Mode of Cell Entry and Host Tropism

AAV appears to have a broad host range and different AAV serotypes replicate in vitro in many human cells and in a variety of simian and rodent cell lines if a helper virus with the appropriate host range is also present. AAV also infects various animal species, and human isolates of AAV will grow in mice or monkeys if the appropriate mouse or monkey ade-novirus is also present. This indicated that cellular receptors for AAV were likely to be relatively common on many cell types.

Recent experiments demonstrated (42) that AAV2 particles can use heparin sulfate proteoglycans (HSPGs) as a receptor, and some cell lines that do not produce HSPGs are impaired for AAV binding and infection. Additional studies suggest that AAV2 also uses a coreceptor for efficient internalization and 2 possible coreceptors, avp5 integrin (43) and human fibroblast growth factor receptor 1 (FGFR1) (44), were identified.

It is of interest that the avps integrin coreceptor, which is used for a similar purpose by adenovirus type 2 and S, is preferentially located on airway epithelial cells in the more distal areas of the conducting airway (45). This maybe important for use of an AAV2 gene therapy vector for cystic fibrosis because the distal airway is the region of the lung most impacted by the disease. It is also noteworthy that FGFR is expressed in most tissues but is of highest abundance in skeletal muscle and neuroblasts and glioblasts in the brain, and these 2 organs appear to be good targets for AAV2 transduction.

The existence of more than one coreceptor suggests that AAV may have multiple mechanisms for cell entry, and there is already some evidence to support this concept (46). Also, real-time imaging of entry into HeLa cells by individual AAV2 particles labeled with the dye CyS (47) showed that endocytosis was rapid and that some particles could reach the nucleus within 15 min of first contacting the cell. However, there was evidence of free diffusion of both endosomes and AAV particles, and also evidence for movement of each of these entities being driven by cellular motor proteins. Furthermore, cellular trafficking events following endocytosis of AAV that may involve the ubiquitin-proteosome pathway appear to play a significant role (48-50), as discussed in Section VIII.A.

Cell entry may also be impacted by the route of delivery, and AAV vectors can transduce airway cells when delivered directly to the lung (51,52), or brain cells (S3) and myocytes (54,55) when delivered directly to these organs. However, when delivered intravenously by tail vein injection in mice (56,57), the vector preferentially accumulated in the liver, and this may reflect both the presence of a much more porous vasculature in the liver and also the small size of the AAV particles. The small size of the AAV particle may also be of advantage in passing through the basal lamina pores in muscle, thus accessing a large number of myoblasts and myotubes.

III. AAV MOLECULAR BIOLOGY A. Particle Structure

AAV is a nonenveloped particle about 20 nm in diameter with icosahedral symmetry, which is stable to heat, mild proteolytic digestion, and nonionic detergents. The AAV particle is comprised of a protein coat, containing the 3 capsid proteins, VP1, VP2, and VP3, which encloses a linear single-stranded DNA genome having a molecular weight (mw) of 1.5 X 106. The VP1, VP2, and VP3 proteins are present in the viral capsid in the ratio of 1:1:8. The DNA represents 25% by mass of the particle that therefore exhibits a high buoyant density (1.41 g/cm3) in cesium chloride. The relative stability of the AAV particle is an important property because it can withstand robust purification procedures, which facilitates scaled-up production of AAV vectors.

The crystal structure of AAV 2, determined at 3.0 A resolution by X-ray crystallography, reveals several interesting features that will be helpful for efforts aimed at modifying the AAV capsid to alter targeting specificity (17). The structure shows that each capsid comprises 60 protein subunits arranged in T = 1 icosahedral symmetry. All the amino acids, except the 14 amino-terminal residues of VP1, could be localized in the structure. The surface of the capsid shows a distinctive topology with 3 peaks clustering around each 3-fold icosahedral axis (Fig. 1). Each 3-fold proximal peak is formed from 2 interacting protein subunits, and the sides of these peaks appear to be the regions that mediate the receptor-binding interactions with heparin sulfate. An additional feature of the AAV capsid is that, like all other parvoviruses, it appears to contain a phospholipase A2 activity that in AAV is located in the unique amino-terminal region of the VP1 protein (58), and this activity appears to be essential for infectivity to mediate exit from endosomes.

A novel feature of AAV is that, although each particle contains only one single-stranded genome, strands of either complementary sense, ''plus'' or ''minus'' strands, arepack-aged into individual particles. Equal numbers of AAV particles contain either a plus or minus strand. Either strand is equally infectious and AAV displays single-hit kinetics for infectivity (59).

When DNA is extracted from AAV particles, the plus and minus strands anneal to generate duplex molecules of 3.0 X

Figure 1 Structure of AAV serotype 2. The structure was determined by X-ray crystallography at a resolution of 3.0 A. The surface topology is shown drawn to scale. The view is down a 2-fold axis (center of the virus) with 3 folds left and right of center, and 5 folds above and below. Overall, the outside surface is positively charged with a prominent ring of symmetry-related positive patches in a depression surrounding the 5-fold axis. See the color insert for a color version of this figure. (Reprinted with permission from Ref. 17.)

Figure 1 Structure of AAV serotype 2. The structure was determined by X-ray crystallography at a resolution of 3.0 A. The surface topology is shown drawn to scale. The view is down a 2-fold axis (center of the virus) with 3 folds left and right of center, and 5 folds above and below. Overall, the outside surface is positively charged with a prominent ring of symmetry-related positive patches in a depression surrounding the 5-fold axis. See the color insert for a color version of this figure. (Reprinted with permission from Ref. 17.)

106 mw. However, Crawford and his colleagues (60) showed that, on the basis of a careful physical characterization of AAV particles, each particle appeared to contain only DNA of 1.5 X 106 mw. They suggested that the only way to reconcile this conundrum was to propose that individual plus and minus strands must be packaged into individual particles. An elegant proof of this conundrum was provided by Rose and his colleagues (61) who made 2 preparations of AAV particles, in which one preparation had thymidine substituted by bromo-deoxyuridine (BudR) and the other was unsubstituted. The preparations of particles were mixed prior to extraction of DNA. Analysis of the duplex DNA obtained upon extraction showed components with intermediate density formed by individual strands from substituted or unsubstituted particles that had annealed during extraction. This constituted formal proof of the novel DNA strand segregation exhibited by AAV during packaging of its DNA. BudR substitution of AAV DNA also permits separation of the plus and minus strands, and this was used along with 5' end-labeling and restriction endonuclease cleavage to determine the strand polarity of the AAV genome (62).

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