Host determinants of adeno-associated virus entry and trafficking

腺相关病毒（Adeno-associated viruses，AAV）是理想的基因递送载体候选者，因为它们是非致病性的、低免疫原性的和广泛嗜性的。尽管它们在包括血友病和肌营养不良症在内的严重疾病的基因治疗临床试验中有很大的希望，但对AAV生命周期的基本方面缺乏了解。已知AAV组织嗜性依赖于病毒生命周期内的某些限速步骤，包括细胞进入和从质膜到细胞核的运输。进一步的阐明是必要的，以解剖的机制，指导这些步骤，并具体确定主机的因素，促进这些过程。为了发现参与病毒进入和贩运的新宿主因素，我们最近进行了一项无偏见的人类遗传筛查。我们鉴定了几种对AAV感染至关重要的蛋白质，包括一种我们命名为AAV受体（AAVR）的特征不明显的跨膜蛋白。我们发现AAVR是必不可少的广泛的AAV血清型在体外和体内的转导。我们观察到，AAVR可以直接结合到AAV颗粒，并且体外过表达AAVR导致AAV易感性增强。特别感兴趣的是AAVR通过与AAV颗粒非常相似的内吞途径再循环的能力。这表明AAVR在颗粒运输中的潜在进入后作用。在这些发现的基础上，我们的长期目标是阐明AAV生命周期中控制限速过程的分子机制，以便更多地了解AAV的生物学，并利用这些知识来提高AAV作为载体的多功能性。我们打算通过确定：（i）受体接合和细胞内病毒运输之间的关系，（ii）AAVR如何有助于体内病毒向性，以及（iii）在我们的筛选中鉴定的其他主要命中如何在介导AAV运输中起作用。

Adeno-associated viruses (AAV) make ideal gene delivery vector candidates because they are non-pathogenic, lowly immunogenic and broadly tropic. Despite their great promise in gene therapy clinical trials for severe diseases including hemophilia and muscular dystrophy, there is a lack of understanding of fundamental aspects of the AAV life cycle. AAV tissue tropism is known to be dependent on certain rate-limiting steps within the virus life cycle, including cellular entry and trafficking from the plasma membrane to the nucleus. Further elucidation is necessary to dissect the mechanisms that direct these steps, and specifically identify the host factors that facilitate these processes. To discover novel host factors that are involved in virus entry and trafficking, we recently performed an unbiased, human genetic screen. We identified several proteins as critically important for AAV infection, including a poorly characterized transmembrane protein that we have since named AAV Receptor (AAVR). We found AAVR to be essential for the transduction of a wide array of AAV serotypes in vitro and in vivo. We observed that AAVR could bind directly to AAV particles, and that overexpression of AAVR in vitro led to enhanced AAV susceptibility. Of particular interest was the capacity of AAVR to recycle through a remarkably similar endocytic path as AAV particles. This suggests a potential post-entry role for AAVR in particle trafficking. Building upon these findings, our long-term goal seeks to elucidate the molecular mechanisms that govern rate-limiting processes within AAV’s life cycle in order to understand more about the biology of AAV, as well as harness this knowledge to improve AAV’s versatility as a vector. We intend to do this by determining: (i) what the relationship is between receptor engagement and intracellular viral trafficking, (ii) how AAVR contributes to viral tropism in vivo, and (iii) how the other major hits identified in our screen function in mediating AAV trafficking.