Capsid protein expression and adeno-associated virus like particles assembly in Saccharomyces cerevisiae.
Capsid protein expression and adeno-associated virus like particles assembly in Saccharomyces cerevisiae.
复制标题
DOI：
10.1186/1475-2859-11-124
复制
发表时间：
2012-09-11
影响因子：
6.4
通讯作者：
Galli A
中科院分区：
文献类型：
--
作者：
Backovic A;Cervelli T;Salvetti A;Zentilin L;Giacca M;Galli A
萌芽酵母支持多种不同的RNA或DNA病毒(如Tombus病毒或乳头瘤病毒)的复制，并提供了可扩展、低成本且具有时间效益的各种病毒样颗粒(例如人类细小病毒B19或轮状病毒)的生产方法。我们最近证明了酿酒酵母可以从一个环状质粒开始形成单链DNA AAV2基因组。在这项工作中，我们研究了在酵母中组装AAV衣壳的可能性。要做到这一点，至少三个AAV结构蛋白中的两个，VP1和VP3，必须在酵母细胞中同时表达，并且它们的细胞内化学计量比必须与从哺乳动物或昆虫细胞中发现的颗粒相似。这是通过两个质粒稳定地共转化酵母细胞实现的，一个表达来自其自然p40启动子的VP3，另一个主要表达在可诱导酵母启动子Gal1控制下的修改的AAV2Cap基因的VP1。在各种诱导方法中，以0.5%葡萄糖+5%半乳糖诱导4.5小时的VP1：VP3为最佳。在诱导后，用两步超速离心法从酵母中分离出AAV病毒样颗粒(VLP)。透射电子显微镜分析表明，它们的形态与人类细胞中产生的空衣壳相似。综上所述，结果首次表明，酵母可以用来组装AAV衣壳，从而作为一种遗传系统来识别AAV生物学中涉及的新的细胞因子。
The budding yeast Saccharomyces cerevisiae supports replication of many different RNA or DNA viruses (e.g. Tombusviruses or Papillomaviruses) and has provided means for up-scalable, cost- and time-effective production of various virus-like particles (e.g. Human Parvovirus B19 or Rotavirus). We have recently demonstrated that S. cerevisiae can form single stranded DNA AAV2 genomes starting from a circular plasmid. In this work, we have investigated the possibility to assemble AAV capsids in yeast. To do this, at least two out of three AAV structural proteins, VP1 and VP3, have to be simultaneously expressed in yeast cells and their intracellular stoichiometry has to resemble the one found in the particles derived from mammalian or insect cells. This was achieved by stable co-transformation of yeast cells with two plasmids, one expressing VP3 from its natural p40 promoter and the other one primarily expressing VP1 from a modified AAV2 Cap gene under the control of the inducible yeast promoter Gal1. Among various induction strategies we tested, the best one to yield the appropriate VP1:VP3 ratio was 4.5 hour induction in the medium containing 0.5% glucose and 5% galactose. Following such induction, AAV virus like particles (VLPs) were isolated from yeast by two step ultracentrifugation procedure. The transmission electron microscopy analysis revealed that their morphology is similar to the empty capsids produced in human cells. Taken together, the results show for the first time that yeast can be used to assemble AAV capsid and, therefore, as a genetic system to identify novel cellular factors involved in AAV biology.