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Adeno-associated virus rep proteins produced in insect and mammalian expression systems: Wild-type and dominant-negative mutant proteins bind to the viral replication origin
Year:
1991
Source of publication :
Virology
Authors :
צ'חנובסקי, נור
;
.
Volume :
184
Co-Authors:
Owens, R.A., Laboratory of Molecular and Cellular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, Building 8, Room 304, National Institutes of Health, Bethesda, Maryland 20892USA
Trempe, J.P., Laboratory of Molecular and Cellular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, Building 8, Room 304, National Institutes of Health, Bethesda, Maryland 20892USA
Chejanovsky, N., Laboratory of Molecular and Cellular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, Building 8, Room 304, National Institutes of Health, Bethesda, Maryland 20892USA
Carter, B.J., Laboratory of Molecular and Cellular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, Building 8, Room 304, National Institutes of Health, Bethesda, Maryland 20892USA
Facilitators :
From page:
14
To page:
22
(
Total pages:
9
)
Abstract:
The adeno-associated virus (AAV)rep gene proteins, Rep78 and Rep68, are required for replication of AAV DNA and bind to the AAV replication origin. An AAV genome having a Lys340 to His (K340H) mutation in the consensus purine nucleotide binding site of therep gene protein exhibited a dominant-negative phenotype for DNA replication. We synthesized both wild-type and the K340H mutant Rep78 protein in a baculovirus expression system. Nuclear extracts of Sf9 cells containing these proteins were examined in gel mobility-shift assays with radiolabeled AAV terminal repeat DNA. Each protein bound specifically to the hairpin configuration of the AAV terminal repeat DNA to yield three shifted components. However the mobility of these components observed with the mutant Rep protein was slightly decreased compared to that with the wild-type Rep78. The addition of an antibody made against an oligopeptide from the car{ballot box}yl terminal region of the Rep78 protein generated novel shifted bands in the presence of either extract. Similar results were observed when the wild-type and mutant Rep proteins were expressed from an inducible expression system employing the human immunodeficiency virus type 1 transcription promoter in human 293 cells. These results suggest that the dominant-negative phenotype of the K340H mutation may be mediated by binding of the mutant protein to the AAV replication origin. © 1991 Academic Press, Inc.
Note:
Related Files :
Animal
animal cell
Baculoviridae
Base Sequence
HeLa cells
human cell
insects
Mammalia
עוד תגיות
תוכן קשור
More details
DOI :
10.1016/0042-6822(91)90817-U
Article number:
Affiliations:
Database:
סקופוס
Publication Type:
מאמר
;
.
Language:
אנגלית
Editors' remarks:
ID:
28191
Last updated date:
02/03/2022 17:27
Creation date:
17/04/2018 00:37
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Scientific Publication
Adeno-associated virus rep proteins produced in insect and mammalian expression systems: Wild-type and dominant-negative mutant proteins bind to the viral replication origin
184
Owens, R.A., Laboratory of Molecular and Cellular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, Building 8, Room 304, National Institutes of Health, Bethesda, Maryland 20892USA
Trempe, J.P., Laboratory of Molecular and Cellular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, Building 8, Room 304, National Institutes of Health, Bethesda, Maryland 20892USA
Chejanovsky, N., Laboratory of Molecular and Cellular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, Building 8, Room 304, National Institutes of Health, Bethesda, Maryland 20892USA
Carter, B.J., Laboratory of Molecular and Cellular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, Building 8, Room 304, National Institutes of Health, Bethesda, Maryland 20892USA
Adeno-associated virus rep proteins produced in insect and mammalian expression systems: Wild-type and dominant-negative mutant proteins bind to the viral replication origin
The adeno-associated virus (AAV)rep gene proteins, Rep78 and Rep68, are required for replication of AAV DNA and bind to the AAV replication origin. An AAV genome having a Lys340 to His (K340H) mutation in the consensus purine nucleotide binding site of therep gene protein exhibited a dominant-negative phenotype for DNA replication. We synthesized both wild-type and the K340H mutant Rep78 protein in a baculovirus expression system. Nuclear extracts of Sf9 cells containing these proteins were examined in gel mobility-shift assays with radiolabeled AAV terminal repeat DNA. Each protein bound specifically to the hairpin configuration of the AAV terminal repeat DNA to yield three shifted components. However the mobility of these components observed with the mutant Rep protein was slightly decreased compared to that with the wild-type Rep78. The addition of an antibody made against an oligopeptide from the car{ballot box}yl terminal region of the Rep78 protein generated novel shifted bands in the presence of either extract. Similar results were observed when the wild-type and mutant Rep proteins were expressed from an inducible expression system employing the human immunodeficiency virus type 1 transcription promoter in human 293 cells. These results suggest that the dominant-negative phenotype of the K340H mutation may be mediated by binding of the mutant protein to the AAV replication origin. © 1991 Academic Press, Inc.
Scientific Publication
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