BCH6415-1 - CH 6415 DNA Virus Classification and...

CH 6415 – DNA Virus Classification and Replication

Basic Virus Structure

Viral Strategies and Replication Cycle

Chromosome 19 integration
requires AAV Rep protein
expression.

AAV Replication
FIG. 1. Model of AAV DNA replication. The boxed region illustrates the steps
involved in the terminal resolution of AAV viral ends. In vitro, Rep68 is
necessary and sufficient for both the site-specific endonuclease and helicase
activities required for terminal resolution. The viral 3' end is indicated with an
arrow. Circles depict Rep covalently attached to the viral 5' end at the
terminal resolution site ( trs ).

ITR Sequence and Genome Map

Promoter Structure

Decoding the AAV Genome
Virology (1989) 173:120-128. Mutagenesis of an AUG codon in the adeno-associated virus rep gene: effects on viral DNA replication.
Chejanovsky N, Carter BJ.

4, 20 no AUG
no Rep 52, 40

Rep Protein Deletion Analysis
J. Virol. 74:2936-2942 (2000). Mutational Analysis of Adeno-Associated Virus Type 2 Rep68 Protein Endonuclease
Activity on Partially Single-Stranded Substrates. Michael D. Davis, Jianwen Wu, and Roland A. Owens *
FIG. 1. AAV ITR hairpin DNA ("flop" configuration). The positions of the primary
Rep68/78 recognition sequence (RRS) (52) and the secondary binding site for
Rep68/78 (RRS') (30, 55) are within the labeled rectangles. The individual
imperfect GAGC repeats of the RRS are indicated by subdivisions of its
rectangle. The positions of the terminal resolution site (trs) (13) and the 3' ends of
unfilled hairpin DNA and partially filled hairpin DNA are also indicated. Fifty-four
base pairs at the right end of the hairpin sequence are indicated by dots.
FIG. 3. Rep68/78 amino acids 3 to
200 are sufficient for trs
endonuclease activity on unfilled
hairpin DNA. trs endonuclease
assays were performed (as detailed in
the text) with the truncated fusion
proteins indicated above each lane.
Samples contained either no protein,
1.0 μg of MBP-LacZ (LacZ), or 1.0 μg
of the fusion protein with the
indicated truncation. The wild-type
(W-type) lanes contained
1.0, 0.5, 0.25, or 0.125 μg of MBP-
Rep68 . All reaction mixtures were
incubated at 37°C for 60 min, boiled,
and resolved by nondenaturing PAGE
(6% polyacrylamide). The positions of
the substrate and released cleavage
product are indicated on the right.
Each sample contained 25,000 cpm of
5' 32 P-end-labeled unfilled (A) or filled
(B) AAV terminal repeat hairpin DNA.
Unfilled
Filled

FIG. 5. The Y156F protein lacks trs endonuclease activity but
retains DNA helicase activity. Assays were performed (as
detailed in the text) with MBP-Rep68 (W-type) or the mutant
fusion proteins indicated above each lane. MBP-LacZ (LacZ)
was included as a negative control. Samples contained either
no protein or the amount of each protein (in micrograms)
indicated below each lane. (A) trs endonuclease assays. Each
sample contained 25,000 cpm of 5' 32 P-end-labeled unfilled
AAV hairpin DNA. All reaction mixtures were incubated at 37°C
for 60 min, boiled, and resolved on a nondenaturing 6%
polyacrylamide gel. The positions of the substrate and