Name: a quantitative dot blot assay for aav titration and its use for functional assessment of the adeno-associated virus assembly-activating proteins
Uploaded: 2018-06-12T13:00:00
Description: Watch this Scientific Journal Video about A Quantitative Dot Blot Assay for AAV Titration and Its Use for Functional Assessment of the Adeno-associated Virus Assembly-activating Proteins at JoVE.com

We thank Xiao Xiao at the University of North Carolina at Chapel Hill for providing us with the pEMBL-CMV-GFP plasmid. We thank Christopher Cheng and Samuel J. Huang for critical reading of the manuscript. This work was supported by Public Health Service grants (NIH R01 NS088399 and T32 EY232113).

NOTE: Recipes for the solutions and buffers needed for this protocol are provided in Table 1. The protocol described below is for the VP-AAP cross-complementation dot blot assay to study the roles of the AAP proteins in capsid assembly. The method for the more generic quantitative dot blot assay for purified AAV vector titration is explained in the Representative Results section.
1. Construction of VP3, AAP, and AAV2 Rep Expressing Plasmids
<ol>
	<li><strong...

<ol>
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In this report, the utility of quantitative dot blot assays to study AAV AAPs and their role in capsid assembly is described. Knowledge gained from these studies can provide detailed insights into the innate differences in the process of AAV capsid assembly and the functional role of AAPs between different serotypes. In this respect, the AAV VP3-AAP cross-complementation dot blot assay revealed that Snake AAV VP3 displayed a strict dependency on the co-expression of its cognate AAP for capsid assembly and that Snake AAP ...

Adeno-associated virus (AAV) is a small, non-enveloped, single-stranded DNA virus with a genome of approximately 4.7 kilobases (kb). The AAV genome contains open-reading frames (ORFs) for the rep and cap genes. In 2010, a previously unidentified nonstructural protein encoded by a +1 frame-shifted ORF within the AAV2 cap gene was discovered by Sonntag et al. and found to play a critical role in the assembly of AAV2 capsid VP monomer proteins into a viral capsid1. This novel protein has been named assembly-activating protein (AAP) after the role it plays in promoting capsid assembly1.
The ORFs for AAP have been identified bioinformatically in the genomes of all parvoviruses within the genus Dependoparvovirus, but not within the genomes of viruses of different genera of the parvovirus family1,2. Functional studies of this novel protein were initially focused on the AAP from the prototype AAV2 (AAP2), which has established the essential role of AAP2 in targeting unassembled VP proteins to the nucleolus for their accumulation and formation into fully assembled capsids1,3,4. The inability of the AAV2 capsids to assemble in the absence of AAP expression has been independently confirmed by multiple groups, including ours1,2,3,4,5. Subsequent studies on AAV serotypes 1, 8, and 9 corroborated the critical role of AAPs in capsid assembly, as VP3 monomer proteins of AAV1, 8, and 9 were unable to form a fully assembled capsid in the absence of co-expression of AAP2.
Recently, through approaches that include the use of quantitative dot blot assays, we investigated the ability of AAV1 to 12 VP3 monomers to assemble into capsids in the absence of AAP expression and the ability of AAP1 to 12 to promote assembly of VP3 monomers from heterologous serotypes. This study has revealed that AAV4, 5, and 11 VP3 monomers can assemble without AAP. Additionally, it was found that eight out of the twelve AAP serotypes we examined (i.e., all but AAP4, 5, 11, and 12) displayed a broad ability to support capsid assembly of heterologous AAV serotype capsids, while AAP4, 5, 11 and 12 displayed a substantially limited ability in this regard6. These four serotypes are phylogenetically distant from the other AAP serotypes2,3. Moreover, the study has uncovered significant heterogeneity in subcellular localizations of different AAPs6. Furthermore, the study has suggested that the tight association of AAP with assembled capsids and the nucleolus, the hallmark of AAV2 capsid assembly, cannot necessarily be extended to other serotypes including AAV5, 8, and 9, which display nucleolar exclusion of assembled capsids6. Thus, the information gained from the study of any particular serotype AAP is not broadly applicable to all AAP biology. Such puzzling nature of AAP biology underscores the need to investigate the role and function of each AAP from both canonical and non-canonical AAV serotypes.
The biological role of AAP in capsid assembly can be assessed by determining the fully-packaged AAV viral particle titers produced in human embryonic kidney (HEK) 293 cells, the most commonly used cell line for AAV vector production, with or without AAP protein expression. The standard methods for AAV quantitation are quantitative PCR (qPCR)-based assays7,8 and quantitative dot blot-based assays9. Other methods for AAV viral particle quantitation such as enzyme-linked immunosorbent assay10,11 or optical density measurement12 are not ideal for samples derived from many different AAV serotypes or samples contaminated with impurities (crude lysates or culture media), which are often the samples used for AAV research. Currently, qPCR is most widely used for AAV quantitation; however, it is necessary to acknowledge potential caveats of the qPCR-based assay, as the assay can result in systemic errors and significant titer variations13,14. PCR-based assays are affected by a number of potentially confounding factors, such as the presence of covalently closed terminal hairpins in PCR templates that inhibit amplification13. Even an experienced individual can introduce potential confounding factors into a qPCR-based assay unknowingly13. In contrast, quantitative dot blot assays are a classical molecular biology technique that does not involve genome amplification and uses a much simpler principle with a minimal risk of errors as compared to qPCR-based assays. The method is less technically challenging; therefore, the assay results are reasonably reproducible even by inexperienced individuals.
In this report, we describe the methodological details of a quantitative dot blot assay we routinely use for AAV vector quantitation and provide an example of how to apply the assay to study the assembly-promoting role of AAPs in common serotypes (AAV5 and AAV9) and a previously uncharacterized AAP from Snake AAV14. In nature, AAV VP proteins and AAP proteins are expressed in cis from a single gene (i.e., VP-AAP cis-complementation), while in the assay described here, VP and AAP proteins are supplied in trans from two separate plasmids (i.e., VP-AAP trans-complementation). Since each VP or AAP protein from different serotypes can be expressed from each independent plasmid, it becomes possible to test heterologous VP-AAP combinations for capsid assembly (i.e., VP-AAP cross-complementation). Briefly, AAV VP3 from various serotypes is expressed in HEK 293 cells by plasmid DNA transfection to package an AAV vector genome in the presence or absence of the cognate serotype AAP, or in the presence of a heterologous serotype AAP. Following production, culture media and cell lysates are subjected to a dot blot assay to quantify the viral genome within the capsid shell. The first step of the dot blot assay is to treat samples with a nuclease to remove contaminating plasmid DNAs and unpackaged AAV genomes in samples. Failure to do so would increase the background signals in particular when unpurified samples are assayed. This is then followed by a protease treatment to break viral capsids and release nuclease-resistant viral genomes into sample solutions. Next, viral genomes are denatured, blotted on a membrane, and hybridized with a viral genome-specific DNA probe for quantitation. In the example assay reported here, we demonstrate that Snake AAV VP3 requires Snake AAP for capsid assembly and that Snake AAP does not promote the assembly of AAV9 capsids unlike many of the AAPs derived from AAP-dependent serotypes that can also promote assembly of heterologous serotype capsids. Lastly, we report an important caveat to qPCR or dot blot-based AAV quantitation assays that the choice of nuclease significantly affects the assay results.

<table>
	<tbody>
		<tr>
			<td>Benzonase</td>
			<td>MilliporeSigma</td>
			<td>1016970001</td>
			<td>Referred to as Serratia marcescen endonuclease in the main text.</td>
		</tr>
		<tr>
			<td>DNase I</td>
			<td>Roche</td>
			<td>4716728001</td>
			<td>Referred to as DNase I Enzyme A in the main text.</td>
		</tr>
		<tr>
			<td>DNase I</td>
			<td>Invitrogen</td>
			<td>18047019</td>
			<td>Referred to as DNase I Enzyme B in the main text.</td>
		</tr>
		<tr>
			<td>DNase I</td>
			<td>New England Biolabs</td>
			<td>M0303L</td>
			<td>Referred to as DNase I Enzyme C in the main text.</td>
		</tr>
		<tr>
			<td>1.5 mL Attached O-Ring Screw Cap Microcentrifuge Tubes</td>
			<td>Corning</td>
			<td>430909</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL Microcentrifuge Tubes</td>
			<td>Thermo Fisher Scientific</td>
			<td>05-408-129</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL Polypropylene Conical Tube</td>
			<td>Corning</td>
			<td>352097</td>
			<td></td>
		</tr>
		<tr>
			<td>3 M Sodium Acetate</td>
			<td>Teknova</td>
			<td>S0298</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL Polypropylene Conical Tube</td>
			<td>Corning</td>
			<td>430291</td>
			<td></td>
		</tr>
		<tr>
			<td>AAV-293 Cells</td>
			<td>Agilent</td>
			<td>240073</td>
			<td>HEK-293 cell line optimized for the packaging of AAV virions.</td>
		</tr>
		<tr>
			<td>AccuGENE 0.5 M EDTA Solution</td>
			<td>Lonza</td>
			<td>51234</td>
			<td></td>
		</tr>
		<tr>
			<td>AccuGENE 1 M Tris-HCl pH 8.0</td>
			<td>Lonza</td>
			<td>51238</td>
			<td></td>
		</tr>
		<tr>
			<td>AccuGENE 10% SDS</td>
			<td>Lonza</td>
			<td>51213</td>
			<td></td>
		</tr>
		<tr>
			<td>AccuGENE 1X TE Buffer</td>
			<td>Lonza</td>
			<td>51235</td>
			<td></td>
		</tr>
		<tr>
			<td>Bio-Dot Apparatus</td>
			<td>Bio-Rad</td>
			<td>1706545</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>MilliporeSigma</td>
			<td>A3294</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Lifter</td>
			<td>Corning</td>
			<td>3008</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>MilliporeSigma</td>
			<td>372978</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA Extractor Kit</td>
			<td>Wako Pure Chemical Industries</td>
			<td>295-50201</td>
			<td>This is used for quantitative dot blots on purified virus. We use only a half volume of all the reagents in each step recommended for the Protocol Scheme 2 in the manual provided by the manufacturer.</td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Modified Eagle Medium (DMEM)-high glucose (4.5 g/L)</td>
			<td>Lonza</td>
			<td>12-614F</td>
			<td></td>
		</tr>
		<tr>
			<td>ElectroMAX DH10B Cells</td>
			<td>Thermo Fisher Scientific</td>
			<td>18290015</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol 200 Proof</td>
			<td>Decon Labs</td>
			<td>2716</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>VWR</td>
			<td>1500-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Ficoll 400</td>
			<td>Alfa Aesar</td>
			<td>B22095</td>
			<td>Referred to as Polysucrose 400.</td>
		</tr>
		<tr>
			<td>Fluorescence&#160;Microscope</td>
			<td>Zeiss</td>
			<td>Axiovert 40 CFL</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycogen</td>
			<td>Roche</td>
			<td>10901393001</td>
			<td></td>
		</tr>
		<tr>
			<td>Herring Sperm DNA</td>
			<td>Invitrogen</td>
			<td>15634-017</td>
			<td></td>
		</tr>
		<tr>
			<td>Hybridization Tubes</td>
			<td>Thermo Fisher Scientific</td>
			<td>13-247-150</td>
			<td></td>
		</tr>
		<tr>
			<td>ImageQuant TL</td>
			<td>GE Healthcare Life Sciences</td>
			<td>ImageQuant TL</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine 200 mM</td>
			<td>Lonza</td>
			<td>17-605E</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium Choloride Hexahydrate</td>
			<td>MilliporeSigma</td>
			<td>M0250</td>
			<td></td>
		</tr>
		<tr>
			<td>MicroPulser Electroporator</td>
			<td>Bio-Rad</td>
			<td>1652100</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini Quick Spin DNA Columns</td>
			<td>MilliporeSigma</td>
			<td>11814419001</td>
			<td></td>
		</tr>
		<tr>
			<td>pAAV-RC2 Vector</td>
			<td>Cell Biolabs Inc</td>
			<td>VPK-422</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin 10K/10K</td>
			<td>Lonza</td>
			<td>17-602E</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline</td>
			<td>Lonza</td>
			<td>17-516F</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphorimaging Exposure Cassette</td>
			<td>GE Healthcare Life Sciences</td>
			<td>Various</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphorimaging Screen</td>
			<td>GE Healthcare Life Sciences</td>
			<td>Various</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasmid Maxi Kit</td>
			<td>QIAGEN</td>
			<td>12162</td>
			<td></td>
		</tr>
		<tr>
			<td>Platinum Pfx DNA Polymerase</td>
			<td>Invitrogen</td>
			<td>11708013</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethylenimine</td>
			<td>Polysciences Inc</td>
			<td>23966-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyvinylpyrrolidone</td>
			<td>MilliporeSigma</td>
			<td>P5288</td>
			<td></td>
		</tr>
		<tr>
			<td>Prime-It II Random Primer Labeling Kit</td>
			<td>Agilent Technologies</td>
			<td>300385</td>
			<td></td>
		</tr>
		<tr>
			<td>Primer AAP Forward (N25 nucleotides from the 4th nucleotide in the AAP ORF, RE1 is a restriction enzyme site for cloning): CTAA-RE1-CACCATGGACTACAAGGACGACGATGACAAA-N25</td>
			<td>MilliporeSigma</td>
			<td>Custom Primer</td>
			<td></td>
		</tr>
		<tr>
			<td>Primer AAP Reverse (N25 is the last 25 nucleotides of the AAP ORF, RE2 is a restriction enzyme site for cloning): TCTT-RE2-N25</td>
			<td>MilliporeSigma</td>
			<td>Custom Primer</td>
			<td></td>
		</tr>
		<tr>
			<td>Primer VP3 Forward (N25 the first 25 nucleotides of the VP3 ORF, RE1 is a restriction enzyme site for cloning): CTAA-RE1-CACC-N25</td>
			<td>MilliporeSigma</td>
			<td>Custom Primer</td>
			<td></td>
		</tr>
		<tr>
			<td>Primer VP3 Reverse (N25 is the last 25 nucleotides of the AAP ORF, RE2 is a restriction enzyme site for cloning): TCTT-RE2-N25</td>
			<td>MilliporeSigma</td>
			<td>Custom Primer</td>
			<td></td>
		</tr>
		<tr>
			<td>Proteinase K Solution</td>
			<td>Invitrogen</td>
			<td>25530-049</td>
			<td></td>
		</tr>
		<tr>
			<td>Restriction Enzymes</td>
			<td>New England Biolabs</td>
			<td>Various</td>
			<td></td>
		</tr>
		<tr>
			<td>Salmon Sperm DNA</td>
			<td>Invitrogen</td>
			<td>15632011</td>
			<td></td>
		</tr>
		<tr>
			<td>Serological Pipettes</td>
			<td>Thermo Fisher Scientific</td>
			<td>13-678-11D / 13-678-11E / 13-678-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Thermo Fisher Scientific</td>
			<td>S271-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Citrate Tribasic Dihydrate</td>
			<td>MilliporeSigma</td>
			<td>C8532</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 DNA Polymerase</td>
			<td>New England Biolabs</td>
			<td>M0203L</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermal Cycler</td>
			<td>Eppendorf</td>
			<td>6321000515</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue-culture Treated 6-well Plate</td>
			<td>Corning</td>
			<td>353046</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris Base</td>
			<td>Thermo Fisher Scientific</td>
			<td>BP152-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Typhoon FLA 7000</td>
			<td>GE Healthcare Life Sciences</td>
			<td>28955809</td>
			<td></td>
		</tr>
		<tr>
			<td>UltraPure Buffer-Saturated Phenol</td>
			<td>Invitrogen</td>
			<td>15513-047</td>
			<td></td>
		</tr>
		<tr>
			<td>UV Crosslinker</td>
			<td>Spectroline</td>
			<td>XLE-1000</td>
			<td>Optimal Crosslink mode is used, providing a UV energy dosage of 120 mJ/cm2.</td>
		</tr>
		<tr>
			<td>Zeta-Probe Membrane</td>
			<td>Bio-Rad</td>
			<td>162-0165</td>
			<td></td>
		</tr>
	</tbody>
</table>

a quantitative dot blot assay for aav titration and its use for functional assessment of the adeno-associated virus assembly-activating proteins

While adeno-associated virus (AAV) is widely accepted as an attractive vector for gene therapy, it also serves as a model virus for understanding virus biology. In the latter respect, the recent discovery of a non-structural AAV protein, termed assembly-activating protein (AAP), has shed new light on the processes involved in assembly of the viral capsid VP proteins into a capsid. Although many AAV serotypes require AAP for assembly, we have recently reported that AAV4, 5, and 11 are exceptions to this rule. Furthermore, we demonstrated that AAPs and assembled capsids of different serotypes localize to different subcellular compartments. This unexpected heterogeneity in the biological properties and functional roles of AAPs among different AAV serotypes underscores the importance of studies on AAPs derived from diverse serotypes. This manuscript details a straightforward dot blot assay for AAV quantitation and its application to assess AAP dependency and serotype specificity in capsid assembly. To demonstrate the utility of this dot blot assay, we set out to characterize capsid assembly and AAP dependency of Snake AAV, a previously uncharacterized reptile AAV, as well as AAV5 and AAV9, which have previously been shown to be AAP-independent and AAP-dependent serotypes, respectively. The assay revealed that Snake AAV capsid assembly requires Snake AAP and cannot be promoted by AAPs from AAV5 and AAV9. The assay also showed that, unlike many of the common serotype AAPs that promote heterologous capsid assembly by cross-complementation, Snake AAP does not promote assembly of AAV9 capsids. In addition, we show that the choice of nuclease significantly affects the readout of the dot blot assay, and thus, choosing an optimal enzyme is critical for successful assessment of AAV titers.

A representative result of quantitative dot blots for quantitation of purified AAV vector stocks produced on a large scale is shown in Figure 1. With this dot blot assay, the titer of a double-stranded AAV2G9-CMV-GFP vector stock was determined. The vector was purified by two rounds of cesium chloride (CsCl) density-gradient ultracentrifugation followed by dialysis as previously described20. In general, for purified AAV vector stocks, ...

Watch this Scientific Journal Video about A Quantitative Dot Blot Assay for AAV Titration and Its Use for Functional Assessment of the Adeno-associated Virus Assembly-activating Proteins at JoVE.com

A Quantitative Dot Blot Assay for AAV Titration and Its Use for Functional Assessment of the Adeno-associated Virus Assembly-activating Proteins

This manuscript details a straightforward dot blot assay for quantitation of adeno-associated virus (AAV) titers and its application to study the role of assembly-activating proteins (AAPs), a novel class of non-structural viral proteins found in all AAV serotypes, in promoting the assembly of capsids derived from cognate and heterologous AAV serotypes.

While adeno-associated virus (AAV) is widely accepted as an attractive vector for gene therapy, it also serves as a model virus for understanding virus ...

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