Mapping interaction sites of the A20R protein component of the vaccinia virus DNA replication complex.
The vaccinia virus A20R protein is required for DNA replication, is associated with the processive form of the viral DNA polymerase, and directly interacts with the viral proteins encoded by the D4R, D5R, and H5R open reading frames as determined by a genome-wide yeast two-hybrid analysis. The purpose of the present study was to further analyze the latter protein-protein interactions. Association of an epitope-tagged A20R protein with an epitope-tagged D4R or H5R protein, expressed in vaccinia virus-infected cells, was demonstrated by binding the complex to one mAb followed by Western blotting with another. Interaction between the A20R and D5R proteins, which was weakest in the yeast two-hybrid analysis, could not be demonstrated by this method. A panel of N- and C-terminal truncated forms of the A20R protein was tested for interaction with the D4R, H5R, and D5R proteins using the yeast two-hybrid system. These studies revealed that nonoverlapping regions of A20R comprising amino acids 1 to 25, 26 to 76, and 201 to 251 were required for binding of D4R, H5R, and D5R, respectively. By contrast, no interaction of A20R with D4R could be detected after deletion of only 25 codons from either end of the latter open reading frame. A fusion protein containing either full-length A20R or only the N-terminal 25 amino acids of A20R was sufficient to capture the D4R protein, whereas the fusion protein containing A20R amino acids 26 to 426 was not, confirming the results of the yeast two-hybrid analysis. The distinct protein binding domains of the A20R protein may contribute to the assembly or stability of the multiprotein DNA replication complex.
Ishii
Koji
K
Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892-0445, USA.
Moss
Bernard
B
D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry; D045647:DNA Glycosylases; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000378:metabolism; D058619:Maltose-Binding Proteins; D009699:N-Glycosyl Hydrolases / Q000378:metabolism; D020798:Two-Hybrid System Techniques; D051981:Uracil-DNA Glycosidase; D014616:Vaccinia virus / Q000502:physiology*; D014764:Viral Proteins / Q000737:chemistry / Q000378:metabolism*; D014779:Virus Replication
C422803:A20R protein, vaccinia virus; D002352:Carrier Proteins; D004268:DNA-Binding Proteins; C091945:H5R protein, Vaccinia virus; D058619:Maltose-Binding Proteins; D014764:Viral Proteins; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; D051981:Uracil-DNA Glycosidase