Replication

In a productive infection in the presence of a helper such as adenovirus (4,64), the infecting parental AAV ss genome is converted to a parental duplex replicating form (RF) by a self-priming mechanism that takes advantage of the ability of the ITR to form a hairpin structure (Fig. 3). The parental RF molecule is then amplified to form a large pool of progeny RF molecules in a process that requires both the helper functions and the AAV rep gene products, Rep78 and Rep68. AAV RF genomes are a mixture of head-to-head or tail-to-tail multimers or concatemers and are precursors to progeny ss DNA genomes, which are packaged into preformed empty AAV capsids (65). Rep52 and Rep40 interact with the preformed capsid, apparently to provide a DNA helicase function for DNA packaging (66)

The kinetics of AAV replication and assembly has been investigated (64,67). In human HeLa or 293 cells simultaneously infected with AAV and adenovirus, there are 3 phases of the growth cycle. In the first 8 to 10 h, the cell becomes permissive for AAV replication as a result of expression of a subset of adenovirus genes, including E1, E2A, E4, and the VA RNA (68). During this period, the infecting AAV genome is converted to the initial parental duplex RF DNA by self-priming from the terminal base-paired 3' hydroxyl group provided by the ability of the ITR to form a self-paired hairpin. This initial generation of a duplex genome also provides a template for transcription and expression of AAV proteins. In a second phase, from about 10 to 20 h after infection, the bulk of the AAV rep and cap proteins are synthesized, and there is a large amplification of monomeric and concatemeric duplex AAV RF genomes to a constant level (67). During the third phase of AAV growth, between 16 and 30 h, ss progeny molecules are synthesized by a strand-displacement replication mechanism and packaged into preformed capsids followed by accumulation of mature, infectious AAV particles (65,67).

The rep proteins perform important biochemical functions (4). Rep68 and Rep78 bind to the ITR and are site- and strand-specific endonucleases that cleave the hairpin in an RF molecule at the site that is the 5' terminus of the mature strand. In addition, these proteins contain an ATP-binding site that is important for the enzymatic activity but not for binding to the ITR. Further, Rep78 and Rep68 have both DNA and RNA helicase activity. These rep proteins also regulate transcription (4,6,7). Rep78 is a negative autoregulator of the p5 promoter (i.e., of its own synthesis), but is an activator of the p40 promoter to enhance capsid protein production. Rep52 and Rep40 do not bind to the ITR but provide a helicase in assembly of mature particles. Also, the smaller rep proteins are antirepres-sors and block the negative autoregulation of p5 by Rep78 (69).

The AAV replication cycle is highly coordinated with respect to expression of rep and cap proteins and the relationship between replication and assembly (67,69). Any vector production process that provides the rep and cap functions by complementation may decrease the efficiency of this highly regulated

Figure 2 Structure of the AAV 2 genome. The AAV2 genome is shown as a single bar with a 100 map unit scale (1 map unit = 47 nucleotides). Stippled boxes indicate inverted terminal repeats (ITRs; replication origins) and solid circles indicate transcription promoters (p5, p19, p40). The poly A site is at map position 96. RNAs from AAV promoters are shown as heavy arrows with introns indicated by the caret. The coding regions for the 4 rep proteins (Rep78, Rep68, Rep52, Rep40) and for the viral capsid proteins (VP1, VP2, VP3) are shown with open boxes, and the numbers indicate the locations of initiation and termination codons. (Reprinted with permission from Ref. 63.)

Figure 2 Structure of the AAV 2 genome. The AAV2 genome is shown as a single bar with a 100 map unit scale (1 map unit = 47 nucleotides). Stippled boxes indicate inverted terminal repeats (ITRs; replication origins) and solid circles indicate transcription promoters (p5, p19, p40). The poly A site is at map position 96. RNAs from AAV promoters are shown as heavy arrows with introns indicated by the caret. The coding regions for the 4 rep proteins (Rep78, Rep68, Rep52, Rep40) and for the viral capsid proteins (VP1, VP2, VP3) are shown with open boxes, and the numbers indicate the locations of initiation and termination codons. (Reprinted with permission from Ref. 63.)

process. Nonetheless, AAV has one of the largest burst sizes of any virus and following infection of cells with AAV and adenovirus as helper, this may be well in excess of 100,000 particles per cell (11). These considerations imply that a high yield of AAV vector particles per cell theoretically is attainable. Attaining high specific productivity is of crucial importance in developing scaled-up vector production because the ability to obtain maximum yields ideally requires high specific productivity (yield of particles per cell) or large biomass (total number of cells). Maximizing the specific productivity may avoid unnecessary increases in biomass.

0 0

Post a comment