2 Curriculum in Applied and Materials Sciences, Program in Cellular and Molecular Biophysics, CB# 3287, University of North Carolina, Chapel Hill, NC 27599; 3 Division of Medicinal Chemistry and Natural Products, School of Pharmacy, CB# 7630, University of North Carolina, Chapel Hill, NC 27599; 4 Gene Therapy Center, CB# 7352, University of North Carolina, Chapel Hill, NC 27599; and 5 Department of Physics and Astronomy, CB# 3255, University of North Carolina, Chapel Hill, NC 27599
Received on March 23, 2004; revised on June 11, 2004; accepted on June 17, 2004
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Abstract |
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Key words: adeno-associated virus / atomic force microscope / binding assay / gene therapy / heparan sulfate
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Introduction |
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Adeno-associated virus (AAV), a single-stranded DNA virus carrying a 4680-base-long DNA molecule, is a promising viral vector for gene therapy. There are at least eight serologically distinct primary isolates (AAV-18) (Gao et al., 2002), of which AAV-2 has been the most extensively characterized. AAV is becoming a major vector to deliver genes to a variety of tissues because of its low toxicity and low immunogenicity (Berns and Giraud, 1996
; Monahan and Samulski, 2000
). The cellular receptors that serve as primary and secondary binding moieties for the virus are currently under intense study, first because they determine the specific tissues that can be treated with these gene delivery vectors, and second, because they appear to influence the intracellular trafficking of the virus, thus effecting the efficiency of its translocation to the nucleus and subsequent expression (Yan et al., 2004
).
Summerford and Samulski (1998) demonstrated that AAV-2 uses HS as a primary receptor for binding to the cell surface and that
Vß 5 integrin is a secondary receptor used for internalization (Summerford et al., 1999
). Human fibroblast growth factor receptor 1 also serves as a secondary receptor for AAV-2 vectors (Qing et al., 1999
). Mutations in the putative HS domains on the capsid abolished the binding of AAV-2 viral particles to heparin-Sepharose (Opie et al., 2003
; Rabinowitz et al., 1999
), and recent crystal structure and molecular modeling studies of AAV-2 are consistent with the HS interacting with the highly positively charged region at the viral threefold axis of symmetry (Kern et al., 2003
; Xie et al., 2002
). In addition, a study by Pruchnic and colleagues (2000)
suggests that HSPG is a primary natural receptor for AAV-2 in muscle fibers, because the AAV-2 infected areas overlap with the regions where HSPGs are highly expressed. It should be noted that herpes simplex virus type 1 uses a unique HS saccharide sequence containing 3-O-sulfated glucosamine residue as an entry receptor, suggesting that viruses recognize a specific polysaccharide-based receptor (Liu et al., 2002
; Shukla et al., 1999
; Xia et al., 2002
). It is unknown, however, whether AAV-2 binds to a specific sugar sequence.
Current methods to study virus and receptor binding include cell-based assays, microtiter plates, and surface plasmon resonance (Lea et al., 1998; Qiu et al., 2000
; Summerford and Samulski, 1998
). Our research attention has been focused on using the atomic force microscope (AFM), a scanning probe imaging system, to study the interaction of viruses and their cell surface receptors. The AFM provides height data on an analyzed object with nanometer resolution and offers the insight to virus capsid structure by imaging the viral particle. Since its invention in 1986, AFM has complemented techniques such has optical microscopy, electron microscopy, and X-ray crystallography (Binnig et al., 1986
). Although the resolution of the AFM cannot compare with the subnanometer resolution of electron microscopy and X-ray crystallography, the AFM has the potential to image biomolecules and biological processes in both air and liquid with minimal sample modification and preparation. Thus the observed results more closely replicate physiological conditions (Bustamante and Keller, 1995
; Engel and Muller, 2000
; Hansma and Hoh, 1994
; Shao et al., 1996
). In addition, manipulation with the AFM probe allows the binding forces between biological entities to be measured, or alternatively, an external force to be applied on the objects (Engel et al., 1999
; Guthold et al., 1999
). For these reasons, the AFM is particularly useful in studying viral interactions with cellular structures and infection processes.
The AFM has been used to distinguish the morphology of lymphoid cells with and without HIV infection (Cricenti et al., 1999). Real-time AFM imaging of live cells has allowed the observation of exocytosis of a virus from an infected cell (Ohnesorge et al., 1997
). Through AFM manipulation of individual viruses, Falvo et al. (1997)
measured the elastic properties of tobacco mosaic virus. The AFM thus far has been primarily used in structural investigations of viruses (Drygin et al., 1998
; Kuznetsov et al., 2001
; Malkin et al., 2003
; Müller et al., 1997
). Recently, Nettikadan et al. (2003)
reported imaging viruses captured on an antibody-coated chip using the AFM.
In this report, an AFM imagingbased assay is used to study the binding between AAV-2 and HS immobilized HS on a silicon surface. As expected for a specific virus-receptor interaction, soluble HS competed the binding of AAV-2 onto the HS surface in a concentration-dependent manner. Using the AFM assay allowed us to determine the binding affinity of AAV-2 to HS to be 3.4 nM.
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Results and discussion |
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Characterization and modification of the functionalized surface
Determination of the surface roughness of the functionalized areas
The functionalized surface for the AFM study was achieved by immobilizing HS on the surface through the biotin-neutravidin interaction. A similar technique has been used to immobilize HS chains on sensor chips to study the interaction of HS and HS-binding proteins using surface plasmon resonance (Hernaiz et al., 2000). The procedure for conjugating biotin to the surface and creating biotin-avidin-biotinylated HS bridges is summarized in Figure 1.
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Analysis of the binding of HS to AAV using AFM
The binding of AAV-2 to the functionalized surface
The binding experiments with AAV-2 viral particles and HS were carried out in the microreaction wells on the HS-functionalized surface. Image analysis using the AFM was performed after removing the silicone layer that was used to make the microreaction wells. The topographic images of the surface are shown in Figure 3A. Numerous spots (in greyscale) with the approximate height of 20 nm were observed, suggesting the presence of viral particles on the surface. Measurement of the number of viral particles in a given area (particle density) was used to quantify the binding of HS and the virus. We also measured the binding of AAV-2 to HS as a function of time. Three topographic images of the functionalized surface at time 0, 30, and 120 min are shown in Figure 3A. A graphic presentation of virus density over time is shown in Figure 3B. It is apparent that the virus density reached its plateau at around 60 min, suggesting that the binding of AAV-2 to HS reached maximum.
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The binding of each of the four serotypes was compared on surfaces that were fully functionalized with either HS or with only neutravidin. In the presence of HS, the density of bound viral particles increased by 40- and 50-fold for AAV-2 and AAV-3, respectively, when compared to surfaces lacking HS. In contrast, we did not observe any increase in binding to the HS-functionalized surface for AAV-1 and AAV-5, consistent with previous viral infection results demonstrating that AAV-1 and AAV-5 do not interact with HS.
Determination of the binding affinity between AAV-2 and HS using the AFM
Our observation of the binding and competition between surfaces immobilized with HS and AAV-2 suggested that this method could be extended to measure the affinity between the two components. Solutions containing different concentrations of virus were spotted in individual microreaction wells on the uniformly HS-functionalized surface. After 120 min incubation, the silicone was removed for AFM imaging analysis. The particle density as a function of the concentration of AAV-2 is shown in Figure 5, where the molar concentration of AAV-2 was calculated using this formula: the titer of virus (viral particles/L) ÷ 6.022 x 1023 (Avogadro's number). Based on this plot, we calculated the binding affinity (Kd) between HS and AAV-2 to be 3.4 ± 0.3 nM. We noted that the highest concentration of AAV-2 used in this experiment was threefold below the Kd value, which may have affected the accuracy of our measurement. Consistent with our determination, the binding affinity of AAV-2 and heparin was determined to be 1.72.5 nM by a radiolabeled binding assay of binding 125I-labeled AAV-2 to a heparin-coated surface (Qiu et al., 2000). Technically, it is very difficult to obtain a highly concentrated solution of AAV-2 because the viruses tend to aggregate. We also noted that the Kd value, 3.4 nM, was significantly lower than the IC50 value of soluble HS, 300 nM (Figure 4). This difference was likely due to the fact that one AAV-2 capsid contains potentially 20 HS binding sites (Xie et al., 2002
).
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It should be noted that we were able to conduct multiple binding experiments on a single surface chip, limited only by the number of microreaction wells that can be formed in the available space. With further development in the design, our technique holds potential for a high throughput array system to study the interaction of viruses and their receptors. Given that each binding reaction was conducted in a volume of less than 12 µl, competition studies with molecules that are difficult to produce or obtain can be conducted efficiently. To our knowledge, this is the first report utilizing AFM to study the interactions between a virus and its receptors. Given that HS is involved in the infections of more than 16 different viruses (Liu and Thorp, 2002), this method could become a common method for the analysis of the bindings of receptors and viruses.
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Materials and methods |
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Preparation of biotinylated HSPG(B*-HSPG)
HSPG was purified from bovine kidney actone powder (ICN, Irvine, CA) using DEAE chromatography as described by Kondo and colleagues (1971), and the chondroitin sulfate was removed by digesting with chondroitin ABCase (from Sigma, St. Louis, MO). HSPG-biotin was prepared by reacting HSPG with EZ-link NHS-biotin (Pierce, Rockford, IL) according to the manufacturer's protocol. Briefly, 3 mg purified HSPG was incubated with 0.1 mg NHS-biotin in 1 ml reaction buffer (0.1 M phosphate, 0.15 M NaCl, pH 7.2) at room temperature for 30 min. The reaction was stopped by adding 200 ml 50 mM Tris-base. The unreacted NHS-biotin was removed by DEAE chromatography, which was equilibrated with 150 mM NaCl, and HSPG was eluted by 1 M NaCl. The sample was dialyzed to remove the salt.
The HSPG-biotin was purified by using Ultralink Immobilized Monomeric Avidin (Pierce) according to the manufacturer's protocol. The HSPG sample was dissolved in 2 ml PBS buffer and applied to the avidin column (1.6 x 2.5 cm). The HSPG without biotin was removed by washing the column with 2 ml PBS buffer six times. The B*-HSPG was eluted by 6 ml of a buffer containing 1 mM of D-biotin (Pierce) in PBS blocking and elution buffer. After dialysis, 160 µg of B*-HSPG was recovered as determined by alcian blue (Bjornsson, 1993
). It should be noted that HS contains low level of N-unsubstituted glucosamine residues, which may react with NHS-biotin (Toida et al., 1997
). As a result, the biotin is coupled to the HS polysaccharide chain directly.
Preparation and functionalization of surface
For the purpose of AFM imaging, fiducial marks on the surface were used to identify the location of each well on the surface. Each mark was assigned a number and a letter signifying the column and row, respectively. Photolithography techniques were used to pattern 21 3 x 3 array fiducial marks on an oxidized silicon wafer (Virginia Semiconductors, Fredericksburg, VA). The freshly prepared wafer was cut into individual 3 x 3 array chips before surface modifications. Each chip was UV cleaned (UV-Ozone Cleaner Model 135500 Boekel Industries, Cambridge, England), rinsed with Biocel water (Millipore, Billerica, MA) and dried under a filtered N2 stream.
For the purpose of silanization, the chips were exposed to 254 nm UV photo-oxidation for 30 min. The oxidized chips were exposed to aminopropyltriethylsilane (APTES) (Sigma-Aldrich, St. Louis, MO) vapor under nitrogen for 2 h to produce amino groups on the surface (as illustrated in Figure 1). The APTES-treated chips were then cured at 120°C for 5 min before submerging the chips into dimethyl sulfoxide containing 0.5 mg/ml NHS-LC-LC-biotin (Pierce) for 1 h at room temperature. The resulting chips, functionalized with biotin, were then washed with purified Biocel water and dried immediately using a filtered N2 stream.
Preparation of microreaction wells
Removable reaction wells were created on the freshly prepared biotinylated chip using a silicone elastomere (as illustrated in Figure 2). PDMS (Sylgard 184, Dow Corning, Midland, MI), was precured with a thickness of 4 mm on a fluoro-silanized silicon wafer (same silanization method as described) in a petri dish. Once the PDMS was cured, 1.5 cm x 1.5 cm PDMS squares were cut out and used for each array. Wells (3 x 3) were punched out into the PDMS squares using 2-mm stainless steel tubing. A Plexiglas hole-punch guide was used to ensure that the wells in the PDMS lined up with the fiducial marks on the chip surface. The PDMS microreaction wells were sonicated in 2% sodium dodecyl sulfate solution for 5 min, rinsed with Milli-Q water, and dried under an N2 stream. This cleaning step ensured a good seal between the PDMS and the chip surfaces. The complete reaction well assembly is shown in Figure 2.
Preparation of HS surface
The HS surface chips were prepared immediately before the experiment for the binding of AAVs. Two steps were involved in the immobilization of B*-HSPG on the biotinylated silicon chip. First, 1.5 µl of 0.25 mg/ml neutravidin (Molecular Probes, Eugene, OR) in PBS and 0.05% v/v Tween was pipetted into the microreaction well and incubated for 30 min at room temperature. After the solution was removed, the wells were washed with PBS to remove the soluble avidin, and the chip was incubated for 10 min at room temperature followed by aspirating the solution from the wells. This process was repeated three times. Second, to the thoroughly washed microreaction well, 1 µl of 20 µg/ml B*-HSPG was added. B*-HSPG was incubated over the neutravidin surface for 1 h at room temperature. Finally, the B*-HSPG solution was aspirated and the wells were similarly washed with PBS to remove the unbound polysaccharides.
The binding of AAV to the functionalized surfaces
Three microliters of AAV in PBS (1011 particles/ml; 0.17 nM) solution was used in each microreaction well. The chips were incubated at room temperature for 2 h so that the binding of AAV to HS reached equilibrium. The unbound AAV were removed by submerging the chip in water and the PDMS layer was peeled off the chip. The chip was then gently dried using a filtered N2 stream.
The conditions were slightly modified depending on the purpose of the experiments. Each experiment was performed on a single chip. For the time-dependent study, AAV-2 was incubated in each well for increasing durations. For the serotype binding study, the viruses were incubated in both wells with immobilized B*-HSPG surface and with only neutravidin surface. For the free HS (ICN) inhibition study, various concentrations of HS were incubated with AAV-2 in solution for 15 min before the samples were pipetted into the wells. For determining the binding affinity of AAV to HS, serial dilutions of AAV-2 were incubated in the microreaction wells at room temperature for 2 h.
AFM imaging and analysis
Chips were imaged in air using an Explorer Scanning Probe Microscope (SPM) (Veeco Instruments, Woodbury, NY). Silicon noncontact mode tips (Nanosensors, Neuchatel, Switzerland) with a spring constant of 3 N/m and resonance frequency of 70 kHz were used to image the virus. All the wells on a single chip were imaged using the same tip and imaging parameters to reduce imaging artifacts. Five scan areas of 25 µm2 with 500 x 500 scan lines were taken from each well, therefore each scan line corresponds to 10 nm and the spatial resolution is of the order of 50 nm, a length scale compatible with the size of AAV but below that of the molecules (biotin, neutravidin, HS) immobilized on the surface. We can detect the presence of double viruses and account for them in our data analysis. Each well was divided into four quadrants and a 25-µm2 area was imaged randomly from each quadrant. The fifth image was taken from the center of the well. Surface roughness measurements and particle densities were averaged from five AFM images.
Image J software (NIH, Bethesda, MD) was used to count the particles in each image. Before the software was used to count the number of particles, each image was converted to an eight-bit grayscale image and was thresholded to highlight the particles, which represented 20 nm viruses and to remove background.
Determination of surface roughness
The Explorer SPM software was used to obtain area RMS surface roughness of the functionalized surfaces. Area RMS surface roughness is the RMS average between the height deviations and the mean surface, taken over the area. The following calculation was performed by the software:
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Determination of the binding constant
The binding constant, Kd, was determined by fitting the binding assay data to the equilibrium isotherm equation
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Acknowledgements |
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Footnotes |
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Abbreviations |
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References |
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