AAV receptor (AAVR) is a key host receptor for multiple serotypes, including the most studied serotype, AAV2. AAVR binds directly to AAV2 particles and is rate limiting for viral transduction. Defining the AAV-AAVR interface in more detail is important to understand how AAV engages with its cellular receptor and how the receptor facilitates the entry process. Here, we further define AAV-AAVR interactions, genetically and biochemically, and show that different AAV serotypes have discrete interactions with the Ig-like PKD domains of AAVR. These findings reveal an unexpected divergence of AAVR engagement within these parvoviruses.