Role of the carboxy terminus of herpes simplex virus type 1 DNA polymerase in its interaction with UL42.
Several recent reports implicate sequences at or near the C terminus of the catalytic subunit (POL) of herpes simplex virus type 1 (HSV-1) DNA polymerase in its interaction with the accessory protein UL42. We have investigated further the involvement of this region by three different approaches: anti-idiotype antibodies, a competition ELISA and inhibition of the interaction by peptides. Antibodies raised in rabbits to peptides corresponding to regions of POL all reacted in Western blots with POL. Surprisingly, the sera raised against C-terminal peptides (amino acids 1221 to 1235 and 1224 to 1235) also reacted with UL42. The UL42 reactivity was shown to be due to the presence of anti-idiotype antibodies, providing direct evidence for complementarity of the structure of the extreme C terminus of POL to a region of UL42. To measure the contribution of the C terminus of POL to UL42 binding we developed a competition ELISA using POL, a truncated polymerase lacking the carboxyl-terminal 27 amino acids (POLd1) and UL42. UL42 binding to immobilized POL was inhibited approximately four times more effectively by competition, in solution, with POL than with POLd1, indicating that the C-terminal 27 amino acids of POL are responsible for at least 75% of the binding energy. A peptide corresponding to these 27 amino acids (residues 1209 to 1235) inhibited both the POL-UL42 interaction and the stimulation of POL by UL42 and did so more effectively than peptides corresponding to amino acids just away from the C terminus (residues 1195 to 1223 and 1177 to 1195).
Marsden
H S
HS
MRC Virology Unit, Institute of Virology, Glasgow, U.K.
Murphy
M
M
McVey
G L
GL
MacEachran
K A
KA
Owsianka
A M
AM
Stow
N D
ND
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D015153:Blotting, Western; D002460:Cell Line; D004279:DNA, Viral / Q000302:isolation & purification / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005090:Exodeoxyribonucleases; D018259:Herpesvirus 1, Human / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis / Q000276:immunology; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D018411:Spodoptera; D014764:Viral Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*
D000906:Antibodies; D004279:DNA, Viral; D010455:Peptides; D011994:Recombinant Proteins; D014764:Viral Proteins; D004259:DNA-Directed DNA Polymerase; D005090:Exodeoxyribonucleases; C043417:DNA polymerase, Simplexvirus