Capsid protein expression and adeno-associated virus like particles assembly in Saccharomyces cerevisiae

Abstract

BackgroundThe 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.ResultsTo 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.ConclusionsTaken 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.

Generation of infectious recombinant Adeno-associated virus in Saccharomyces cerevisiae

“…Our system recapitulates major aspects of the AAV life cycle observed in other systems: 1) capsids are formed in the nucleus and require efficient expression of AAP; 2) A DNA flanked by ITRs can be rescued form a plasmid and replicated; 3) Some of the ITR-flanked DNA can be encapsidated forming rAAV virions capable of transducing HEK293 cells. Formation of AAV capsids in yeast has been reported before [34] using yeast transformed with the natural AAV2 cap gene plus an additional cassette for VP1 expression driven by a galactose inducible promoter. Although VP3 expression from the natural p40 promoter was detectable in yeast, it was likely suboptimal since VP1 expression from GAL1 promoter had to be induced only partially to achieve close to optimal VP1 to VP3 ratio.…”

“…Our results also showed that efficient expression of AAP is essential for capsid assembly. Since the system described by Backovic et al [34] showed formation of capsids, it is conceivable that some AAP was expressed in yeast from the AAV2 cap gene by translation initiation at the non-conventional CTG start codon [6]. Expression of VP2, which is also dependent on translation initiation at a non-conventional ACG codon, was detectable after capsid purification [34].…”

“…Yeast has been used also for production of virus-like particles (VLPs) of a number of viruses [29], illustrating the capability of yeast to fold proteins correctly and support multimerization and capsid assembly [30–32]. The utility of yeast as model system for AAV has been explored before [33,34]. Cervelli et al [33], showed formation of ssDNA in yeast from a plasmid containing an ITR-flanked URA3 gene that was dependent on the presence of ITRs and expression of Rep proteins.…”

“…Cervelli et al [33], showed formation of ssDNA in yeast from a plasmid containing an ITR-flanked URA3 gene that was dependent on the presence of ITRs and expression of Rep proteins. Backovic et al [34], showed formation of AAV capsids in yeast transformed with the AAV2 Cap gene plus a VP1 expression cassette driven by a GAL1 promoter. In the present work, six AAV2 proteins (VP1, VP2, VP3, AAP, Rep78 and Rep52) were expressed simultaneously in yeast together with a plasmid carrying a GFP gene flanked by ITRs.…”

Production of Human Parvovirus 4 VP2 Virus-Like Particles in Yeast and Their Evaluation as an Antigen for Detection of Virus-Specific Antibodies in Human Serum

“…The formation of recombinant parvoviral capsid protein VLPs in yeast has been recently demonstrated [19,22]. Yeast-derived B19 VLPs can resemble native virus or recombinant VP2-VLPs produced by baculovirus systems in respect of size, molecular weight and antigenicity.…”