AAV virions

AAV virions are small nonenveloped particles (20-25 nm) that carry a linear single-stranded DNA (ssDNA) genome, which is 4.7 kb in size. Two open reading frames (ORFs), rep and cap, have been identified in the viral genome and are flanked by T-shaped inverted terminal repeats (ITRs). The cap ORF encodes for the structural proteins that form the capsid, whereas the regulatoiy proteins are produced from the rep ORF (Figure L4). [For more details see (1).]... 

Collect AAV from the gradient by dripping 1-ml fractions, and verify the density of the fractions by refractometry. The density of infectious AAV virions is 1.40-1.42 g/cm Empty AAV particles and DI particles have a density of 1.32-1.35 g/cm. ... 

Fig. 2A—E. Isopycnic CsCl centrifugation demonstrating the separation of two classes of BUdR-labeled AAV virions with different densities. A Unfractionated vims. B Fractions 9-12 (heavy) from A were pooled and resedimented. C Fractions 16-19 from A (light) were pooled and resedimented. D Fractions 11 and 12 (heavy) from B were pooled, and a sample was resedimented in the presence of unfractionated BUdR-labeled AAV as a density marker. E Fractions 16 and 17 (light) from C were pooled, and a sample resedimented in the presence of unfractionated H BUdR-labeled AAV as a density marker. Symbols Reprinted by permission of J. Virol. 9,...

It is not known whether one or both of the two types of AAV virions is individually capable of initiating a productive infection in the presence of adenovirus or whether both types of AAV particles are required. Titration studies of AAV infectivity in the presence of excess adenovirus have suggested single-hit kinetics for infection this would imply that it is not essential for both types of co-infect a cell (Blacklow et al., 1967). Attempts to productively infect cells with physically separated particles (Berns and Adler, 1972) have been made but the data have not been of sufficient precision to resolve this problem (Hoggan and Berns, unpublished data). Purified AAV DNA has been shown to be infectious in the presence of helper adenovirus (Hoggan et al., 1968). The infectivity of the DNA was increased by the addition of... 

The development of AAV-based gene-therapy vectors is presently focused on the analysis of the properties of the different AAV serotypes, especially the host range of the virion shells of the different serotypes. By packaging... 

Production of vector virions requires transfection of the vector construct in plasmid form together with the introduction on separate plasmids and/or helper viruses of both the AAV genes and the helper virus genes required for a productive AAV infection (Xiao et al., 1998) (Fig. 1.1). Variations have included producing vectors... 

The structure of AAV DNA has proved to be unusual and complex. There was initial uncertainty as to whether the DNA was single- or double-stranded. Early studies on purified DNA seemed to indicate it was duplex in nature, but staining of viral preparations with acridine dyes seemed to indicate that the DNA within the virion might be single-stranded (Mayor and Melnick,... 

AAV 1-4 are serologically distinct although AAV 2 and 3 cross-react (Hoggan, 1970). Because the DNAS of the four serotypes have 30-50% of nucleotide sequences in common it might be expected that there would be some amino acid sequences of the structural proteins of the different serotypes which would be similar. Common amino acid sequences may not be exposed in the tertiary structure assumed by the capsid proteins. Support for this suggestion has been provided by the data of Johnson et al. (1972). These authors prepared antisera to the three structural proteins of AAV-3 after SDS treatment. Antisera to the SDS-polypeptides did not interact with intact virions, but the antisera raised against specific AAV-3 SDS-polypeptides did react with the analogous SDS-polypeptides of AAV-1 and AAV-2. 

AAV is usually recovered from infected cells using trypsin and deoxy-cholate. Sedimentation in CsCl leads to a major AAV band at a buoyant density of 1.40 g/cm and a minor band at 1.467 g/cm. When sarkosyl was substituted for deoxycholate, the major AAV band in CsCl occurred at the same density as that of AAV purified in the usual manner. However, the amount of material in the denser minor band was increased. Virions in the minor band have been characterized as being smaller than AAV and probably have had some of the capsid stripped away (Hoggan, 1971). After sarkosyl treatment even the AAV from the major normal density band in CsCl was unable to adsorb to KB cells (less than 10% of normal) implying that an intact capsid is required for adsorption (Berns and Adler, unpublished data). 

As already indicated adsorption, penetration, and uncoating of AAV do not require helper virus and purified DNA is infectious but requires helper. However, in the absence of helper no DNA or RNA synthesis is detected. Therefore, a helper function (s) appears to be required to initiate AAV-specific nucleic acid synthesis. Co-infection with herpes simplex virus type 1 (HSV-1) also serves to allow AAV DNA or RNA synthesis (Boucher et al., 1971 Rose and Koczot, 1972). The extent of AAV nucleic acid synthesis appeared to be comparable with either HSV or adenovirus as the helper. AAV DNA synthesized in the presence of HSV has been reported to be infectious (Boucher et al., 1971). Additionally, AAV antigens in cells co-infected with HSV are detectable by means of a fluorescent antibody staining technique (Atchison, 1970 Blacklow et al, 1970 Blacklow et al, 1971 Johnson et al., 1972). However, no infectious particles can be recovered with HSV as the helper. Interestingly, antisera to both AAV-specific SDS-polypeptides and virion protein stain the infected cells. 

Major distinct problems for experimental study in the near future include (1) the question of whether both types of virions are capable of or necessary for productive infection (2) a mechanism of DNA replication which will account for the fine structure of the DNA and the observation that approximately equal numbers of plus and minus strands are separately encapsidated (3) determination of the portion of the amino acid sequence of the structural proteins which can be or are coded for by the viral genome (4) the manner of association of the viral genome with latently infected cells and (5) further studies on the defectiveness of AAV. 

Lytie and latent life cycles are two distinctive phases of AAV replication and persistence, respectively. Productive infection occurs in the presence of helper virus (such as adenovims, herpes virus, vaccinia virus) or genotoxic stimuli (such as ultraviolet [UV] irradiation, chemical carcinogens) (29). The propagation of both helper and AAV vimses leads to cell lysis and release of mature infectious virions. Up to 10 infectious AAV particles can be produced fiom a single cell (79). 

The latest developments in AAV assembly have arisen fiom studies establishing in vitro packaging systems (90,91). In these protocols, infectious AAV particles are generated in a cell-free system. Biological and physical characterizations indicate that virions produced in this manner have similar properties to those obtained fiom cell culture. Although, the current yield fiom in vitro systems is still quite low, further optimization of this system may eventually lead to industrial-scale cell- fiee AAV production. 

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