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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

Concentration of AAV2 capsid like-structures by high-speed ultracentrifugation through 40% sucrose-cushion. (A): YEplacRepCap + pYESVP1KM (RepCap + VP1KM)co-transformed yeast cells induced for 4.5 h (lanes 1–4) or 7 h (lanes 5 and 6) in 0.5% Glu + 5% Gal medium were subjected to a small scale (2 x 108 cells) - protein extraction yielding total cell lysate (lanes 1 and 5) and to a large scale extraction (~400 x 108 cells) (lanes 2, 3, 4 and 6) under non-denaturing conditions yielding the “native extract“ which was subjected to ultracentrifugation. As indicated on the bottom, the total cell lysate (lanes 1 and 5) and the resulting ultracentrifugation fractions, the supernatant (lane 3) and the pellet (lanes 2 and 6), were analyzed for the presence of VP proteins by Western blot. The antibody mAb B1 recognized only VP1 ad VP3 in the total cell lysate and all three VPs in the pellet fraction. The VP ratios in the total cell lysate were 1:8 (lane 1) and 1:3.3 (lane 5). The VP ratios in the pellet resulting from ultracentrifugation were 1:1.2:6.5 (lane 2), 1:3 (lane 6). The pellet obtained by ultracentrifugation of the extracts derived from cells co-transformed with empty vectors, YEplac181 and pYES2, was used as negative control (lane 4). Positive control is loaded in lane 7. (B): Cells transformed with YEplacRepCap (RepCap, lane 1 and 3) or with pYESVP1KM (VP1KM, lane 2 and 4) were induced for 7 h in 0.5% glucose + 5% galactose medium. All cells were processed as described in (A). VP proteins in the total cell lysates (lanes 1 and 2) and the ultracentrifugation pellet (lanes 3 and 4) were identified by Western blot analysis with mAb B1. The relative VP ratio is 1:0.25:0.9 for sample loaded in lane 4. No proteins were loaded in lanes marked with *. In the lane 5 the positive control was loaded.
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Figure 6: Concentration of AAV2 capsid like-structures by high-speed ultracentrifugation through 40% sucrose-cushion. (A): YEplacRepCap + pYESVP1KM (RepCap + VP1KM)co-transformed yeast cells induced for 4.5 h (lanes 1–4) or 7 h (lanes 5 and 6) in 0.5% Glu + 5% Gal medium were subjected to a small scale (2 x 108 cells) - protein extraction yielding total cell lysate (lanes 1 and 5) and to a large scale extraction (~400 x 108 cells) (lanes 2, 3, 4 and 6) under non-denaturing conditions yielding the “native extract“ which was subjected to ultracentrifugation. As indicated on the bottom, the total cell lysate (lanes 1 and 5) and the resulting ultracentrifugation fractions, the supernatant (lane 3) and the pellet (lanes 2 and 6), were analyzed for the presence of VP proteins by Western blot. The antibody mAb B1 recognized only VP1 ad VP3 in the total cell lysate and all three VPs in the pellet fraction. The VP ratios in the total cell lysate were 1:8 (lane 1) and 1:3.3 (lane 5). The VP ratios in the pellet resulting from ultracentrifugation were 1:1.2:6.5 (lane 2), 1:3 (lane 6). The pellet obtained by ultracentrifugation of the extracts derived from cells co-transformed with empty vectors, YEplac181 and pYES2, was used as negative control (lane 4). Positive control is loaded in lane 7. (B): Cells transformed with YEplacRepCap (RepCap, lane 1 and 3) or with pYESVP1KM (VP1KM, lane 2 and 4) were induced for 7 h in 0.5% glucose + 5% galactose medium. All cells were processed as described in (A). VP proteins in the total cell lysates (lanes 1 and 2) and the ultracentrifugation pellet (lanes 3 and 4) were identified by Western blot analysis with mAb B1. The relative VP ratio is 1:0.25:0.9 for sample loaded in lane 4. No proteins were loaded in lanes marked with *. In the lane 5 the positive control was loaded.

Mentions: To test whether yeast assembled VP1 and VP3 into virus-like particles when these proteins are expressed at the optimal ratio, 3 g of the yeast cell biomass (~400 × 108 cells) carrying the YEplacRepCap and the pYESVP1KM plasmid were lysed and processed under non-denaturing conditions. Cell lysates were subjected to high speed centrifugation in 40% sucrose cushion (as reported in the materials and methods) and the resulting fractions, the pellet (Figure6A, lane 2) and the supernatant (Figure6A, lane 3) were analyzed for the presence of VP proteins by Western blot. VPs were found in the pellet (Figure6A, lane 2) and not in the supernatant fraction (Figure6A, lane 3). Surprisingly, beside VP1 and VP3, Western blot analysis for the first time revealed the presence of VP2 protein. This result was interpreted in terms of low overall VP2 expression whose concentration in the total cell-lysate (Figure6A, lane 1) was under Western blot detection limit. To confirm that the ratio of VPs found in the pellet after the ultracentifugation through 40% sucrose cushion, resembled their relative intracellular levels, cells were induced under conditions that yielded VP1 and VP3 in the ratio different from the “optimal” one. For instance, after 7 h of induction in 0.5% glucose + 5% galactose medium, VP1:VP3 in the ultracentrifugation pellet was 1:3 (Figure6A, lane 6) being almost identical to the ratio in the total cell lysate, 1:3.3 (Figure6A, lane 5). Again, in the ultracentrifugation pellet is detected also VP2 protein (Figure6A, lane 6).


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)

Concentration of AAV2 capsid like-structures by high-speed ultracentrifugation through 40% sucrose-cushion. (A): YEplacRepCap + pYESVP1KM (RepCap + VP1KM)co-transformed yeast cells induced for 4.5 h (lanes 1–4) or 7 h (lanes 5 and 6) in 0.5% Glu + 5% Gal medium were subjected to a small scale (2 x 108 cells) - protein extraction yielding total cell lysate (lanes 1 and 5) and to a large scale extraction (~400 x 108 cells) (lanes 2, 3, 4 and 6) under non-denaturing conditions yielding the “native extract“ which was subjected to ultracentrifugation. As indicated on the bottom, the total cell lysate (lanes 1 and 5) and the resulting ultracentrifugation fractions, the supernatant (lane 3) and the pellet (lanes 2 and 6), were analyzed for the presence of VP proteins by Western blot. The antibody mAb B1 recognized only VP1 ad VP3 in the total cell lysate and all three VPs in the pellet fraction. The VP ratios in the total cell lysate were 1:8 (lane 1) and 1:3.3 (lane 5). The VP ratios in the pellet resulting from ultracentrifugation were 1:1.2:6.5 (lane 2), 1:3 (lane 6). The pellet obtained by ultracentrifugation of the extracts derived from cells co-transformed with empty vectors, YEplac181 and pYES2, was used as negative control (lane 4). Positive control is loaded in lane 7. (B): Cells transformed with YEplacRepCap (RepCap, lane 1 and 3) or with pYESVP1KM (VP1KM, lane 2 and 4) were induced for 7 h in 0.5% glucose + 5% galactose medium. All cells were processed as described in (A). VP proteins in the total cell lysates (lanes 1 and 2) and the ultracentrifugation pellet (lanes 3 and 4) were identified by Western blot analysis with mAb B1. The relative VP ratio is 1:0.25:0.9 for sample loaded in lane 4. No proteins were loaded in lanes marked with *. In the lane 5 the positive control was loaded.
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Figure 6: Concentration of AAV2 capsid like-structures by high-speed ultracentrifugation through 40% sucrose-cushion. (A): YEplacRepCap + pYESVP1KM (RepCap + VP1KM)co-transformed yeast cells induced for 4.5 h (lanes 1–4) or 7 h (lanes 5 and 6) in 0.5% Glu + 5% Gal medium were subjected to a small scale (2 x 108 cells) - protein extraction yielding total cell lysate (lanes 1 and 5) and to a large scale extraction (~400 x 108 cells) (lanes 2, 3, 4 and 6) under non-denaturing conditions yielding the “native extract“ which was subjected to ultracentrifugation. As indicated on the bottom, the total cell lysate (lanes 1 and 5) and the resulting ultracentrifugation fractions, the supernatant (lane 3) and the pellet (lanes 2 and 6), were analyzed for the presence of VP proteins by Western blot. The antibody mAb B1 recognized only VP1 ad VP3 in the total cell lysate and all three VPs in the pellet fraction. The VP ratios in the total cell lysate were 1:8 (lane 1) and 1:3.3 (lane 5). The VP ratios in the pellet resulting from ultracentrifugation were 1:1.2:6.5 (lane 2), 1:3 (lane 6). The pellet obtained by ultracentrifugation of the extracts derived from cells co-transformed with empty vectors, YEplac181 and pYES2, was used as negative control (lane 4). Positive control is loaded in lane 7. (B): Cells transformed with YEplacRepCap (RepCap, lane 1 and 3) or with pYESVP1KM (VP1KM, lane 2 and 4) were induced for 7 h in 0.5% glucose + 5% galactose medium. All cells were processed as described in (A). VP proteins in the total cell lysates (lanes 1 and 2) and the ultracentrifugation pellet (lanes 3 and 4) were identified by Western blot analysis with mAb B1. The relative VP ratio is 1:0.25:0.9 for sample loaded in lane 4. No proteins were loaded in lanes marked with *. In the lane 5 the positive control was loaded.
Mentions: To test whether yeast assembled VP1 and VP3 into virus-like particles when these proteins are expressed at the optimal ratio, 3 g of the yeast cell biomass (~400 × 108 cells) carrying the YEplacRepCap and the pYESVP1KM plasmid were lysed and processed under non-denaturing conditions. Cell lysates were subjected to high speed centrifugation in 40% sucrose cushion (as reported in the materials and methods) and the resulting fractions, the pellet (Figure6A, lane 2) and the supernatant (Figure6A, lane 3) were analyzed for the presence of VP proteins by Western blot. VPs were found in the pellet (Figure6A, lane 2) and not in the supernatant fraction (Figure6A, lane 3). Surprisingly, beside VP1 and VP3, Western blot analysis for the first time revealed the presence of VP2 protein. This result was interpreted in terms of low overall VP2 expression whose concentration in the total cell-lysate (Figure6A, lane 1) was under Western blot detection limit. To confirm that the ratio of VPs found in the pellet after the ultracentifugation through 40% sucrose cushion, resembled their relative intracellular levels, cells were induced under conditions that yielded VP1 and VP3 in the ratio different from the “optimal” one. For instance, after 7 h of induction in 0.5% glucose + 5% galactose medium, VP1:VP3 in the ultracentrifugation pellet was 1:3 (Figure6A, lane 6) being almost identical to the ratio in the total cell lysate, 1:3.3 (Figure6A, lane 5). Again, in the ultracentrifugation pellet is detected also VP2 protein (Figure6A, lane 6).

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