Host determinants of adeno-associated virus entry and trafficking

腺相关病毒(AAV)是理想的基因递送载体，因为它们无致病性、低免疫原性和广嗜性。尽管AAV在血友病和肌营养不良等严重疾病的基因治疗临床试验中前景光明，但对AAV生命周期的基本方面缺乏了解。已知AAV的组织嗜性依赖于病毒生命周期中的某些限速步骤，包括细胞进入和从质膜运输到细胞核。有必要进一步阐明，以剖析指导这些步骤的机制，并具体确定促进这些过程的宿主因素。为了发现与病毒进入和贩运有关的新宿主因素，我们最近进行了一次无偏见的人类基因筛查。我们确定了几种对AAV感染至关重要的蛋白质，包括一种特性不佳的跨膜蛋白，我们后来将其命名为AAV受体(AAVR)。我们发现AAVR在体内和体外对广泛的AAV血清型的转导是必不可少的。我们观察到AAVR可以直接与AAV颗粒结合，在体外过表达AAVR可以增强AAV的敏感性。特别令人感兴趣的是AAVR通过与AAV颗粒非常相似的内吞途径进行循环的能力。这表明AAVR在粒子贩运中具有进入后的潜在作用。在这些发现的基础上，我们的长期目标是阐明AAV生命周期中控制速率限制过程的分子机制，以便更多地了解AAV的生物学，以及利用这一知识来提高AAV作为载体的多功能性。我们打算通过确定：(I)受体参与和细胞内病毒转运之间的关系，(Ii)AAVR如何在体内促进病毒趋向性，以及(Iii)在我们的屏幕上发现的其他主要靶点如何在介导AAV转运中发挥作用来实现这一点。

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.