Site-directed mutagenesis of adeno-associated virus type 2 structural protein initiation codons: effects on regulation of synthesis and biological activity
Open accessAbstract
It has been shown that two of the three adeno-associated virus type 2 capsid proteins, B and C, are synthesized from a single spliced transcript. Protein C arises from an AUG codon at nucleotide 2810, whereas protein B is initiated by a unique eucaryotic initiation codon (ACG) that lies 65 triplets upstream from the C origin. The third capsid component, protein A, is synthesized from a second spliced transcript which uses an alternative 3' acceptor site. In this study we used oligonucleotide-directed mutagenesis to confirm the positions of the B initiation codon and the 3' acceptor sites for the alternatively spliced B/C and A protein messages. We also located definitively the protein A initiation codon, an AUG triplet mapping to nucleotide 2203. Mutagenesis of the B initiator permitted a direct test of the effect of increased B initiator strength on the translational efficiencies of the B and C proteins. It was found that conversion of the relatively inefficient protein B initiator (ACG) to an AUG enhanced the level of B synthesis while abolishing the synthesis of C from its downstream AUG initiator. Protein C synthesis thus depends on the strength of the B initiator, i.e., the relatively higher levels of C (approximately 20-fold greater than B) must result from frequent readthrough of the weak B initiator. Finally, we examined the abilities of mutants deficient in the synthesis of A, B, or C to produce infectious virions. We found that at least two of the structural protein…
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Wild-type AAV virions are composed of three capsid proteins, Vp1, Vp2, and Vp3, produced in a 1:1:10 ratio from the same open reading frame by differential splicing and alternative translational start sites.
“One primary difference is the fact that wild-type (wt) AAV utilizes three capsid subunits instead of two to form the virion shell.”
From Packaging Capacity of Adeno-Associated Virus Serotypes: Impact of Larger Genomes on Infectivity and Postentry Steps (2005) · cites this paper as [30]
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Grieger, J. C., & Samulski, R. J. (2005) · Journal of Virology · doi:10.1128/jvi.79.15.9933-9944.2005 · Cited by 419 · see citing works
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- Authors
- Muralidhar, S., Becerra, S. P., & Rose, J. A.
- Affiliations
- National Institute of Allergy and Infectious Diseases (US)
- ORCID
- Muralidhar, S
- Venue
- Journal of Virology
- Publisher
- American Society for Microbiology
- Published
- 1994-01
- Volume
- 68
- Issue
- 1
- Pages
- 170-176
- ISSN
- 0022-538X (ISSN-L)
- 1098-5514
- PMC
- PMC236275
- OpenAlex
- W2141917844
- Cited by
- 52
- References
- 2
- Citations by year
- 2026: 2 · 2024: 1 · 2023: 1 · 2021: 2 · 2019: 1 · 2017: 2 · 2015: 2 · 2013: 2 · 2012: 3
- FWCI
- 2.32
- Type
- journal article
- Language
- en
- Open access
- green
- europepmc.org
- Locations
- Journal of Virology (publishedVersion)
- Europe PMC (PubMed Central) (publishedVersion)
- PubMed Central (publishedVersion)
- PubMed (publishedVersion)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Virus-based gene therapy research
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA and protein synthesis mechanisms
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Research and Splicing
- Keywords
- adeno-associated virus type 2, capsid protein, initiation codon, alternative splicing, translation efficiency, readthrough, protein A, protein B, protein C, site-directed mutagenesis
- MeSH terms
- Dependovirus, Capsid, Cell Line, Transformed, Codon, Fluorescent Antibody Technique, Humans, Peptide Chain Initiation, Translational, RNA Splicing, Transcription, Genetic, Virulence, Blotting, Southern, Blotting, Northern, Viral Structural Proteins, Gene Expression Regulation, Viral, Mutagenesis, Site-Directed
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