Abstract
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.
Highlights
Adeno-associated viruses (AAV) are single stranded DNA, non-enveloped, icosahedral viruses belonging to the genus Dependoparvovirus, family Parvoviridae
In natural hosts the AAV Rep proteins, VP proteins and activating protein (AAP) are expressed from the Rep and Cap genes at specific ratios through a combination of promoters, alternative splicing and leaky translation start at non-AUG codons
Rep78, Rep52 and VP3 were cloned under the control of the GAL1 promoter, while VP1, VP2 and AAP were cloned under the control of the GAL10 promoter
Summary
Adeno-associated viruses (AAV) are single stranded DNA, non-enveloped, icosahedral viruses belonging to the genus Dependoparvovirus, family Parvoviridae. First discovered as contaminants of adenovirus preparations, they require co-infection with adenovirus or herpes virus for efficient replication and are asymptomatic in humans [1]. Several characteristics have made it possible to develop gene delivery vectors based on AAV and a number of gene therapy treatments for human diseases are being developed using recombinant AAV (rAAV) vectors [2,3,4,5]. AAV capsids are composed of 60 units of VP1, VP2 and VP3 in an approximate ratio of 1:1:10. The three proteins are produced from the cap gene through a combination of alternative splicing and leaky scanning of transcripts from the p40 promoter and they all share the C.
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