Generation of infectious recombinant Adeno-associated virus in Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae has been successfully employed to establish model systems for a number of viruses. Such model systems are powerful tools to study the virus biology and in particular for the identification and characterization of host factors playing a role in the viral infection cycle. Adeno-associated viruses (AAV) are heavily studied due to their use as gene delivery vectors. AAV relies on other helper viruses for successful replication and on host factors for several aspects of the viral life cycle. However the role of host and helper viral factors is only partially known. Production of recombinant AAV (rAAV) vectors for gene delivery applications depends on knowledge of AAV biology and the limited understanding of host and helper viral factors may be precluding efficient production, particularly in heterologous systems. Model systems in simpler eukaryotes like the yeast S. cerevisiae would be useful tools to identify and study the role of host factors in AAV biology. Here we show that expression of AAV2 viral proteins VP1, VP2, VP3, AAP, Rep78, Rep52 and an ITR-flanked DNA in yeast leads to capsid formation, DNA replication and encapsidation, resulting in formation of infectious particles. Many of the AAV characteristics observed in yeast resemble those in other systems, making it a suitable model system. Future findings in the yeast system could be translatable to other AAV host systems and aid in more efficient production of rAAV vectors.

酿酒酵母（Saccharomyces cerevisiae）已被成功用于构建多种病毒的模型系统。这类模型系统是研究病毒生物学的有力工具，尤其可用于鉴定与表征在病毒感染周期中发挥功能的宿主因子。腺相关病毒（adeno-associated viruses, AAV）作为基因递送载体被广泛研究：AAV的复制依赖其他辅助病毒，而病毒生命周期的多个环节均需宿主因子参与，但目前人们对宿主因子与辅助病毒因子的具体作用仅部分了解。用于基因递送的重组腺相关病毒（recombinant AAV, rAAV）载体的生产，依赖对AAV生物学的认知；而对宿主因子与辅助病毒因子的认知不足，可能阻碍其高效生产，在异源系统中这一问题尤为突出。在酿酒酵母这类简单真核生物中构建的模型系统，可用于鉴定并研究宿主因子在AAV生物学中的作用，具备较高应用价值。本研究证实，在酵母中表达AAV2的病毒蛋白VP1、VP2、VP3、AAP、Rep78、Rep52以及侧翼带有ITR的DNA后，可触发衣壳形成、DNA复制与衣壳包裹过程，最终生成感染性颗粒。在酵母系统中观察到的诸多AAV特性与其他系统中的表现高度相似，表明该系统是合适的AAV研究模型。未来通过酵母系统获得的研究成果，可推广应用于其他AAV宿主系统，助力rAAV载体的高效生产。