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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.

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Related in: MedlinePlus

Expression of AAV2 structural proteins from yeast the galactose-inducible promoter Gal1. (A): pYESIntronCap-transformed cells were first grown for 12 h in glucose and then transferred to galactose medium for 4 and 8 h of induction. Mid-log phase cells were collected at each of these time points and equal amounts of the total cell lysates (~50 μg), were analyzed for Cap protein expression by Western blot using mAb B1. VP3 was the only Cap protein detected and only in the extract 2 (insoluble fraction). Its relative amount was the highest in 12 h glucose samples, diminished after 4 h induction and was no more detectable upon 8 h of galactose induction. (B): pYESVP1KM-transformed yeast cells were exposed to galactose for different times, as indicated on the top, and equal amount of corresponding protein (~50 μg) extracts was analyzed for Cap protein expression using mAb B1. The majority of VP1 proteins were found in extract 2 (insoluble fraction). Extracts from cells transformed with empty vector, pYES2 were used as negative controls (−control).
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Figure 3: Expression of AAV2 structural proteins from yeast the galactose-inducible promoter Gal1. (A): pYESIntronCap-transformed cells were first grown for 12 h in glucose and then transferred to galactose medium for 4 and 8 h of induction. Mid-log phase cells were collected at each of these time points and equal amounts of the total cell lysates (~50 μg), were analyzed for Cap protein expression by Western blot using mAb B1. VP3 was the only Cap protein detected and only in the extract 2 (insoluble fraction). Its relative amount was the highest in 12 h glucose samples, diminished after 4 h induction and was no more detectable upon 8 h of galactose induction. (B): pYESVP1KM-transformed yeast cells were exposed to galactose for different times, as indicated on the top, and equal amount of corresponding protein (~50 μg) extracts was analyzed for Cap protein expression using mAb B1. The majority of VP1 proteins were found in extract 2 (insoluble fraction). Extracts from cells transformed with empty vector, pYES2 were used as negative controls (−control).

Mentions: The galactose inducible promoter of the GAL1 gene (pGal1) can be finely regulated by the amount of inducting agent in the growth medium and by varying the induction time[25,26]. Since VP2 was shown to be non essential for infectivity of the AAV virions[27,28], simultaneous expression of VP1 and VP3 in yeast cells was the principal condition for developing yeast-cell based system for production of the wt like AAV-capsids. We made various constructs based on the yeast expression vector pYES2 which contains two potent gene expression regulators, the pGal1 and the cyc1 terminator (Table1). To check weather all AAV capsid proteins could be detected simultaneously, these vectors were transformed in yeast and transformants were selected as already reported. Moreover, we made the vector named pYESCap, where the Cap from the VP1 start codon was cloned downstream pGal1 (Figure1 scheme “c”). Surprisingly, neither VP1 nor VP3 was detected in the extracts from yeast cells carrying the pYESCap ( Figure1 scheme “c”; data not shown). On the other hand, extracts from yeast cells carrying the vector pYESIntronCap containing the intron sequence upstream VP1 start codon (Figure1 scheme “d” ), the only protein detected was again VP3 and its highest amount was detected in the extract of the cells grown on glucose (Figure3A). After the carbon source switch to galactose, some background VP3 level was still observed after 4 h of galactose growth and completely vanished after 8 h in galactose (Figure3A). The decrease of the amount of VP3 in galactose could not be explained in terms of toxicity since no toxicity-related phenotype (e.g. decreased growth rate or abolished growth) was observed in galactose-grown cells. More likely, carbon source exchange could be quite stressful for the yeast cells.


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 structural proteins from yeast the galactose-inducible promoter Gal1. (A): pYESIntronCap-transformed cells were first grown for 12 h in glucose and then transferred to galactose medium for 4 and 8 h of induction. Mid-log phase cells were collected at each of these time points and equal amounts of the total cell lysates (~50 μg), were analyzed for Cap protein expression by Western blot using mAb B1. VP3 was the only Cap protein detected and only in the extract 2 (insoluble fraction). Its relative amount was the highest in 12 h glucose samples, diminished after 4 h induction and was no more detectable upon 8 h of galactose induction. (B): pYESVP1KM-transformed yeast cells were exposed to galactose for different times, as indicated on the top, and equal amount of corresponding protein (~50 μg) extracts was analyzed for Cap protein expression using mAb B1. The majority of VP1 proteins were found in extract 2 (insoluble fraction). Extracts from cells transformed with empty vector, pYES2 were used as negative controls (−control).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Expression of AAV2 structural proteins from yeast the galactose-inducible promoter Gal1. (A): pYESIntronCap-transformed cells were first grown for 12 h in glucose and then transferred to galactose medium for 4 and 8 h of induction. Mid-log phase cells were collected at each of these time points and equal amounts of the total cell lysates (~50 μg), were analyzed for Cap protein expression by Western blot using mAb B1. VP3 was the only Cap protein detected and only in the extract 2 (insoluble fraction). Its relative amount was the highest in 12 h glucose samples, diminished after 4 h induction and was no more detectable upon 8 h of galactose induction. (B): pYESVP1KM-transformed yeast cells were exposed to galactose for different times, as indicated on the top, and equal amount of corresponding protein (~50 μg) extracts was analyzed for Cap protein expression using mAb B1. The majority of VP1 proteins were found in extract 2 (insoluble fraction). Extracts from cells transformed with empty vector, pYES2 were used as negative controls (−control).
Mentions: The galactose inducible promoter of the GAL1 gene (pGal1) can be finely regulated by the amount of inducting agent in the growth medium and by varying the induction time[25,26]. Since VP2 was shown to be non essential for infectivity of the AAV virions[27,28], simultaneous expression of VP1 and VP3 in yeast cells was the principal condition for developing yeast-cell based system for production of the wt like AAV-capsids. We made various constructs based on the yeast expression vector pYES2 which contains two potent gene expression regulators, the pGal1 and the cyc1 terminator (Table1). To check weather all AAV capsid proteins could be detected simultaneously, these vectors were transformed in yeast and transformants were selected as already reported. Moreover, we made the vector named pYESCap, where the Cap from the VP1 start codon was cloned downstream pGal1 (Figure1 scheme “c”). Surprisingly, neither VP1 nor VP3 was detected in the extracts from yeast cells carrying the pYESCap ( Figure1 scheme “c”; data not shown). On the other hand, extracts from yeast cells carrying the vector pYESIntronCap containing the intron sequence upstream VP1 start codon (Figure1 scheme “d” ), the only protein detected was again VP3 and its highest amount was detected in the extract of the cells grown on glucose (Figure3A). After the carbon source switch to galactose, some background VP3 level was still observed after 4 h of galactose growth and completely vanished after 8 h in galactose (Figure3A). The decrease of the amount of VP3 in galactose could not be explained in terms of toxicity since no toxicity-related phenotype (e.g. decreased growth rate or abolished growth) was observed in galactose-grown cells. More likely, carbon source exchange could be quite stressful for the yeast cells.

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