Sequences at the C-terminus of the herpes simplex virus type 1 UL30 protein are dispensable for DNA polymerase activity but not for viral origin-dependent DNA replication.
The UL30 protein of herpes simplex virus type 1 (HSV-1) is a catalytically active DNA polymerase which is present in virus infected cells in a heterodimeric complex with an accessory subunit, the UL42 polypeptide. Both proteins are essential for viral DNA synthesis but because the UL42 protein is much more abundant it has been difficult to determine whether its role is related to, or independent of, its interaction with the UL30 protein in vivo. Since the C-terminal region of UL30 has been shown to be important for interaction with the UL42 protein but dispensable for DNA polymerase activity, a recombinant baculovirus which overexpresses a UL30 protein truncated by 27 amino acids at its C-terminus was constructed and used to assess the significance of the protein-protein interaction. The mutated protein was as active as wildtype (wt) UL30 in a DNA polymerase assay in which activated calf thymus DNA was used as template. However, in contrast to the wt protein, the activity of the truncated polymerase on this template was not stimulated by addition of purified UL42. A monoclonal antibody against the UL42 protein co-precipitated the full length but not truncated polymerase from extracts of cells which had been co-infected with a UL42-expressing recombinant baculovirus. Finally, the truncated protein was not active in a transient assay for HSV-1 origin-dependent DNA replication performed in insect cells in tissue culture. These results indicate that sequences at the C-terminus of the UL30 protein which are dispensable for DNA polymerase activity play essential roles both in viral DNA replication and interaction with the UL42 protein, and strongly suggest that the interaction between the proteins is important in vivo.
Stow
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ND
Medical Research Council Virology Unit, Institute of Virology, Glasgow, UK.
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002482:Cellulose / Q000031:analogs & derivatives; D002845:Chromatography; D004261:DNA Replication; D004279:DNA, Viral / Q000096:biosynthesis*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005090:Exodeoxyribonucleases; D008969:Molecular Sequence Data; D009036:Moths; D011233:Precipitin Tests; D018139:Simplexvirus / Q000201:enzymology / Q000235:genetics*; D014764:Viral Proteins / Q000378:metabolism*
D004279:DNA, Viral; D014764:Viral Proteins; D002482:Cellulose; C008881:phosphocellulose; D004259:DNA-Directed DNA Polymerase; D005090:Exodeoxyribonucleases; C043417:DNA polymerase, Simplexvirus