Site-directed mutagenesis of adeno-associated virus type 2 structural protein initiation codons: effects on regulation of synthesis and biological activity.
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 proteins, B and C, are required for the production of infectious virions and that sequestration of single-stranded adeno-associated virus genomes from the pool of replicating DNA molecules does not occur in the absence of either of these proteins.
Muralidhar
S
S
Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland 20892.
Becerra
S P
SP
Rose
J A
JA
D015152:Blotting, Northern; D015139:Blotting, Southern; D002213:Capsid / Q000235:genetics*; D002461:Cell Line, Transformed; D003062:Codon; D000229:Dependovirus / Q000235:genetics* / Q000472:pathogenicity; D005455:Fluorescent Antibody Technique; D015967:Gene Expression Regulation, Viral; D006801:Humans; D016297:Mutagenesis, Site-Directed; D010442:Peptide Chain Initiation, Translational / Q000235:genetics; D012326:RNA Splicing; D014158:Transcription, Genetic; D015678:Viral Structural Proteins / Q000235:genetics*; D014774:Virulence
D003062:Codon; D015678:Viral Structural Proteins