1. AAV interactions with host-cell
receptor AAVR
MaLi Dong
Chapman Lab
QBB Engineering Internship Program
26 August 2015

2. Adeno-associated virus (AAV2)
• Single stranded DNA
• Not known to cause disease
• Enters host cell through receptor-
mediated endocytosis
• Has clinical success as recombinant DNA
in gene therapy
 Can infect without integrating into genome of
host cell Fig.1. 3D crystal structure of AAV2
centered on viral 5-fold axis1
1. Weisman, J, 2015. Surface of the AAV-2 serotype of the adeno-associated virus, with one of the five fold axes centered. Derived from the 3-A crystal structure,1LP3. Image.
Wikipedia (2015).

3. Adeno-associated virus receptor (AAVR)
• Direct binding
• Resistant mammalian cells to AAV2 infection
• Universal receptor
• E.coli as host cells for AAVR protein
• Express with His-tags to test with other receptors
 6 histidine residue

4. Gel electrophoresis
• Resulting test expressions
• Isopropyl β-D-1-
thiogalactopyranoside (IPTG)
• Little difference between 3 hour
and overnight induction
• 1-5 His-tag constructs were used
in electron microscopy

5. Sample preparation
Fig.2. 300 mesh copper grid Fig.3. Size comparison Fig.4. PELCO easiGlowTM

6. Tecnai iCorr
• Integrated light and electron
microscope
 Combines fluorescence light
microscope and electron
microscope
• Accelerates CLEM experiments
• High resolution EM images
sample

7. Grid 4
• AAV2 VLP –KL-His complex
in HN buffer
• 1/10 dilution
• Aggregated binding around
virus
• Imidazole to free His-
tagged proteins from Nickel
ions

8. Grid 6
• Same mix as Grid 4
• 1/100 dilution
• Aggregated binding around
virus
• Nanogold appears to be
binding

9. Thanks to
• Dr. Michael Chapman
• Dr. Qing Xie
• Dr. Omar Davulcu
• Nancy Meyer
• Andrew Trzynka
• Jazz Weisman