Sequence and symmetry requirements within the internal palindromic sequences of the adeno-associated virus terminal repeat.
The defective parvovirus, adeno-associated virus (AAV), contains a single-stranded DNA genome of 4681 bases with inverted terminal repeats of 145 bases. The distal 125 bases of the repeat are palindromic allowing a hairpin to form for initiation of DNA synthesis. The palindromic region contains three palindromes, two smaller internal palindromes flanked by a larger palindrome, which allow the hairpinned DNA to assume a T-shaped conformation during DNA replication. Deletion of an internal palindrome forming one of the crossarms of the T results in the inability of the AAV genome to be rescued from plasmid sequences and replicated. Restoration of the crossarm sequences with DNA that differs in primary sequence but maintains the symmetry of the palindrome results in viable AAV and propagation of the mutant sequences. In this paper we report further studies on the nature of mutants made within the crossarm of the T. Two types of substitution mutants were analyzed. Symmetrical sequence substitution mutants were viable as previously reported. An analysis of the kinetics of AAV DNA accumulation showed that the symmetrical sequence substitution mutants were indistinguishable from wild-type AAV. This was true if the AAV DNA was introduced into the cells either as plasmid DNA or as DNA extracted from virions. In contrast, intermolecular competition experiments showed either a dominance of the wild-type sequence or codominance of both sequences when both alleles were cotransfected into helper virus-infected cells. A preference for the wild-type sequence may also exist but is not required for efficient AAV replication. The second type of mutation studied was an asymmetrical sequence substitution mutant. This mutant was replicated but at a level too low to be propagated. These data suggest that symmetry is required in the internal palindromic region, presumably for the formation of the crossarm structure in the T-shape.
Bohenzky
R A
RA
Department of Immunology and Medical Microbiology, College of Medicine, University of Florida, Gainesville 32610.
LeFebvre
R B
RB
Berns
K I
KI
D003001:Cloning, Molecular; D004252:DNA Mutational Analysis; D004260:DNA Repair; D004261:DNA Replication; D004279:DNA, Viral / Q000235:genetics*; D000229:Dependovirus / Q000235:genetics*; D006860:Hydrogen Bonding; D007700:Kinetics; D015394:Molecular Structure; D012091:Repetitive Sequences, Nucleic Acid
D004279:DNA, Viral