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

Backovic A, Cervelli T, Salvetti A, Zentilin L, Giacca M, Galli A - Microb. Cell Fact. (2012)

Bottom Line: We have recently demonstrated that S. cerevisiae can form single stranded DNA AAV2 genomes starting from a circular plasmid.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.The transmission electron microscopy analysis revealed that their morphology is similar to the empty capsids produced in human cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratorio di Tecnologie Genomiche, Istituto di Fisiologia Clinica, CNR, Pisa, Italy.

ABSTRACT

Background: 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.

Results: 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.

Conclusions: 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.

Show MeSH

Related in: MedlinePlus

Expression of AAV2 Cap and Rep proteins from natural promoters. Transformed RSY12 cells were grown in liquid selective medium until cultures reached different densities determining different growth phases (early → late log) indicated on the top. Equal amounts of total cell lysate, extract 1 and 2 (~50 μg each), obtained from 2 × 108 cells at each phase, were analyzed by Western blot analysis with antibodies indicated on the left: mAb B1 to detect Cap proteins (A and C), mAb 303.9 to detect Rep proteins (B) and mAb Anti-3PGK to detect constitutive yeast protein PGK (Phospho-Glicerate-Kinase). (A): VP3 was the only capsid protein detected in the samples from YEplacp40Cap transformed cells;thehighest level was detected in the mid-log phase extracts. (B): All four Rep proteins were detected in the samples from YEplacRepCap transformed cells; the highest level was obtained in the late-log phase extracts. (C): late-log phase extracts from YEplacRepCap cells were also analyzed for Cap protein expression and compared with late-log phase extracts from YEplacp40Cap cells; only VP3 was detected in both samples. Denatured, 293 T-cell-derived, AAV2 capsids were used as positive control (+ control) for defining VPs (A, C).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3539887&req=5

Figure 2: Expression of AAV2 Cap and Rep proteins from natural promoters. Transformed RSY12 cells were grown in liquid selective medium until cultures reached different densities determining different growth phases (early → late log) indicated on the top. Equal amounts of total cell lysate, extract 1 and 2 (~50 μg each), obtained from 2 × 108 cells at each phase, were analyzed by Western blot analysis with antibodies indicated on the left: mAb B1 to detect Cap proteins (A and C), mAb 303.9 to detect Rep proteins (B) and mAb Anti-3PGK to detect constitutive yeast protein PGK (Phospho-Glicerate-Kinase). (A): VP3 was the only capsid protein detected in the samples from YEplacp40Cap transformed cells;thehighest level was detected in the mid-log phase extracts. (B): All four Rep proteins were detected in the samples from YEplacRepCap transformed cells; the highest level was obtained in the late-log phase extracts. (C): late-log phase extracts from YEplacRepCap cells were also analyzed for Cap protein expression and compared with late-log phase extracts from YEplacp40Cap cells; only VP3 was detected in both samples. Denatured, 293 T-cell-derived, AAV2 capsids were used as positive control (+ control) for defining VPs (A, C).

Mentions: The AAV capsid is composed of 60 subunits with a constant stoichiometry of the proteins VP1, VP2 and VP3 corresponding to 1:1:10[19,20]. By densitomeric analysis of protein bands resolved by Western blot, the ratio of the AAV structural proteins VP1:VP2:VP3 has been shown to fluctuate from 1:1:8 to 1:1:20[21]. Moreover, in crude extracts, an average ratio ranging from 1:1:5 to 1:1:20 has been observed[18]. In order to assess the best promoter combination to express the AAV structural proteins at the optimal ratio, we constructed several plasmids carrying yeast constitutive or inducible promoter, or the natural AAV promoter (Figure1, Table1). First of all, we studied the expression of the capsid proteins under control of the AAV p40 promoter. We have previously shown that the AAV p5 and p19 promoters can be recognized by the yeast transcriptional machinery[8]. This may imply that also p40 could be functional in this simple eukaryote. We constructed a yeast multi-copy plasmid containing unmodified AAV cap gene with all regulative elements: p40 promoter, intron element and polyadenylation signal (Figure1 scheme “a”, Table1). To do this, the entire VP expression cassette located between 1428 and 4495 nucleotides of AAV genome (numbering is as for the sequence under GenBank accession no. AF043303.1) was cloned into YEplac181. The resulting plasmid named YEplacp40Cap has the yeast 2-micron origin of replication which constantly provides 20–50 copies of the recombinant gene per haploid yeast genome[22]. Plasmid YEplacp40Cap was transformed in the haploid yeast strain RSY12. Growth curves carried out at 30°C under constant shaking of YEplacp40Cap and YEplac181 control plasmid-transformed cells were similar, with doubling time of about 2.7 h, when 2% glucose was used as the carbon source. At 4 different time points, cells were collected and subjected to protein extraction. The first two time points correspond to early exponential/logarithmic (log) growth phase, the third one to mid-log and the fourth one to the late-log phase. Cap protein expression was analyzed by Western blot analysis of total cell lysates at each of these time points (Figure2A). As shown in the Figure2A, the only capsid protein detected is VP3. Since the concentration of VP1 and VP2 is expected to be 10 times lower than that of VP3, it is likely that the proteins are present in an amount too low to be detected by western blot. VP3 accumulated with time, following exponential cell growth and biomass accumulation (the maximal amount of protein was extracted from mid-late exponential growth phases). The majority of VP3 protein was found in insoluble fraction from which it was extracted (see materials and methods). This fraction of the total cellular proteins was designated as “extract 2”.


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

Backovic A, Cervelli T, Salvetti A, Zentilin L, Giacca M, Galli A - Microb. Cell Fact. (2012)

Expression of AAV2 Cap and Rep proteins from natural promoters. Transformed RSY12 cells were grown in liquid selective medium until cultures reached different densities determining different growth phases (early → late log) indicated on the top. Equal amounts of total cell lysate, extract 1 and 2 (~50 μg each), obtained from 2 × 108 cells at each phase, were analyzed by Western blot analysis with antibodies indicated on the left: mAb B1 to detect Cap proteins (A and C), mAb 303.9 to detect Rep proteins (B) and mAb Anti-3PGK to detect constitutive yeast protein PGK (Phospho-Glicerate-Kinase). (A): VP3 was the only capsid protein detected in the samples from YEplacp40Cap transformed cells;thehighest level was detected in the mid-log phase extracts. (B): All four Rep proteins were detected in the samples from YEplacRepCap transformed cells; the highest level was obtained in the late-log phase extracts. (C): late-log phase extracts from YEplacRepCap cells were also analyzed for Cap protein expression and compared with late-log phase extracts from YEplacp40Cap cells; only VP3 was detected in both samples. Denatured, 293 T-cell-derived, AAV2 capsids were used as positive control (+ control) for defining VPs (A, C).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3539887&req=5

Figure 2: Expression of AAV2 Cap and Rep proteins from natural promoters. Transformed RSY12 cells were grown in liquid selective medium until cultures reached different densities determining different growth phases (early → late log) indicated on the top. Equal amounts of total cell lysate, extract 1 and 2 (~50 μg each), obtained from 2 × 108 cells at each phase, were analyzed by Western blot analysis with antibodies indicated on the left: mAb B1 to detect Cap proteins (A and C), mAb 303.9 to detect Rep proteins (B) and mAb Anti-3PGK to detect constitutive yeast protein PGK (Phospho-Glicerate-Kinase). (A): VP3 was the only capsid protein detected in the samples from YEplacp40Cap transformed cells;thehighest level was detected in the mid-log phase extracts. (B): All four Rep proteins were detected in the samples from YEplacRepCap transformed cells; the highest level was obtained in the late-log phase extracts. (C): late-log phase extracts from YEplacRepCap cells were also analyzed for Cap protein expression and compared with late-log phase extracts from YEplacp40Cap cells; only VP3 was detected in both samples. Denatured, 293 T-cell-derived, AAV2 capsids were used as positive control (+ control) for defining VPs (A, C).
Mentions: The AAV capsid is composed of 60 subunits with a constant stoichiometry of the proteins VP1, VP2 and VP3 corresponding to 1:1:10[19,20]. By densitomeric analysis of protein bands resolved by Western blot, the ratio of the AAV structural proteins VP1:VP2:VP3 has been shown to fluctuate from 1:1:8 to 1:1:20[21]. Moreover, in crude extracts, an average ratio ranging from 1:1:5 to 1:1:20 has been observed[18]. In order to assess the best promoter combination to express the AAV structural proteins at the optimal ratio, we constructed several plasmids carrying yeast constitutive or inducible promoter, or the natural AAV promoter (Figure1, Table1). First of all, we studied the expression of the capsid proteins under control of the AAV p40 promoter. We have previously shown that the AAV p5 and p19 promoters can be recognized by the yeast transcriptional machinery[8]. This may imply that also p40 could be functional in this simple eukaryote. We constructed a yeast multi-copy plasmid containing unmodified AAV cap gene with all regulative elements: p40 promoter, intron element and polyadenylation signal (Figure1 scheme “a”, Table1). To do this, the entire VP expression cassette located between 1428 and 4495 nucleotides of AAV genome (numbering is as for the sequence under GenBank accession no. AF043303.1) was cloned into YEplac181. The resulting plasmid named YEplacp40Cap has the yeast 2-micron origin of replication which constantly provides 20–50 copies of the recombinant gene per haploid yeast genome[22]. Plasmid YEplacp40Cap was transformed in the haploid yeast strain RSY12. Growth curves carried out at 30°C under constant shaking of YEplacp40Cap and YEplac181 control plasmid-transformed cells were similar, with doubling time of about 2.7 h, when 2% glucose was used as the carbon source. At 4 different time points, cells were collected and subjected to protein extraction. The first two time points correspond to early exponential/logarithmic (log) growth phase, the third one to mid-log and the fourth one to the late-log phase. Cap protein expression was analyzed by Western blot analysis of total cell lysates at each of these time points (Figure2A). As shown in the Figure2A, the only capsid protein detected is VP3. Since the concentration of VP1 and VP2 is expected to be 10 times lower than that of VP3, it is likely that the proteins are present in an amount too low to be detected by western blot. VP3 accumulated with time, following exponential cell growth and biomass accumulation (the maximal amount of protein was extracted from mid-late exponential growth phases). The majority of VP3 protein was found in insoluble fraction from which it was extracted (see materials and methods). This fraction of the total cellular proteins was designated as “extract 2”.

Bottom Line: We have recently demonstrated that S. cerevisiae can form single stranded DNA AAV2 genomes starting from a circular plasmid.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.The transmission electron microscopy analysis revealed that their morphology is similar to the empty capsids produced in human cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratorio di Tecnologie Genomiche, Istituto di Fisiologia Clinica, CNR, Pisa, Italy.

ABSTRACT

Background: 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.

Results: 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.

Conclusions: 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.

Show MeSH
Related in: MedlinePlus