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

Summary

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.

Abstract

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.

Introduction

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.

Protocol

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

1. Construction of pCMV-AAVx-VP3 (x = serotypes)
   1. PCR-amplify the entire VP3 ORF (1.6 kb) using a high-fidelity DNA polymerase and t....

Results

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, .......

Discussion

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 .......

Materials

List of materials used in this article

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