Multiple cellular proteins are recognized by the adeno-associated virus Rep78 major regulatory protein and the amino-half of Rep78 is required for many of these interactions.
Adeno-associated virus (AAV) encoded Rep78 is a multi-functional protein which is required for AAV DNA replication, is able to regulate both AAV and heterologous gene expression at the transcriptional level, and appears necessary for site specific integration of AAV DNA into human chromosome 19. By comparison with the analogous replication protein of the polyomaviruses, large T antigen, it seemed likely that Rep78 would interact with cellular proteins to carry out at least some its functions. This study demonstrates that Rep78 is able to interact with multiple cellular proteins, from cellular extracts as measured by West(far)-western, coimmunoprecipitation, and Rep78-affinity chromatography analysis. Eight cellular proteins of approximately 180, 140, 120, 95, 75, 55, 45, and 35 kDa (+/- 10%), were observed to bind Rep78 in all three assay systems. Two others, of 30 and 24 kDa, were observed in two of three assay systems. Furthermore, using truncated Rep78 proteins, it is demonstrated that the amino-terminus is required for most Rep78-cellular protein interactions. However, the extreme carboxy-terminus of Rep78 was found to be all that is required for binding to the 55 kDa cellular protein.
Hermonat
P L
PL
Department of Obstetrics and Gynecology, University of Arkansas for Medical Sciences, Little Rock 72205, USA.
Santin
A D
AD
Carter
C A
CA
Parham
G P
GP
Quirk
J G
JG
D001665:Binding Sites; D015153:Blotting, Western; D002352:Carrier Proteins / Q000378:metabolism; D002846:Chromatography, Affinity; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D000229:Dependovirus / Q000502:physiology*; D006367:HeLa Cells; D006801:Humans; D007163:Immunosorbent Techniques; D058619:Maltose-Binding Proteins; D008970:Molecular Weight; D010446:Peptide Fragments / Q000378:metabolism; D010449:Peptide Mapping; D011506:Proteins / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013462:Sulfur Radioisotopes; D014764:Viral Proteins / Q000378:metabolism*
D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D058619:Maltose-Binding Proteins; D010446:Peptide Fragments; D011506:Proteins; D011993:Recombinant Fusion Proteins; D013462:Sulfur Radioisotopes; D014764:Viral Proteins; C068734:rep proteins, Adeno-associated virus 2; D004247:DNA