SUMO-1 modification regulates the protein stability of the large regulatory protein Rep78 of adeno associated virus type 2 (AAV-2).
The large Rep proteins Rep78 and Rep68 of the helper-dependent adeno associated virus type 2 (AAV-2) are essential for both site-specific integration of AAV DNA in the absence of helpervirus and productive AAV replication in the presence of helpervirus. We have identified UBC9, the E2 conjugating enzyme for the small ubiquitin-related polypeptide SUMO-1, as binding partner of the large Rep proteins in yeast two-hybrid analysis and in GST pulldown assays. Modification of the large Rep proteins with SUMO-1 could be demonstrated in immunoblot analysis and in immunoprecipitations, with the lysine residue at amino acid position 84 serving as the major attachment site. The largely sumolation-deficient Rep78 lysine to arginine point mutant showed a strongly reduced half-life as compared to the wild-type protein. This finding implicates a role for sumolation in the regulation of Rep78 protein stability that is assumed to be critical for the establishment and maintenance of AAV latency.
Weger
Stefan
S
Institut für Infektionsmedizin, Abteilung Virologie, Charité Campus Benjamin Franklin, Freie Universität Berlin, 12203 Berlin, Germany. stefan.weger@charite.de
Hammer
Eva
E
Heilbronn
Regine
R
D003001:Cloning, Molecular; D004265:DNA Helicases / Q000378:metabolism; D004279:DNA, Viral / Q000235:genetics / Q000302:isolation & purification; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D000229:Dependovirus / Q000378:metabolism*; D005982:Glutathione Transferase / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D016366:Open Reading Frames; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D025842:SUMO-1 Protein / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
D004279:DNA, Viral; D004268:DNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D025842:SUMO-1 Protein; D014764:Viral Proteins; C068734:rep proteins, Adeno-associated virus 2; D005982:Glutathione Transferase; D004265:DNA Helicases