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Sequence and symmetry requirements within the internal palindromic sequences of the adeno-associated virus terminal
R A Bohenzky1, R B LeFebvre, K I Berns
1Department of Immunology and Medical Microbiology, College of Medicine, University of Florida, Gainesville 32610.
Abstract:
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.
Insights
Symmetry in the internal palindromic region of adeno-associated virus (AAV) DNA is crucial for efficient replication. Maintaining this symmetry, even with sequence changes, allows for viable AAV propagation, unlike asymmetrical mutations.
Area of Science:
- Molecular Biology
- Virology
- Genetics
Background:
- Adeno-associated virus (AAV) has a single-stranded DNA genome with palindromic terminal repeats.
- These repeats form hairpin structures essential for DNA synthesis initiation.
- A T-shaped DNA conformation arises from palindromes during replication, with crossarms critical for genome rescue.
Purpose of the Study:
- To investigate the role of symmetry within the internal palindromic region of AAV DNA.
- To analyze the impact of symmetrical and asymmetrical sequence substitution mutants on AAV replication and propagation.
Main Methods:
- Construction and analysis of AAV mutants with sequence substitutions in the internal palindromic region.
- Assessment of AAV DNA accumulation kinetics via plasmid or virion DNA introduction.
- Intermolecular competition experiments with cotransfected wild-type and mutant alleles.
Main Results:
- Symmetrical sequence substitution mutants were viable and replicated similarly to wild-type AAV, regardless of DNA source.
- Competition experiments showed wild-type dominance or codominance, suggesting a potential preference but not a requirement for wild-type sequence.
- Asymmetrical substitution mutants replicated at significantly lower levels, hindering propagation.
Conclusions:
- The symmetry of the internal palindromic region is essential for efficient AAV replication, likely for proper T-shaped structure formation.
- Sequence variations that maintain palindromic symmetry do not impede AAV viability.
- Asymmetrical alterations disrupt critical structural features, severely impairing viral replication.