Mutations in the C terminus of herpes simplex virus type 1 DNA polymerase can affect binding and stimulation by its accessory protein UL42 without affecting basal polymerase activity.
We have analyzed the effects of mutations in the herpes simplex virus type 1 DNA polymerase (Pol) C-terminal UL42 binding domain on the activity of Pol and its ability to form complexes with and be stimulated by UL42 in vitro. Wild-type Pol expressed in Saccharomyces cerevisiae was both bound and stimulated by UL42 in vitro. C-terminal truncations of 19 and 40 amino acids (aa) did not affect the ability of Pol to be stimulated by UL42 in vitro. This stimulation as well as basal Pol activity in the presence of UL42 was inhibited by polyclonal anti-UL42 antiserum, thus indicating a physical interaction between Pol and UL42. Removal of the C-terminal 59 aa of Pol and internal deletions of 72 aa within the Pol C terminus eliminated stimulation by UL42. None of the truncations or deletions within Pol affected basal polymerase activity. In contrast with their ability to be stimulated by UL42, only wild-type Pol and Pol lacking the C-terminal 19 aa bound UL42 in a coimmunoprecipitation assay. These results demonstrate that a functional UL42 binding domain of Pol is separable from sequences necessary for basal polymerase activity and that the C-terminal 40 aa of Pol appear to contain a region which modulates the stability of the Pol-UL42 interaction.
Department of Virology, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, New Jersey 08543-4000.
D J
DJ
Tenney
P A
PA
Micheletti
J T
JT
Stevens
R K
RK
Hamatake
J T
JT
Matthews
A R
AR
Sanchez
W W
WW
Hurlburt
M
M
Bifano
M G
MG
Cordingley
D000595:Amino Acid Sequence; D000914:Antibodies, Viral; D004252:DNA Mutational Analysis; D004259:DNA-Directed DNA Polymerase / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D004789:Enzyme Activation; D005090:Exodeoxyribonucleases; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009500:Neutralization Tests; D011233:Precipitin Tests; D012441:Saccharomyces cerevisiae / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D018139:Simplexvirus / Q000201:enzymology / Q000235:genetics*; D013329:Structure-Activity Relationship; D014764:Viral Proteins / Q000276:immunology / Q000378:metabolism* / Q000494:pharmacology
D000914:Antibodies, Viral; D046911:Macromolecular Substances; D014764:Viral Proteins; D004259:DNA-Directed DNA Polymerase; D005090:Exodeoxyribonucleases; C043417:DNA polymerase, Simplexvirus