cis effects in adeno-associated virus type 2 replication.
We have utilized deletion mutants of adeno-associated virus (AAV) to investigate which elements of the AAV genome are required in cis for high yields of the wild-type virus in a plasmid transfection assay and in addition whether these elements affect primarily AAV DNA replication or encapsidation. All tested deletions from within the Rep region demonstrated a modest, approximately threefold, decrease in viral production. Deletions within the cap region resulted in markedly less virus. Previous observations suggested that in cells in which recombinant AAV (rAAV) was produced, as in our assay with the helper plasmid pDG, there is a substantial excess of empty capsids. Co-transfections of high- and low-yielding constructs demonstrated that under conditions where Cap is abundant, the constructs with cap deletions did not package efficiently. These observation suggest that the lower yields of rAAV cannot be entirely due to lack of capsids but that elements within the cap region of the wild-type genome are important for efficient encapsidation. The production of virus by the mutants we tested was, however, not consistent with the disruption of a cis-acting packaging signal. Apparently, when Cap is provided "in trans," encapsidation is inefficient. A second observation is that there were equivalent amounts of replicated but unencapsidated viral DNA in cells transfected with each of our constructs. We propose that, in accord with the previously proposed link between DNA replication and encapsidation, the total amount of AAV DNA replication can be limited by the efficiency of encapsidation.
Ward
Peter
P
Division of Hematology/Oncology, Department of Geneand Cell Medicine, Mount Sinai School of Medicine, One Gustave Levy Place, New York, NY 10029, USA. peter.ward@mssm.edu
Clément
Nathalie
N
Linden
R Michael
RM
D002213:Capsid / Q000378:metabolism; D002460:Cell Line; D004261:DNA Replication / Q000502:physiology; D004279:DNA, Viral / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D000229:Dependovirus / Q000235:genetics / Q000378:metabolism*; D005816:Genetic Complementation Test; D016679:Genome, Viral / Q000502:physiology*; D006801:Humans; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*; D019065:Virus Assembly / Q000502:physiology*; D014779:Virus Replication / Q000502:physiology*
D004279:DNA, Viral; D004268:DNA-Binding Proteins; D014764:Viral Proteins; C068734:rep proteins, Adeno-associated virus 2