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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

VP1-VP3 expression pattern in co-transformed yeast clones. Cells co-transformed with YEplacp40Cap and pYESVP1KM (Cap + VP1KM clone) (A) and Rep expressing cells co-transformed with YEplacRepCap and pYESVP1KM (RepCap + VP1KM) (B), were grown on glucose and then transferred to galactose for induction. Equal amounts of total cellular proteins (extracts 1 + 2) were analyzed by Western blot, using mAb B1 to detect VP proteins. (A): VP3 was detected in both yeast clones after 12 h growth in glucose and it decreased along with VP1 induction upon 7 h in galactose. (B): Extracts from RepCap + VP1KM clones were analyzed for Rep protein expression before (12 h glucose) and after 7 h galactose growth, using mAb 303.9. Similar amounts and distribution of Rep isoforms to extracts 1/2 were detected in glucose and galactose samples. Extracts from cells co-transformed with empty vectors, YEplac181 and pYES2 were used as -control. (C): Lanes 0–5: VP1-VP3 expression pattern in total cell-extracts derived from RepCap + VP1KM clones before induction (“0” time point) and at various times of galactose induction. VP1:VP3 ratios are determined band densitometry and shown in the table below. Numbers represent the density expressed in arbitrary unit detected by the analysis software described in materials and methods. Results are reported as mean of at least three independent experiments ± standard error. The best ratio was obtained after 40 minutes of galactose induction.
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Figure 4: VP1-VP3 expression pattern in co-transformed yeast clones. Cells co-transformed with YEplacp40Cap and pYESVP1KM (Cap + VP1KM clone) (A) and Rep expressing cells co-transformed with YEplacRepCap and pYESVP1KM (RepCap + VP1KM) (B), were grown on glucose and then transferred to galactose for induction. Equal amounts of total cellular proteins (extracts 1 + 2) were analyzed by Western blot, using mAb B1 to detect VP proteins. (A): VP3 was detected in both yeast clones after 12 h growth in glucose and it decreased along with VP1 induction upon 7 h in galactose. (B): Extracts from RepCap + VP1KM clones were analyzed for Rep protein expression before (12 h glucose) and after 7 h galactose growth, using mAb 303.9. Similar amounts and distribution of Rep isoforms to extracts 1/2 were detected in glucose and galactose samples. Extracts from cells co-transformed with empty vectors, YEplac181 and pYES2 were used as -control. (C): Lanes 0–5: VP1-VP3 expression pattern in total cell-extracts derived from RepCap + VP1KM clones before induction (“0” time point) and at various times of galactose induction. VP1:VP3 ratios are determined band densitometry and shown in the table below. Numbers represent the density expressed in arbitrary unit detected by the analysis software described in materials and methods. Results are reported as mean of at least three independent experiments ± standard error. The best ratio was obtained after 40 minutes of galactose induction.

Mentions: Since we obtained detectable expression of VP3 from the vector YEplacp40Cap, and VP1 from pYESVP1KM, we transformed yeast cells with these plasmids to achieve simultaneous high level expression of AAV capsid proteins. We modulated their relative amount by growing the co-transformed clones, first in glucose and, then, in galactose for different induction times. In parallel, we also tested the clones co-transformed with pYESVP1KM and YEplacRepCap to assess if Rep could affect the VP expression pattern. As expected, Western blot analysis showed that both co-transformed cell clones (Rep positive and Rep negative) produced VP3 protein after growth in glucose, while VP1 expression was induced only after the cell growth in galactose containing medium (Figure4A). The induction was initially done for 7 h since VP1 was previously shown (Figure3B) to reach its maximum level at this time point. However, at the end of galactose induction, VP3 protein is not detectable in the cells that do not express Rep (Figure4A, Cap + VP1KM co-transformed clones) and was hardly detectable in the presence of Rep protein (Figure4A, RepCap + VP1KM co-transformed clones). This slight difference cannot be only attributed to Rep proteins which, indeed, were efficiently expressed throughout the whole culture (at least Rep78 and 52), both during glucose and galactose growth (Figure4B). The decrease in the VP3 protein level after the growth in galactose may imply that de novo VP3 synthesis is either prevented or reduced when galactose is used as a carbon source, resulting in VP3 “dilution” in the growing cell population. To overcome VP3 decrease and set up the best conditions for production of VP3 and VP1 proteins in the optimal ratio (similar to the one found in the wild type AAV capsids), we gradually decreased the induction time in galactose and analyzed VP1:VP3 ratio at different time points as indicated in the Figure4C (the densitometry for each induction time is reported in the table below the western blot). VP1 expression was observed already 40 minutes after galactose induction (Figure4C, lane 2) and increased with time. After 4 hours of induction, gradual increase of VP1 was followed by decrease in the VP3 level (Figure4C, lane 4), which was no longer detectable after 8 h of induction, when VP1 expression reached its maximum level (Figure4C, lane 5). The relative VP1:VP3 ratios were calculated from corresponding band intensities at each time point and the values are presented in the Figure4C. By decreasing induction time to 40 minutes, we obtained the VP1:VP3 ratio of 1:9 (Figure4C, lane 2). This VP stoichiometry is reported to be in the optimal range to form AAV capsids[18]. Nevertheless, such a short induction time makes the experimental reproducibility very difficult to achieve, so we tried the strategy of “glucose + galactose mixed cultures” which enabled fine tuning of VP1:VP3 ratio. When both nutrients are present in high concentration, glucose is used by cells as preferable carbon source. In other words, yeast starts utilizing galactose after the glucose concentration in the medium is completely exhausted[30]. After 12 h growth in 2% glucose medium, cells were transferred to the medium containing 1.5% glucose and 2.5% galactose (named “high glucose, high galactose”) and VP1:VP3 ratios tested at different induction times. The best ratio was reached after 9 h (Figure5A). After 18 hour induction the VP1 protein level increased and the VP3 decrease. Based on these results, we hypothesized that during the VP1 induction in 5% galactose, glucose should be kept at residual concentration to ensure the constitutive expression of VP3. Therefore, by decreasing the glucose concentration to 0.5%, the optimal VP1:VP3 ratio could be obtained at earlier time points with respect to “high glucose (glu)- high galactose (gal)” conditions. In particular, yeast grown in 2% glucose for 18 hours were transferred in the medium containing 0.5% glucose and 5% galactose, designated as “low glu-high gal” medium. The relative VP1 and VP3 protein level was analyzed by Western blot in extracts from yeast cells collected at different induction time points (Figure5B). The best VP1:VP3 ratio (1:8) was obtained after 4.5 h of induction (Figure5B, lane 1, the densitometry for each induction time is reported in the table below the Western blot). When we increased the induction time, VP1 started to accumulate while VP3 decreased leading to a non optimal ratio (Figure5B, lane 5).


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)

VP1-VP3 expression pattern in co-transformed yeast clones. Cells co-transformed with YEplacp40Cap and pYESVP1KM (Cap + VP1KM clone) (A) and Rep expressing cells co-transformed with YEplacRepCap and pYESVP1KM (RepCap + VP1KM) (B), were grown on glucose and then transferred to galactose for induction. Equal amounts of total cellular proteins (extracts 1 + 2) were analyzed by Western blot, using mAb B1 to detect VP proteins. (A): VP3 was detected in both yeast clones after 12 h growth in glucose and it decreased along with VP1 induction upon 7 h in galactose. (B): Extracts from RepCap + VP1KM clones were analyzed for Rep protein expression before (12 h glucose) and after 7 h galactose growth, using mAb 303.9. Similar amounts and distribution of Rep isoforms to extracts 1/2 were detected in glucose and galactose samples. Extracts from cells co-transformed with empty vectors, YEplac181 and pYES2 were used as -control. (C): Lanes 0–5: VP1-VP3 expression pattern in total cell-extracts derived from RepCap + VP1KM clones before induction (“0” time point) and at various times of galactose induction. VP1:VP3 ratios are determined band densitometry and shown in the table below. Numbers represent the density expressed in arbitrary unit detected by the analysis software described in materials and methods. Results are reported as mean of at least three independent experiments ± standard error. The best ratio was obtained after 40 minutes of galactose induction.
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Related In: Results  -  Collection

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Figure 4: VP1-VP3 expression pattern in co-transformed yeast clones. Cells co-transformed with YEplacp40Cap and pYESVP1KM (Cap + VP1KM clone) (A) and Rep expressing cells co-transformed with YEplacRepCap and pYESVP1KM (RepCap + VP1KM) (B), were grown on glucose and then transferred to galactose for induction. Equal amounts of total cellular proteins (extracts 1 + 2) were analyzed by Western blot, using mAb B1 to detect VP proteins. (A): VP3 was detected in both yeast clones after 12 h growth in glucose and it decreased along with VP1 induction upon 7 h in galactose. (B): Extracts from RepCap + VP1KM clones were analyzed for Rep protein expression before (12 h glucose) and after 7 h galactose growth, using mAb 303.9. Similar amounts and distribution of Rep isoforms to extracts 1/2 were detected in glucose and galactose samples. Extracts from cells co-transformed with empty vectors, YEplac181 and pYES2 were used as -control. (C): Lanes 0–5: VP1-VP3 expression pattern in total cell-extracts derived from RepCap + VP1KM clones before induction (“0” time point) and at various times of galactose induction. VP1:VP3 ratios are determined band densitometry and shown in the table below. Numbers represent the density expressed in arbitrary unit detected by the analysis software described in materials and methods. Results are reported as mean of at least three independent experiments ± standard error. The best ratio was obtained after 40 minutes of galactose induction.
Mentions: Since we obtained detectable expression of VP3 from the vector YEplacp40Cap, and VP1 from pYESVP1KM, we transformed yeast cells with these plasmids to achieve simultaneous high level expression of AAV capsid proteins. We modulated their relative amount by growing the co-transformed clones, first in glucose and, then, in galactose for different induction times. In parallel, we also tested the clones co-transformed with pYESVP1KM and YEplacRepCap to assess if Rep could affect the VP expression pattern. As expected, Western blot analysis showed that both co-transformed cell clones (Rep positive and Rep negative) produced VP3 protein after growth in glucose, while VP1 expression was induced only after the cell growth in galactose containing medium (Figure4A). The induction was initially done for 7 h since VP1 was previously shown (Figure3B) to reach its maximum level at this time point. However, at the end of galactose induction, VP3 protein is not detectable in the cells that do not express Rep (Figure4A, Cap + VP1KM co-transformed clones) and was hardly detectable in the presence of Rep protein (Figure4A, RepCap + VP1KM co-transformed clones). This slight difference cannot be only attributed to Rep proteins which, indeed, were efficiently expressed throughout the whole culture (at least Rep78 and 52), both during glucose and galactose growth (Figure4B). The decrease in the VP3 protein level after the growth in galactose may imply that de novo VP3 synthesis is either prevented or reduced when galactose is used as a carbon source, resulting in VP3 “dilution” in the growing cell population. To overcome VP3 decrease and set up the best conditions for production of VP3 and VP1 proteins in the optimal ratio (similar to the one found in the wild type AAV capsids), we gradually decreased the induction time in galactose and analyzed VP1:VP3 ratio at different time points as indicated in the Figure4C (the densitometry for each induction time is reported in the table below the western blot). VP1 expression was observed already 40 minutes after galactose induction (Figure4C, lane 2) and increased with time. After 4 hours of induction, gradual increase of VP1 was followed by decrease in the VP3 level (Figure4C, lane 4), which was no longer detectable after 8 h of induction, when VP1 expression reached its maximum level (Figure4C, lane 5). The relative VP1:VP3 ratios were calculated from corresponding band intensities at each time point and the values are presented in the Figure4C. By decreasing induction time to 40 minutes, we obtained the VP1:VP3 ratio of 1:9 (Figure4C, lane 2). This VP stoichiometry is reported to be in the optimal range to form AAV capsids[18]. Nevertheless, such a short induction time makes the experimental reproducibility very difficult to achieve, so we tried the strategy of “glucose + galactose mixed cultures” which enabled fine tuning of VP1:VP3 ratio. When both nutrients are present in high concentration, glucose is used by cells as preferable carbon source. In other words, yeast starts utilizing galactose after the glucose concentration in the medium is completely exhausted[30]. After 12 h growth in 2% glucose medium, cells were transferred to the medium containing 1.5% glucose and 2.5% galactose (named “high glucose, high galactose”) and VP1:VP3 ratios tested at different induction times. The best ratio was reached after 9 h (Figure5A). After 18 hour induction the VP1 protein level increased and the VP3 decrease. Based on these results, we hypothesized that during the VP1 induction in 5% galactose, glucose should be kept at residual concentration to ensure the constitutive expression of VP3. Therefore, by decreasing the glucose concentration to 0.5%, the optimal VP1:VP3 ratio could be obtained at earlier time points with respect to “high glucose (glu)- high galactose (gal)” conditions. In particular, yeast grown in 2% glucose for 18 hours were transferred in the medium containing 0.5% glucose and 5% galactose, designated as “low glu-high gal” medium. The relative VP1 and VP3 protein level was analyzed by Western blot in extracts from yeast cells collected at different induction time points (Figure5B). The best VP1:VP3 ratio (1:8) was obtained after 4.5 h of induction (Figure5B, lane 1, the densitometry for each induction time is reported in the table below the Western blot). When we increased the induction time, VP1 started to accumulate while VP3 decreased leading to a non optimal ratio (Figure5B, lane 5).

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