Capsid Assembly and Single Stranded DNA Genome Formation of Adeno-Associated Virus Type2 in Yeast Cells

Saccharomyces cerevisiae has provided an array of genetic tools to study unknown aspects of viral life cycles, supporting replication of many different RNA or DNA viruses (e.g. Tombusviruses or Papillomaviruses). It also provides means for up-scalable, cost- and time-effective production of various virus-like particles (e.g. Human Parvovirus B19 or Rotavirus) and as such represents a useful tool for vaccine development. To extend the utility of the S. cerevisiae expression system, we expressed AAV2 structural and nonstructural proteins in yeast cells, using both authentic AAV2 and heterologous yeast promoters. For the first time, we described the assembly of AAV2 virus-like particles from yeast-expressed AAV2 structural proteins. To do this we used AAV p40 promoter, whose activity in yeast cells resembled the one of yeast glycolytic promoters, resulting in the synthesis of the most abundant capsid protein VP3 when transformed yeast cells were grown on glucose as a carbon source. The expression of other two VPs was induced from yeast, galactose inducible pGal1 promoter. Simultaneous production of all three VPs was achieved by growing the yeast cells in the medium containing both glucose and galactose, while their relative production levels were further optimized by varying amounts of each carbon source in the induction medium, followed by the fine tuning of the induction time. Moreover, we investigated the ability of the yeast Saccharomyces cerevisiae to carry out the replication of a recombinant rAAV2. When a plasmid harboring the rAAV2 genome in which the cap gene was replaced with the S. cerevisiae URA3 gene, was co-transformed in yeast with a plasmid expressing Rep68 from constitutive yeast promoter pADH, a significant number of URA3+ clones were scored (more than 30-fold over controls). Molecular analysis of low molecular weight DNA revealed that the single stranded DNA is formed, in Rep68 and ITR dependent manner, and that the plasmid is entirely replicated. The ss DNA contained the ITRs, URA3 gene and also vector sequences suggesting that ss rAAV genomes were not obtained by the canonical AAV replication mechanism. These results could open new prospects for using yeast cell in two ways: (i) as a model system for studying viral and cellular factors involved in AAV2 capsid assembly and packaging of rAAV ss genomes; and (ii) as a novel cell factory for developing superior recombinant rAAV production technologies.