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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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Isolation of AAV2 capsid like-structures by ultracentrifugation in CsCl-gradient. Native protein extracts derived from ~ 0.5x1012 YEplacRepCap + pYESVP1KM (RepCap + VP1KM)co-transformed yeast cells, induced under optimal conditions, were subjected to 40% sucrose cushion-ultracentrifugation and the pelleted material was further fractionated in CsCl gradient by 48 h,. (A): 12 CsCl fractions of increasing densities were recovered and analyzed for the presence of VP proteins by Western blot with mAb B1. Only VP positive fractions are presented. Structures recovered in fractions 8–11 had VP compositions that most closely resembled the one of wt capsids. Denatured 293 T-cell derived AAV2 capsids were used as positive control for defining VPs. (B): Fractions of similar densities were united and subjected to TEM analysis. (i) Capsid-like structures of ~20 nm size identified in fraction f8 + f9 are shown and compared with 293 T –derived AAV2 empty capsids (ii). Scale bar is 40 nm. (C): 3 fraction pairs that gave positive results in TEM were spotted on the nitrocellulose membrane in three quantities indicated on the right side bar and analyzed for the presence of AAV capsids with the capsid-specific mAb A20 antibody. The strongest signal (which indicates the greatest number of capsids) was detected in the fraction f8 + f9. As negative control of the assay, the same number of cells co-transformed with empty vectors, YEplac181 and pYES2, were processed as described in (A) and the obtained CsCl fractions of the corresponding densities were incubated with A20 antibody. The name of fractions and relative density are indicated.
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Figure 7: Isolation of AAV2 capsid like-structures by ultracentrifugation in CsCl-gradient. Native protein extracts derived from ~ 0.5x1012 YEplacRepCap + pYESVP1KM (RepCap + VP1KM)co-transformed yeast cells, induced under optimal conditions, were subjected to 40% sucrose cushion-ultracentrifugation and the pelleted material was further fractionated in CsCl gradient by 48 h,. (A): 12 CsCl fractions of increasing densities were recovered and analyzed for the presence of VP proteins by Western blot with mAb B1. Only VP positive fractions are presented. Structures recovered in fractions 8–11 had VP compositions that most closely resembled the one of wt capsids. Denatured 293 T-cell derived AAV2 capsids were used as positive control for defining VPs. (B): Fractions of similar densities were united and subjected to TEM analysis. (i) Capsid-like structures of ~20 nm size identified in fraction f8 + f9 are shown and compared with 293 T –derived AAV2 empty capsids (ii). Scale bar is 40 nm. (C): 3 fraction pairs that gave positive results in TEM were spotted on the nitrocellulose membrane in three quantities indicated on the right side bar and analyzed for the presence of AAV capsids with the capsid-specific mAb A20 antibody. The strongest signal (which indicates the greatest number of capsids) was detected in the fraction f8 + f9. As negative control of the assay, the same number of cells co-transformed with empty vectors, YEplac181 and pYES2, were processed as described in (A) and the obtained CsCl fractions of the corresponding densities were incubated with A20 antibody. The name of fractions and relative density are indicated.

Mentions: To better understand if yeast forms VLPs, samples corresponding to 10 g of yeast biomass (YEplacRepCap and pYESVP1KM co-transformed cells grown under “optimal conditions”) were analyzed upon fractionation by high speed ultracentrifugation in CsCl density gradient. After ultracentrifugation, 12 fractions were collected, dialyzed against PBS and analyzed by the Western blot (Figure7A). In the 1st fraction (of the lowest density), only VP3 protein was detected (Figure7A, f1). Fractions 2, 3 and 4 (Figure7A, f2, f3, f4) contained almost equal amounts of VP2 and VP3, but VP1 is below the detection limit. Fractions 8–11 (Figure7A, f8, f9, f10, f11) contained all three VPs. Only VP3 was detected in f12. Finally, the VP proteins were also detected in the pellet CsCl fraction (f13) (data not shown) and hence not recovered in the other fractions. Importantly, the staining of the nitrocellulose membrane after blotting, showed very low level of VP proteins and no contaminant proteins corresponding to the fractions f8-f12 (data not shown). This result suggest that a very low amount of VLPs was purified. Altogether, our results indicate that yeast is able to assemble AAV VLPs.


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)

Isolation of AAV2 capsid like-structures by ultracentrifugation in CsCl-gradient. Native protein extracts derived from ~ 0.5x1012 YEplacRepCap + pYESVP1KM (RepCap + VP1KM)co-transformed yeast cells, induced under optimal conditions, were subjected to 40% sucrose cushion-ultracentrifugation and the pelleted material was further fractionated in CsCl gradient by 48 h,. (A): 12 CsCl fractions of increasing densities were recovered and analyzed for the presence of VP proteins by Western blot with mAb B1. Only VP positive fractions are presented. Structures recovered in fractions 8–11 had VP compositions that most closely resembled the one of wt capsids. Denatured 293 T-cell derived AAV2 capsids were used as positive control for defining VPs. (B): Fractions of similar densities were united and subjected to TEM analysis. (i) Capsid-like structures of ~20 nm size identified in fraction f8 + f9 are shown and compared with 293 T –derived AAV2 empty capsids (ii). Scale bar is 40 nm. (C): 3 fraction pairs that gave positive results in TEM were spotted on the nitrocellulose membrane in three quantities indicated on the right side bar and analyzed for the presence of AAV capsids with the capsid-specific mAb A20 antibody. The strongest signal (which indicates the greatest number of capsids) was detected in the fraction f8 + f9. As negative control of the assay, the same number of cells co-transformed with empty vectors, YEplac181 and pYES2, were processed as described in (A) and the obtained CsCl fractions of the corresponding densities were incubated with A20 antibody. The name of fractions and relative density are indicated.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 7: Isolation of AAV2 capsid like-structures by ultracentrifugation in CsCl-gradient. Native protein extracts derived from ~ 0.5x1012 YEplacRepCap + pYESVP1KM (RepCap + VP1KM)co-transformed yeast cells, induced under optimal conditions, were subjected to 40% sucrose cushion-ultracentrifugation and the pelleted material was further fractionated in CsCl gradient by 48 h,. (A): 12 CsCl fractions of increasing densities were recovered and analyzed for the presence of VP proteins by Western blot with mAb B1. Only VP positive fractions are presented. Structures recovered in fractions 8–11 had VP compositions that most closely resembled the one of wt capsids. Denatured 293 T-cell derived AAV2 capsids were used as positive control for defining VPs. (B): Fractions of similar densities were united and subjected to TEM analysis. (i) Capsid-like structures of ~20 nm size identified in fraction f8 + f9 are shown and compared with 293 T –derived AAV2 empty capsids (ii). Scale bar is 40 nm. (C): 3 fraction pairs that gave positive results in TEM were spotted on the nitrocellulose membrane in three quantities indicated on the right side bar and analyzed for the presence of AAV capsids with the capsid-specific mAb A20 antibody. The strongest signal (which indicates the greatest number of capsids) was detected in the fraction f8 + f9. As negative control of the assay, the same number of cells co-transformed with empty vectors, YEplac181 and pYES2, were processed as described in (A) and the obtained CsCl fractions of the corresponding densities were incubated with A20 antibody. The name of fractions and relative density are indicated.
Mentions: To better understand if yeast forms VLPs, samples corresponding to 10 g of yeast biomass (YEplacRepCap and pYESVP1KM co-transformed cells grown under “optimal conditions”) were analyzed upon fractionation by high speed ultracentrifugation in CsCl density gradient. After ultracentrifugation, 12 fractions were collected, dialyzed against PBS and analyzed by the Western blot (Figure7A). In the 1st fraction (of the lowest density), only VP3 protein was detected (Figure7A, f1). Fractions 2, 3 and 4 (Figure7A, f2, f3, f4) contained almost equal amounts of VP2 and VP3, but VP1 is below the detection limit. Fractions 8–11 (Figure7A, f8, f9, f10, f11) contained all three VPs. Only VP3 was detected in f12. Finally, the VP proteins were also detected in the pellet CsCl fraction (f13) (data not shown) and hence not recovered in the other fractions. Importantly, the staining of the nitrocellulose membrane after blotting, showed very low level of VP proteins and no contaminant proteins corresponding to the fractions f8-f12 (data not shown). This result suggest that a very low amount of VLPs was purified. Altogether, our results indicate that yeast is able to assemble AAV VLPs.

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