Data from: Generation of infectious recombinant Adeno-associated virus in Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae has been successfully employed to establish model systems for a number of viruses. Such model systems are powerful tools to study the virus biology and in particular for the identification and characterization of host factors playing a role in the viral infection cycle. Adeno-associated viruses (AAV) are heavily studied due to their use as gene delivery vectors. AAV relies on other helper viruses for successful replication and on host factors for several aspects of the viral life cycle. However the role of host and helper viral factors is only partially known. Production of recombinant AAV (rAAV) vectors for gene delivery applications depends on knowledge of AAV biology and the limited understanding of host and helper viral factors may be precluding efficient production, particularly in heterologous systems. Model systems in simpler eukaryotes like the yeast S. cerevisiae would be useful tools to identify and study the role of host factors in AAV biology. Here we show that expression of AAV2 viral proteins VP1, VP2, VP3, AAP, Rep78, Rep52 and an ITR-flanked DNA in yeast leads to capsid formation, DNA replication and encapsidation, resulting in formation of infectious particles. Many of the AAV characteristics observed in yeast resemble those in other systems, making it a suitable model system. Future findings in the yeast system could be translatable to other AAV host systems and aid in more efficient production of rAAV vectors

Codon optimized sequences

AAP and Rep coding sequences from AAV2, codon optimized for expression in Saccharomyces cerevisiae

Generation of infectious recombinant Adeno-associated virus in <i>Saccharomyces cerevisiae</i>

<div><p>The yeast <i>Saccharomyces cerevisiae</i> has been successfully employed to establish model systems for a number of viruses. Such model systems are powerful tools to study the virus biology and in particular for the identification and characterization of host factors playing a role in the viral infection cycle. Adeno-associated viruses (AAV) are heavily studied due to their use as gene delivery vectors. AAV relies on other helper viruses for successful replication and on host factors for several aspects of the viral life cycle. However the role of host and helper viral factors is only partially known. Production of recombinant AAV (rAAV) vectors for gene delivery applications depends on knowledge of AAV biology and the limited understanding of host and helper viral factors may be precluding efficient production, particularly in heterologous systems. Model systems in simpler eukaryotes like the yeast <i>S</i>. <i>cerevisiae</i> would be useful tools to identify and study the role of host factors in AAV biology. Here we show that expression of AAV2 viral proteins VP1, VP2, VP3, AAP, Rep78, Rep52 and an ITR-flanked DNA in yeast leads to capsid formation, DNA replication and encapsidation, resulting in formation of infectious particles. Many of the AAV characteristics observed in yeast resemble those in other systems, making it a suitable model system. Future findings in the yeast system could be translatable to other AAV host systems and aid in more efficient production of rAAV vectors.</p></div

AAV capsid formation in yeast.

<p>(A) Yeast lysates (undiluted or diluted 1/10 and 1/100 fold) were spotted onto nitrocellulose membranes. Left panel: Detection of capsids with A20 capsid-specific antibody. Right panel: Detection of VP proteins with B1 antibody. Samples from yeast carrying plasmids pESC-HIS, DB022, DB023 and DB040 (No VPs); DB046, DB027, DB029 and DB040 (AAP-op) or DB025, DB027, DB029 and DB040 (AAP). (B) Western blot analysis of VP proteins in subcellular fractions from yeast carrying plasmids DB046, DB027, DB029 and DB040. VP proteins were detected using B1 antibody. GAPDH was detected using a specific antibody. (C) Electron microscopy of purified AAV capsids from yeast carrying plasmids DB046, DB027, DB029 and DB040. Examples of individual AAV capsids are pointed by arrows. Scale bar corresponds to 200 nm.</p

Detection of AAV proteins expressed in yeast.

<p>Yeast samples were processed for SDS-PAGE and western blot. (A) Detection of VP capsid proteins with B1 antibody. Samples from yeast carrying plasmids pESC-HIS, DB022, DB023 and DB040 (left lane, No VPs) or DB046, DB027, DB029 and DB040 (right lane, VPs). (B) Detection of HA-tagged AAP with anti-HA antibody. Samples from yeast carrying plasmids DB232, DB138, DB029 and DB040 (left lane, AAP-HA); DB046, DB228, DB029 and DB040 (middle lane, AAP no HA) or DB233, DB138, DB029 and DB040 (right lane, AAP-op-HA). (C) Detection of Rep proteins with 303.1 anti-Rep antibody. Samples from yeast carrying plasmids DB046, DB026, DB028 and DB040 (left lane, No Rep) or DB046, DB027, DB029 and DB040 (right lane, Rep78/Rep52).</p

Encapsidation of AAV DNA.

<p>(A) Analysis of GFP DNA co-purified with AAV capsids. AAV capsids were purified by AVB affinity chromatography from yeast carrying plasmids DB021, DB027, DB029 and DB040 (No AAP); DB155, DB149, DB029 and DB040 (All AAV); pESC-HIS, DB022, DB023, DB040 (No VPs); DB155, DB149, DB028 and DB040 (No Rep78). GFP DNA was quantified by ddPCR while capsids were quantified by ELISA. Asterisks (*) indicate capsid content bellow the limit of detection. (B) Detection by western blot of VP proteins in affinity purified capsids from yeast carrying plasmids DB155, DB149, DB029 and DB040 (All AAV) or from yeast carrying plasmids pESC-HIS, pESC-LEU, DB081 and DB040 (Rep78). Note that plasmid DB081 contains a VP2 expression cassette, however VP2 alone does not form capsids and is not purified by AVB affinity chromatography. (C) Detection by southern blot of GFP DNA co-purified with AAV capsids. Samples were treated with benzonase (b) or not treated before DNA extraction and run as single stranded DNA on alkaline gels. A band of the expected size for the ITR-GFP-ITR rAAV DNA product was observed (white arrow) as well as additional material of smaller size (black arrow) which is likely the result of partial encapsidation [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173010#pone.0173010.ref008" target="_blank">8</a>]. Refer to panel B for sample descriptions.</p

Infective rAAV virions in affinity purified fractions from yeast.

<p>(A) Detection of infectious rAAV virions in affinity purified fractions from yeast carrying plasmids DB155, DB149, DB029 and DB040 (AAV, GAL10p-VP1); pESC-HIS, DB102, DB023 and DB040 (No CAP); DB155, DB205, DB029 and DB040 (AAV, GAL7p-VP1); DB155, DB220, DB029 and DB040 (AAV, GAL7p-VP1-op). Affinity purified fraction treated at 90°C to denature capsids prior to addition to HEK293 cultures (AAV, 90°C). rAAV2-GFP produced in Sf9/baculovirus was used as a reference (AAV, Sf9/bac). HEK293 cells were treated with etoposide and transduced with 1.2E+8 viral genomes from each of the preparations. GFP expressing cells were observed 4 days after on a UV microscope. (B) Analysis with B1 anti-VP antibody of VP protein content in affinity purified fractions. rAAV produced in Sf9/baculovirus (Sf9/bac) was loaded in three fold serial dilutions for comparison. (C) Flow cytometry histograms showing percentage of cells expressing GFP.</p

Replication of AAV DNA in yeast.

<p>(A) Southern blot with a GFP specific probe of yeast samples carrying plasmids DB046, DB026, DB028 and DB040 (No Rep) or DB046, DB027, DB029 and DB040 (Rep78, Rep52). The top band (black arrow) corresponds to plasmid DB040-pAAV-GFP-2mic-URA3 (pAAV-GFP/URA3). The bottom band (white arrow) likely corresponds to the rescued ITR-GFP-ITR DNA. The middle band (*) could be a dimer of the ITR-GFP-ITR DNA. (B) Western blot with 303.9 anti-Rep antibody of samples from yeast carrying plasmids DB046, DB026, DB028 and DB040 (No Rep); DB046, DB027, DB029 and DB040 (Rep78, Rep52); DB046, DB027, DB135 and DB040 (Rep78-op, Rep52); DB046, DB138, DB029 and DB040 (Rep78, Rep52-op); DB046, DB138, DB135 and DB040 (Rep78-op, Rep52-op). Additional bands not consistent with the expected size for Rep78 and Rep52 were also observed (*). (C) Southern blot with a GFP specific probe of yeast samples described above. See band descriptions in panel A and sample descriptions in panel B. (D) quantification by ddPCR of GFP and URA3 DNA species. ddPCR was performed with primers and TaqMan probes specific for GFP, URA3 and 18S ribosomal DNA sequences. Black bars indicate the average (2 to 3 samples) GFP copy number relative to 18S rDNA copy number. White bars indicate the average URA3 copy number relative to 18S rDNA. The ratio of GFP to URA3 copies is indicated below the diagram. See sample descriptions in panel B.</p