Four-Dimensional Visualization of the Simultaneous Activity of Alternative Adeno-Associated Virus Replication Origins
| Autor: | N. Alexander Balsiger, Irma Heid, Daniel L. Glauser, R. Michael Linden, Okay Saydam, Cornel Fraefel, Mathias Ackermann |
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| Rok vydání: | 2005 |
| Předmět: |
DNA Replication
Concatemer viruses Immunology Replication Origin Herpesvirus 1 Human Biology Origin of replication medicine.disease_cause Microbiology Viral Proteins chemistry.chemical_compound Plasmid Genes Reporter Virology Chlorocebus aethiops DNA Packaging medicine Animals Humans Promoter Regions Genetic Vero Cells Gene Adeno-associated virus Microscopy Confocal Terminal Repeat Sequences DNA replication Dependovirus Genome Replication and Regulation of Viral Gene Expression Herpes simplex virus chemistry Insect Science Origin recognition complex HeLa Cells Plasmids |
| Zdroj: | Journal of Virology. 79:12218-12230 |
| ISSN: | 1098-5514 0022-538X |
| Popis: | The adeno-associated virus (AAV) inverted terminal repeats (ITRs) contain the AAV Rep protein-binding site (RBS) and the terminal resolution site (TRS), which together act as a minimal origin of DNA replication. The AAV p5 promoter also contains an RBS, which is involved in Rep-mediated regulation of promoter activity, as well as a functional TRS, and origin activity of these signals has in fact been demonstrated previously in the presence of adenovirus helper functions. Here, we show that in the presence of herpes simplex virus type 1 (HSV-1) and AAV Rep protein, p5 promoter-bearing plasmids are efficiently amplified to form large head-to-tail concatemers, which are readily packaged in HSV-1 virions if an HSV-1 DNA-packaging/cleavage signal is provided in cis . We also demonstrate simultaneous and independent replication from the two alternative AAV replication origins, p5 and ITR, on the single-cell level using multicolor-fluorescence live imaging, a finding which raises the possibility that both origins may contribute to the AAV life cycle. Furthermore, we assess the differential affinities of Rep for the two different replication origins, p5 and ITR, both in vitro and in live cells and identify this as a potential mechanism to control the replicative and promoter activities of p5. |
| Databáze: | OpenAIRE |
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