DNA POLYMERASES 0F MAREK‘S
DiSEASE HERPESVIRUS AND
HERPESVIRUS 0F TURKEYS:

CWTERIZATION AND MECHANESM
0F INHIBW BY WINE

Dissertation for the Degree of Ph. D.
MICHIGAN, STATE UNIVERSiTY
SUSAN SlNGLEY LEINBACH
1 976

 

LIBRARY

i. i».- Michigan Stab
University ,

 

This is to certify that the

thesis entitled

DNA POLYMERASES OF MAREK'S DISEASE HERPESVIRUS AND
HERPESVIRUS OF TURKEYS: CHARACTERIZATION AND
MECHANISM OF INHIBITION BY PHOSPHONOACETATE

presented by

Susan Singley Leinbach

has been accepted towards fulfillment
of the requirements for

Ph. D. degree in Biochemistry

 

(Ms A M 4~
0 Major professor 0

Date 0d. lqj I976

0-7639

 
 
   

 
 

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ABSTRACT
DNA POLYMERASES OF MAREK'S DISEASE HERPESVIRUS AND

HERPESVIRUS OF TURKEYS: CHARACTERIZATION AND
MECHANISM OF INHIBITION BY PHOSPHONOACETATE

BY

Susan Singley Leinbach

Infection of duck embryo fibroblasts (DEF) by Marek's disease
herpesvirus (MDHV) led to the induction of a novel DNA polymerase.
Infection of DEF by herpesvirus of turkeys (HVT) also led to the
induction of a novel DNA polymerase. The properties of these
enzymes have been examined. One unique property of herpesvirus-
induced DNA polymerases is their sensitivity to the compound
phosphonoacetate. Studies to elucidate the mechanism of phosphono-
acetate inhibition have been performed and a model to explain this
inhibition has been developed.

The MDHV-induced and HVT-induced DNA polymerase activities were
partially purified from extracts of infected DEF by DEAD-cellulose
chromatography and/or phosphocellulose chromatography and were
characterized. The properties of these enzymes were virtually
identical. Both required 1-2 mM MgCl2 and 200-250 mM KCl for maxi-
mal activity. When 200 mM KCl was added to DNA polymerization

reaction mixtures, DNA polymerase activity was 10- to 60-fold

greater than in its absence. Both induced DNA polymerases were

Susan Singley Leinbach
inhibited by phosphonoacetate such that in the presence of l-3 uM
phosphonoacetate 50% inhibition of DNA polymerization occurred.

Both enzymes were determined to be DNA-dependent DNA polymerases and
to have sedimentation coefficients in the presence of 0.25 M KCl of
about 75. These properties distinguished the MDHVeinduced and HVT-
induced DNA polymerases from the DNA polymerases of uninfected DEF.

A phosphonoacetate—resistant mutant of HVT (HVTPA) was isolated.
It was able to replicate in the presence of 1.6 mM phosphonoacetate
while wild type HVT (HVTWT) was unable to replicate in the presence
of 0.3 mM phosphonoacetate. The properties of the DNA polymerase

induced by infection of DEF by HVT were compared with those of

PA
the wild type induced DNA polymerase. The HVTPA-induced DNA
polymerase had an apparent inhibition constant for phosphonoacetate
of about 10-fold greater than the HVTWT-induced DNA polymerase. In
vitro thermal inactivation studies demonstrated that the HVTPA-
induced DNA polymerase was more temperature-sensitive than the wild
type enzyme. Other catalytic properties of the two enzymes were
similar. These results suggest that the HVT-induced DNA polymerase
is viral coded and that inhibition by phosphonoacetate occurs by
direct interaction with the herpesvirus-induced DNA polymerase.

The inhibition of the HVT-induced DNA polymerase by phosphono-
acetate was examined in detail using steady state enzyme kinetics.
In the DNA polymerization reaction phosphonoacetate was a noncom-
petitive inhibitor when the four dNTPs were the variable substrate
and activated DNA concentration was 200 pg per ml. Phosphonoacetate

was also a noncompetitive inhibitor when activated DNA was the

variable substrate and the concentration of each of the four dNTPs

Susan Singley Leinbach
was 2.5 pM. When the concentration of each of the four dNTPs was
raised to 100 uM, inhibition by phosphonoacetate became nearly
uncompetitive. Phosphonoacetate was a competitive inhibitor of
perphosphate in the dNTP-pyrophosphate exchange reaction catalyzed
by the HVT-induced DNA polymerase. In the DNA polymerization reac-
tion multiple inhibition analysis of phosphonoacetate and pyro-
phosphate demonstrated that the two were mutually exclusive inhibitors.
The inhibition patterns obtained for pyr0phosphate in the polymeri-
zation reaction were similar to those obtained for phosphonoacetate.
These results indicate that phosphonoacetate inhibits the herpesvirus-
induced DNA polymerase by binding to the pyrophosphate binding site
of the enzyme.

Rate equations were derived for kinetic mechanisms which
described phosphonoacetate inhibition as occurring at the pyro-
phosphate binding site of the DNA polymerase. The mechanism which
was consistent with all the phosphonoacetate kinetic inhibition
data was the alternate product inhibition model. In this case the
binding of phosphonoacetate to the pyrophosphate binding site of
the enzyme would create an alternate reaction pathway. Phosphono—
acetate as an alternate product of the DNA polymerization reaction
would promote an exchange reaction in which a nucleotide analogue
of phosphonoacetate would be generated as an alternate substrate.

The alternate product inhibition model predicted that the
nucleotide analogue of phosphonoacetate should be a substrate for
the DNA polymerization reaction and a competitive inhibitor of
dNTP and should be generated in the reaction mixture during phos-

phonoacetate inhibition. Deoxythymidine 5'-phosphophosphonoacetate

Susan Singley Leinbach
was synthesized and was tested as an alternate substrate. It could
not substitute for dTTP in activated DNA polymerization reactions.
It.was a noncompetitive inhibitor when the four dNTPs were the
variable substrate in the DNA polymerization reaction. No nucleo-
tide analogue of phosphonoacetate was generated when [3H]phosphono-
acetate was incubated in the presence of HVT-induced DNA polymerase,
activated DNA, and the four dNTPs.

Therefore, no direct evidence was obtained in support of the
alternate product inhibition model for phosphonoacetate inhibition.
Nevertheless, the conclusion from the kinetic inhibition studies
remains that phosphonoacetate inhibits the herpesvirus-induced DNA
polymerase by binding to the pyrophosphate binding site of the
enzyme. These studies also indicate that the herpesvirus-induced
DNA polymerase has a high apparent affinity for phosphonoacetate.
Apparent inhibition constants for phosphonoacetate were 1-2 uM
while apparent inhibition constants for pyrophosphate were l-2 mM.

Twenty analogues of phosphonoacetate were tested as inhibitors
of the HVTbinduced DNA polymerase. Only 2-phosphonopropionate was
an inhibitor although its apparent inhibition constant was about 50
times greater than that of phosphonoacetate. Thus the structural
requirements for inhibition at the pyrophosphate binding site of

herpesvirus-induced DNA polymerases are very rigorous.

DNA POLYMERASES OF MAREK'S DISEASE HERPESVIRUS AND
HERPESVIRUS OF TURKEYS: CHARACTERIZATION AND

MECHANISM OF INHIBITION BY PHOSPHONOACETATE

BY

Susan Singley Leinbach

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Biochemistry

1976

DEDICATION

To my parents and husband Ed, who supported
me with patience and understanding in my
goal to become a biochemist.

ii

ACKNOWLEDGEMENTS

This work is the result of a collaboration between Dr. John
Boezi and Dr. Lucy Lee of the USDA, ARS, Regional Poultry Laboratory.
I wish to express my sincere thanks to Dr. Boezi and Dr. Lee for
their continued assistance and guidance during my graduate studies.
My thanks also goes to the members of my guidance committee: Dr.
Fritz Rottman, Dr. Leland Velicer, Dr. Allan Morris, and Dr.

William Deal.

I would also like to thank John Reno, who was directly involved
in some of these studies,for his helpfulness and advice. Apprecia-
tion is also expressed to Dr. Clarence Suelter for helpful discus-
sions and to Betty Baltzer for her technical assistance.

Thanks is also given to Dr. Keyvan Nazerian of the USDA, ARS,
Regional Poultry Laboratory for providing me with valuable cell
samples. I acknowledge the National Institutes of Health for

financial support.

iii

 

 

 

TABLE OF CONTENTS

Page
INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . 1

LITERATURE REVIEW. . . . . . . . . . . . . . . . . . . . . . . 5

Marek's Disease . . . . . . . . . . . . . . . . . . . . 5
Marek's Disease Herpesvirus . . . . . . . . . . . . . . 7
Comparative Aspects of Marek’s Disease and Epstein-

Barr Virus-Induced Human Proliferative Diseases . . . 8

Vertebrate DNA Polymerases. . . . . . . . . . . . . . . 10
DNA Polymerase a . . . . . . . . . . . . . . . . 10
DNA Polymerase B . . . . . . . . . . . . . . . . 12
DNA Polymerase y . . . . . . . . . . . . . . . . l4
Physiological Function of Vertebrate DNA
Polymerases. . . . . . . . . . . . . . . . . . 14
Herpesvirus-Induced DNA Polymerases . . . . . . . . . . 16
Herpes Simplex-Induced DNA Polymerase. . . . . . 16
Marek's Disease Herpesvirus-Induced DNA
Polymerase . . . . . . . . . . . . . . . . . . 21
Cytomegalovirus-Induced DNA Polymerase . . . . . 21
Phosphonoacetate and Herpesvirus Infections . . . . . . 24
Effect of Phosphonoacetate on Herpesvirus
Infections in Tissue Culture . . . . . . . . . 26
Phosphonoacetate as an Inhibitor of Herpesvirus-
Induced DNA Polymerases. . . . . . . . . . . . 27
Animal Studies with Phosphonoacetate . . . . . . 29
Effect of Phosphonoacetate on a Cell Line
Transformed by Epstein-Barr Virus. . . . . . . 30

“TERM AND mmos O O O C O C O C O O O O O O O O O O O O O 32

Materials . . . . . . . . . . . . . . . . . . . . . . . 32
Preparation and Growth of Cells . . . . . . . . . . . . 34
Purification of DNA Polymerases . . . . . . . . . . . . 36
Assay of DNA Polymerases. . . . . . . . . . . . . . . . 38
Assay of the dNTP-Pyrophosphate Exchange Reaction . . . 40
Analytical Methods. . . . . . . . . . . . . . . . . . . 41
Kinetic Analysis. . . . . . . . . . . . . . . . . . . . 43

RESULTS 0 O O O I O O O O O O O O O O I I O O O O O O 0 O O O O 44

Marek's Disease Herpesvirus-Induced DNA Polymerase. . . 44

iv

Page

Identification of an Induced DNA Polymerase
from Marek' 5 Disease Herpesvirus-Infected
Duck Embryo Fibroblasts. . . . . . . . . . . 44
Characterization of the MDHV-Induced DNA
Polymerase . . . . . . . . . . . . . . . . . . 48
Herpesvirus of Turkeys-Induced DNA Polymerase . . . . . 57
Purification of the HVT-Induced DNA Polymerase . 57
Characterization of the HVT-Induced DNA
Polymerase . . . . . . . . . . . . . . . . . . 58
Inhibition of HVT-Induced DNA Polymerase by
Phosphonoacetate. . . . . . . . . . . . . . . . . . . 59
Phosphonoacetate Inhibition Patterns for the
DNA Polymerization Reaction Catalyzed by
HVT-Induced DNA Polymerase . . . . . . . . . 62
Phosphonoacetate Inhibition Pattern for the
dNTP-Pyrophosphate Exchange Reaction Catalyzed
by HVT-Induced DNA Polymerase. . . . . . . . . 67
Multiple Inhibition Analysis of Phosphonoacetate
and Pyrophosphate in the DNA Polymerization
Reaction Catalyzed by HVT-Induced DNA
Polymerase . . . . . . . . . . . . . . . . . 67
Pyrophosphate Inhibition Patterns for the DNA
Polymerization Reaction Catalyzed by HVT-
Induced DNA Polymerase . . . . . . . . . . . . 72
Effect of MgC12 Concentration on Inhibition
of HVT-Induced DNA Polymerase by

Phosphonoacetate . . . . . . . . . . . . . . . 72
Inhibition by Structural Analogues of
Phosphonoacetate . . . . . . . . . . . . . 75

The Effect of Phosphonoacetate on the DNA
Polymerization Reaction Catalyzed by Other
DNA Polymerases. . . . . . . . . . . . . . . . 76
Investigation of Phosphonoacetate as an Alternate
Product Inhibitor of the HVT-Induced DNA Polymeri-

zation Reaction . . . . . . . . . . . . . . . . 76
Synthesis of Deoxythymidine S'Phosphophos-
phonoacetate . . . . . . . . . . . . . . . . . 77

Structural Properties of Deoxythymidine
5'Phosphophosphonoacetate. . . . . . . . . . . 78
Deoxythymidine 5'Phosphophosphonoacetate as
a Substrate of the HVT-Induced DNA Polymeri-
zation Reaction. . . . . . . . . . . . . . . . 81
Deoxythymidine 5'Phosphophosphonoacetate as
an Inhibitor of the HVT-Induced DNA Polymeri-
zation Reaction. . . . . . . . . . . . . . . . 84
Generation of Deoxythymidine S'Phosphophos-
phonoacetate in HVT-Induced DNA Polymeriza-
tion Reactions Inhibited by Phosphonoacetate . 87
Characterization of a Phosphonoacetate-Resistant
HVT-Induced DNA Polymerase. . . . . . . . . . . . . 90
Presence of MDHV-Induced DNA Polymerase in the MSB- 1
Lymphoblastoid Cell Line. . . . . . . . . . . . . . . 97

DIstS ION . O O O O O O O O O O C O 0 O O O O C O 0 O O O O 0 1 o4

 

 

 

 

Page
Characterization of DNA Polymerases of Marek's
Disease Herpesvirus and Herpesvirus of Turkeys. . . . 104
Mechanism of Phosphonoacetate Inhibition of
Herpesvirus-Induced DNA Polymerase. . . . . . . . . . 108
APP‘EmICES O O O O O O O O O O I C C O O O O O O O O O I O O O 119

A NOMENCLATURE USED IN STEADY STATE KINETIC ANALYSES
OF THE HVT-INDUCED DNA POLYMERIZATION REACTION. . . . . 119

B PROCEDURE FOR THE DERIVATION OF STEADY STATE RATE
EQmT IONS O O O O O O O O O O O O O O O O O O O O O O O 1 2 3

C DETERMINATION OF THE KINETIC MECHANISM FOR THE HVT-
INDUCED DNA POLYMERIZATION REACTION . . . . . . . . . . 124

D MULTIPLE INHIBITION ANALYSIS TO DETERMINE THAT TWO
INHIBITORS ARE MUTUALLY EXCLUSIVE . . . . . . . . . . . 130

REFERENCES . . . . . . . . . . . . . . . . . . . . . . . . . . 133

vi

Table

LIST OF TABLES
Page

Intracellular distributions of DNA polymerase
activities in duck embryo fibroblasts . . . . . . . . . 4S

Template-primer specificities of MDHV-induced DNA
polymerase. . . . . . . . . . . . . . . . . . . . . . . 54

Purification of HVT-induced DNA polymerase. . . . . . . 58

Characterization of deoxythymidine S'phosphophosphono—
acetate by paper chromatography and electrophoresis . . 79

Ability of the HVT-induced DNA polymerase to utilize
dTMP-PA in an activated DNA polymerization reaction . . 84

Kinetic constants of PA—resistant HVT-induced DNA
polymerase and of wild-type HVT-induced DNA polymerase. 91

Kinetic constants for HVT-induced DNA polymerase. . . . 127

vii

Figure

10

11

12

13

LIST OF FIGURES

Structure of phosphonoacetic acid . . . . . . . . . .

DEAR-cellulose chromatography of DNA polymerase
activities from nuclear extracts of MDHV-infected
duck embryo fibroblasts and uninfected duck embryo
fibroblasts . . . . . . . . . . . . . . . . . . . . .

Effect of M9012 concentration and MnClz concentra-
tion on the activity of the MDHV-induced DNA
mlwerase O O O O O O O O O O O O O O O O O O I O O 0

Effect of KCl concentration on the activity of the
mW-induced DNA POlymerase o o o o o o o o o o o o o

Glycerol gradient centrifugation of MDHV-induced
DNA polymerase. . . . . . . . . . . . . . . . . . . .

Initial velocity pattern for HVT-induced DNA polymer-
ase with the four dNTPs as the variable substrate
and activated DNA as the changing fixed substrate . .

Double reciprocal plots with the four dNTPs as the
variable substrate and phosphonoacetate as inhibitor.

Double reciprocal plots with activated DNA as the
variable substrate and phosphonoacetate as inhibitor.

Double reciprocal plots of the dNTP-pyrophosphate
exchange reaction with pyrophosphate as the variable
substrate and phosphonoacetate as inhibitor . . . . .

Multiple inhibition of the HVT-induced DNA polymerase
by phosphonoacetate and perphosphate . . . . . . . .

Effect of MgClz concentration on inhibition of HVT-
induced DNA polymerase by phosphonoacetate. . . . . .

Structure of deoxythymidine 5'phosphophosphonoacetic

aCid O C O O O O O O O I O O O O O I O O O O O O O O

Proton decoupled 13C nuclear magnetic resonance
spectra of phosphonoacetate, dTMP, and dTMP-PA. . . .

viii

Page

25

47

50

52

56

61

64

66

69

71

74

80

83

Figure Page

14 Double reciprocal plots with the four dNTPs as
variable substrate and dTMP-PA as inhibitor . . . . . . 86

15 Analysis of the effect of [3H]phosphonoacetate on
HVT-induced DNA polymerization reactions by Dowexrl
(Cl') chromatography. . ... . . . . . . . . . . . . . . 89

16 Inhibition of the HVTPA-induced DNA pOIymerase and
the HVTWT-induced DNA polymerase by phosphonoacetate. . 93

17 Thermal inactivation of the HVTpA-induced DNA poly-
merase and the HVTWT DNA polymerase at 52°. . . . . . . 96

18 DEAE-cellulose chromatography of DNA polymerase
activities from nuclear extracts of uninfected CEF. . . 100

19 DEAE-cellulose chromatography of DNA polymerase
activities from nuclear extracts of MSB~1 cells . . . . 102

20 Alternate product inhibition model of phosphono-

acetate inhibition of herpesvirus-induced DNA
polymerase. . . . . . . . . . . . . . . . . . . . . . . 111

ix

HSV‘l

HSV-Z

DEF

PA

PP'

dTMP-PA

DEAE

Tris

LIST OF ABBREVIATIONS

Marek's Disease Herpesvirus
herpesvirus of turkeys
Epstein-Barr virus

herpes simplex virus type 1
herpes simplex virus type 2
human cytomegalovirus

duck embryo fibroblasts

chick embryo fibroblasts
phosphonoacetate
pyrophosphate

deoxynucleoside triphosphate
deoxynucleoside monophosphate
deoxythymidine 5' phosphophosphonoacetate
diethylaminoethyl

tris-(hydroxymethyl)-methy1amine

INTRODUCTION

Marek's disease is a highly contagious lymphoproliferative
disease of chickens whose etiological agent is a herpesvirus,
Marek's disease herpesvirus (MDHV). In lymphoid tumor cells of the
infected chicken, MDHV genomes are present but no intracellular
viral antigens or virus particles are produced. In the feather
follicle epithelial cells of the infected chicken, productive
infection occurs and cell-free infectious MDHV is produced. Thus,
different virus-cell interactions exist within the MDHV—infected
chicken. In each type MDHV DNA replicates. The mechanism by
which MDHV DNA replicates in these different cells is central to
understanding the molecular basis for different virus-cell inter—
actions between herpesviruses and their hosts. Virus-cell inter-
actions similar to those found for MDHV have also been found for
the Epstein-Barr virus which causes the human lymphoproliferative
disease infectious mononucleosis and is found closely associated
with another human lymphoproliferative disease, Burkitt's lymphoma.

Evidence will be presented in this dissertation that, in pro-
ductive infections, herpesvirus DNA is replicated by novel induced-
DNA polymerases. Study of such DNA polymerases is important for
two major reasons. First, with the knowledge of the properties of
the herpesvirus-induced DNA polymerases, a search can be made in

herpesvirus-derived tumors for the same enzyme to determine if the

l

2
herpesvirus DNA replicates by the same mechanism as in productive
infections. The MDHV system is an excellent one for such a study
because productive infections are readily established in tissue
culture and tumors are readily established in chickens. Second,
with the knowledge of the properties of the herpesvirus-induced DNA
polymerase, a search can be made for a specific inhibitor of the
enzyme which could have potential for controlling and perhaps elimi-
nating herpesvirus infections. Such a compound, phosphonoacetate,
does exist and has been shown to be effective in the treatment of
herpes simplex virus infections in rabbits and mice. Elucidation
of the mechanism of inhibition of phosphonoacetate at the site of
the herpesvirus-induced DNA polymerase might provide information
for the development of other anti-herpesvirus compounds as well as
information about the structure of herpesvirus-induced DNA
polymerases.

This dissertation describes research concerning the DNA
polymerases of Marek's disease herpesvirus and herpesvirus of
turkeys, a related virus, and the mechanism of inhibition of
phosphonoacetate.

It begins with a literature review which describes: (l) the
basic characteristics of Marek's disease and MDHV, (2) the basic
characteristics of vertebrate DNA polymerases and of herpesvirus-
induced DNA polymerases in other systems, and (3) studies by others
with herpesviruses and phosphonoacetate.

Following the literature review, the results of work done with
MDHV- and HVT-induced DNA polymerases are presented. Before this

work was initiated, Boezi et a1. had published a characterization

3
of a DNA polymerase induced by infection of duck embryo fibroblasts
by MDHV, strain GA. The properties of this enzyme were different
from those previously reported for the herpes simplex-induced DNA
polymerase. Therefore, duck embryo fibroblasts infected by MDHV,
strain BC-l, and duck embryo fibroblasts infected by herpesvirus of
turkeys were examined for the presence of a more typical herpesvirus-
induced DNA polymerase. Such an activity was found in these cells.
The characterization of these enzymes is described. Much of this
work was presented by Dr. Lucy Lee at the 3rd International Congress
for Virology.

One property common to all herpesvirus-induced DNA polymerases
is their sensitivity to phosphonoacetate. The next section of this
dissertation describes results obtained from a steady state kinetic
analysis of phosphonoacetate inhibition of the HVT—induced DNA
polymerase undertaken in an effort to elucidate the mechanism of
phosphonoacetate inhibition. A model describing the mechanism of
phosphonoacetate inhibition consistent with the kinetic data was
developed. This work has been published (Leinbach, S. 8., J. M.
Reno, L. F. Lee, A. F. Isbell, J. D. Boezi. 1976. Biochemistry
15: 426-430). This proposed model was tested directly after a
nucleotide analogue of phosphonoacetate was synthesized and labelled
phosphonoacetate of high Specific radioactivity was prepared. A
preliminary report of the results of these experiments was pre-
sented at the 67th Annual Meeting of the Federation of American
Societies for Experimental Biology (Leinbach, S. S., L. F. Lee, and
J. A. Boezi. 1976. Federation Proceedings 35: 1186).

The sensitivity of herpesviruses to phosphonoacetate was used

by Dr. Lucy Lee to isolate a mutant of HVT which grew in the

4

presence of phosphonoacetate. The DNA polymerase induced by this
mutant was characterized for its catalytic, structural and kinetic
properties. These were compared with those of the DNA polymerase
induced by wild-type HVT. This work has been submitted to Virology
for publication.

Finally, using those properties of the MDHV-induced DNA
polymerase which describe it uniquely, a chicken lymphoblastoid
cell line transformed by MDHV was examined for the presence of the

MDHV-induced DNA polymerase.

LITERATURE REVIEW

Marek's Disease

 

Marek's disease is a highly contagious lymphoproliferative
disease of chickens whose etiological agent is a herpesvirus,
Marek's disease herpesvirus (MDHV) (1-5). Unlike other neoplastic
diseases, Marek's disease can be prevented with a live vaccine (6).

Two forms of Marek's disease have been observed to exist in
the chicken: the classical form and the acute form (2). In the
classical form, lymphoid cells infiltrate, proliferate and cause
lesions in peripheral and autonomic nerves. The result is a
paralysis of that part of the body innervated. In the acute form,
lymphoid tumors develop in one or several organs, including gonad,
liver, kidney, lung, muscle, and skin. These tumors are composed
of lymphoblasts, small and medium lymphocytes, and Marek's disease
cells, which are degenerate blast cells having little nuclear detail
and a vacuolated cytoplasm.

The extent of expression of the MDHV genome varies in the cells
of different tissues of the infected chicken (7). In tumors MDHV
DNA is present (8) but no virus particles or intracellular antigens
are observed. In the bursa of Fabricius and the thymus an abortive
infection exists. Cellular necrosis, intranuclear inclusion bodies,
and virus antigens are observed, but virus maturation does not occur.
In the feather follicle epithelium a productive infection occurs.

Infectious cell-free enveloped virus is produced.

5

6

Although Marek's disease was first described in 1907 (l),
unequivocal proof of MDHV as its etiological agent has just recently
been established because of the cell-associated nature of the virus.
When tumor cells from MDHV-infected chickens were used to infect
cultured chick or duck embryo fibroblasts, cytopathic effects and
noninfectious unenveloped herpesvirus particles developed in the
cultures. When these infected culture cells were inoculated into
susceptible chickens, the chickens developed Marek's disease (4,5).
Thus, a good correlation was established between the presence of
the herpesvirus in cell culture and the ability of these cultures
to produce Marek's disease in chickens. When the feather follicle
epithelium was discovered to be the site of MDHV replication and
dissemination in the chicken, infectious MDHV’was isolated and was
used successfully in producing Marek's disease in susceptible
chickens (10,11). Most recently, the demonstration of the presence
of MDHV genomes in tumors of diseased chickens has identified MDHV
as the etiological agent of Marek's disease (8).

Although MDHV has been established as the etiological agent
of Marek's disease, the mechanism.by which MDHV induces prolifera-
tion of lymphoid cells is still unclear. Payne proposed that
Marek's disease lymphomas might be caused by direct transformation
of lymphoid cells (intrinsic mechanism) or by an immune response of
lymphoid cells to viral antigens expressed in other cells (extrinsic
mechanism) (12). Several lines of evidence support the intrinsic
mechanism for MDHV-induced proliferation and suggest that T lympho-
cytes are the target cells of MDHV infection: (1) Marek's disease
tumors were shown by membrane inmunofluorescence to be composed of

77% T cells and 11% B cells (13); (2) thymectomy of susceptible

7
chickens decreased the number of birds with lymphomas (12); (3) the
recently established lymphOblastoid cell lines derived from Marekhs
disease lymphomas carry T cell surface antigens (14,15). The final
proof that MDHV causes Marek's disease by transformation of T cells
requires the demonstration of in vitro transformation of normal
chicken lymphoid cells by MDHV. However, it is becoming apparent
that two types of T cells are involved in Marek's disease: those
which are transformed and those which participate in inmune surveil-
lance against lymphoma development (16).

Three types of live vaccines are used to protect chickens from
Marek's disease: an avirulent strain of MDHV, an attenuated strain
of MDHV, and herpesvirus of turkeys (6,16). Herpesvirus of turkeys
(HVT) is antigenically related to MDHV but is nonpathogenic. It is
used.commercially in the vaccination of chickens. Although vaccina-
tion protects chickens from contracting Marek's disease when chal-
lenged with MDHV, MDHV replicates in the feather follicle epithelium
of the chicken. This suggests that the vaccine acts as an antitumor

agent rather than as an antiviral agent (17).

Marek's Disease Herpesvirus

 

Marek's disease herpesvirus is considered to be a member of
the genus Herpesvirus because: (1) its nucleocapsid is 100-110 nm
in diameter, is an icosahedron composed of 162 hollow capsomeres
and is surrounded by a lipid membrane (16); (2) its DNA is a linear
duplex with a molecular weight of 103 :_S x 106 daltons and a base
composition of 46 mole% GC (18,19): (3) its main site of develop-
ment is the nucleus of the cell (9). Marek's disease herpesvirus

is a group B, cell-associated, herpesvirus.

8

Marek's disease herpesvirus may be grown in culture in duck
embryo fibroblasts (DEF), chick embryo fibroblasts (CEF) and chicken
kidney cells (9). Infection of these cells with MDHV results in
the formation of plaques of altered cells. Plaque morphology and
size vary for each cell type and virus strain, but in all cases
small rounded refractile cells and giant multinucleated cells are
observed. In culture MDHV is highly cell associated. Since cell-
free infectious virus does not exist in MDHV-infected cultures,
virus transfer from cell to cell is probably achieved via temporary
intercellular bridges. Evidence for this comes from the fact that
ultraviolet-inactivated Sendai virus increased the efficiency of
MDHV infection (20).

Herpesvirus of turkeys may also be readily grown in avian cell
cultures. It is not so highly cell associated as MDHV and produces
low levels of cell-free infectious virus (16).

Comparative Aspects of Marek's Disease and Epstein-Barr
Virus-Induced Human Proliferative Diseases

Epstein-Barr virus has been established as the causative agent
of the human lymphoproliferative disease, infectious mononucleosis.
It is also found in close association with Burkitt's lymphoma,
another human lymphOproliferative disease (21). A number of parallels
may be drawn between these diseases and Marek's disease (12).

Infectious mononucleosis is a self-limiting lymphoproliferative
disease. In certain cases Marek's disease becomes a self-limiting
disease where lesions in nerves and organs regress. The organ distri-
bution of lymphomas in Marek's disease and Burkitt's lymphoma is
similar. In each case it is unknown why proliferation of lymphoid

cells initiates or terminates. Perhaps this stimulation of lymphoid

9
cells depends on viral replication at extralymphoid sites (extrinsic
mechanism). If immunity develops and limits viral replication,
lymphoid cell stimulation would cease and.lesions could regress.

In chickens with Marek's disease, MDHV replicates at a single
site in the chicken, in nonlymphoid epidermal cells. Epstein-Barr
virus is regularly found in the throat washings obtained from
patients with infectious mononucleosis. This finding suggests that
a cell type of the salivary glands, perhaps epithelial cells, is
a site of replication of EBV (22).

Finally, in Marek's disease, a chicken contracts the classical
form or the acute form of the disease depending on a number of
variables, including strain of virus, susceptibility of host and
age of host. With Epstein-Barr virus, infectious mononucleosis
could be considered as a classical form of disease and Burkitt's
lymphoma as an acute form of disease. Therefore, perhaps the same
variables which determine the pathology of MDHV infections also
determine the pathology of EBV infections.

Until recently, comparative research between MDHV and EBV was
difficult because of the lack of tumor derived cell lines in the
Marek's disease system. However, four such lines have now been
reported (14,23,24).

The MSB-l cell line derived from an ovarian tumor of a chicken
infected.with MDHV, strain BC-l, has been characterized extensively
(24,25). The cells grow in suspension at 41° with a rapid doubling
time (8-12 hr). They display T cell surface markers and the
morphology of typical lymphoblasts. The cells contain MDHV DNA
(26). The MSB-l cell line is a producer cell line. Virus-specific

antigens and naked nucleocapsids typical of herpesviruses are

10

produced in a low percentage of the cell pOpulation. The percentage
of these positive cells seems to increase with age in subculture.
Infection of DEF or CEF with MSB-l cells produced typical plaques.
Inoculation of chickens with MSB—l cells caused typical lesions.
No C-type virus particles or antigens were found associated with
the cells. All of these observations indicate that these cells
were transformed by MDHV.

One important difference between these MDHV-derived cell lines
and EBVBderived cell lines is that the former are of T cell origin

and the latter are of B cell origin (15).

Vertebrate DNA Polymerases

Before herpesvirus-induced DNA polymerases will be discussed,
the properties of vertebrate DNA polymerases will be reviewed, since
herpesvirus-induced DNA polymerases must be distinguished from
these. Three general classes of DNA polymerases are found in cells
of all vertebrates: DNA polymerase a, DNA polymerase B, and DNA
polymerase y (27). The Greek letters designate their order of
discovery. A mitochondrial DNA polymerase has also been reported
(28,29) but it is not pertinent to this discussion. Each of these
enzymes has been reported from numerous sources, as has been reviewed
by Weissbach (30). In this discussion the general properties of

these enzymes will be noted.

DNA Polymerase a

DNA polymerase a is the vertebrate high molecular weight DNA
polymerase which, when disaggregated, exhibits a sedimentation
coefficient of 6-8S. In its native state it exists as a dimer.

The monomeric molecular weight for the enzyme from calf thymus and

11
from rat liver and spleen was reported to be 53,000-54,000 daltons
(31). That from human KB cells was 87,000 daltons (32). DNA
polymerasetu exhibits anomalous gel filtration behavior probably
due to assymetry of the molecule (31). DNA polymerase a is an
acidic protein. Isoelectric points for this polymerase from human
lymphocytes, human KB cells, and calf thymus fall in the range of
4.5-5.5 (33,34,35). In growing cells DNA polymerase a may often
comprise 80-90% of the total DNA polymerase activity (30).

DNA polymerase a has often been referred to as the "cytoplasmic
DNA polymerase" because it has been found predominantly in the
cytoplasmic fraction when cells are subjected to normal extraction
procedures. This localization of the enzyme may be an artifact,
however. Lynch et al. report that when rat liver is homogenized in
0.3 M sucrose and 4 mM CaClZ, only 1.4% of the total DNA polymerase
a is found in the cytoplasmic fraction (36). If the homogenization
solution contained Tris buffer and/or KCl, a polymerase leached out
of the nuclei. If glycerol was used to prepare the nuclei, approxi—
mately 85% of a polymerase activity was found in the nuclear frac-
tion. Herrick et al. have also demonstrated 85% of a polymerase
activity to be associated with nuclei by subjecting mouse L cells
to cytochalasin-B enucleation (37).

DNA polymerase a is particularly sensitive to high ionic
strength (44). Significant inhibition of enzyme activity can be
Observed with 25 mM sodium chloride. DNA polymerase a is also
strongly inhibited by reagents which block sulfhydryl groups (30).
Pyrophosphate inhibits a polymerase but, at best, a minimal dNTP—

pyrophosphate exchange reaction occurs (32,45). The polymerase

12
from KB cells promotes an exchange reaction which is only about
0.8% of the polymerase activity (32).

No nuclease activity has been found associated with the most
highly purified preparations of DNA polymerase a. For a polymerase
from KB cells, the limit of detection for nuclease activity was
0.003% of the polymerase activity based on production of acid
soluble radioactive nucleotide fragments (32). It did not excise
mispaired primer termini. When a highly sensitive endonuclease
assay, which measured the conversion of supercoiled SV40 DNA to
SV40 DNA with single strand nicks, was used to assay the most highly
purified fraction of DNA polymerase a from regenerating rat liver,
no nuclease activity was detected (38).

DNA polymerase a utilizes gapped duplex DNA (activated DNA) at
a high rate. Spadari and Weissbach have reported that a polymerase
from HeLa cells can utilize DNA primed with natural RNA (39). It
cannot use ribohomopolymers as templates.

A number of investigators have reported that DNA polymerase a
appears to be heterogeneous (31,35,40). In each case several peaks
of activity were obtained by DEAE-cellulose or DEAE-Sephadex

chromatography.

DNA Polymerase B

 

DNA polymerase B is the vertebrae low molecular weight DNA
polymerase which, when disaggregated, exhibits a sedimentation
coefficient of 3-4S. It is a single polypeptide of molecular weight
43,000—45,000 daltons in KB cells (41) and 32,000 daltons in
Novikoff hepatoma cells (42). At low ionic strength, 8 polymerase

forms aggregates comparable in size to a polymerase (41).

13
Consequently, a mistaken structural relationship was first reported
for these two polymerases. DNA polymerase a and DNA polymerase B
are also not antigenically related (43). DNA polymerase B is a
basic protein. Isoelectric points for the enzyme from human lympho-
cytes, KB cells, calf thymus and Novikoff hepatoma cells are in the
range of 8.5-9.5 (33,41,42,44). The primary intracellular location
of DNA polymerase B is nuclear. In growing cells it comprises about
5-15% of the total DNA polymerase activity.

The sensitivity of DNA polymerase B to high ionic strength
varies with the source of the enzyme. Activities in the presence
of 0.2 M sodium chloride may range from 0.5-4-fold of those in its
absence (44). DNA polymerase B is insensitive to sulfhydryl group
inhibitors (30). It has no detectable nuclease activities associated
with it and consequently does not excise misplaced primer termini
(41). Although B polymerase is inhibited by pyrophosphate, no dNTP-
pyroPhosphate exchange has been demonstrated (45,46).

DNA polymerase 8 requires the four dNTPs for maximal activity
but shows activity in the absence of one or more dNTPs. This is
probably a reflection of the fact that activated DNA may contain at
least 1013 3'OH termini per ug of DNA (30). DNA polymerase B
utilizes activated DNA at a high rate and can also utilize poly(A)-

oligo(dT) to a lesser extent (47). It cannot use ribonucleo-

12-18
tide primers.
Chang has conducted a phylogenetic survey for DNA polymerase B
activity using its low molecular weight and insensitivity to N-
ethylmaleimide as primary identifying characteristics (48). This

type of polymerase has a widespread occurrence in multicellular

animals beginning with the sponge, the most primitive animal surveyed.

14
This type of polymerase was not found in prokaryotes, protozoa or

plants.

DNA Polymerase y

 

DNA polymerase y utilizes poly(A)-oligo(dT) at a higher

12—18
rate (5-10 times) than activated DNA. It does not utilize natural
RNA (49). It is not antigenically related to reverse transcriptase
of the RNA tumor viruses (49,50). DNA polymerase y has a molecular
weight of approximately 110,000 daltons, is an acidic protein, and
requires sulfhydryl-groups for maximum activity (30). In growing
cells it comprises about 1-2% of the total DNA polymerase activity
(51). In HeLa cells two forms of DNA polymerase y are resolved by
phosphocellulose or hydroxyapatite chromatography (49). Both can
use poly(A) as a template but only one can use poly(C), poly(I) and
poly(U). They also exhibit differential sensitivities to (NH4)ZSO4
and ethanol. With DNA polymerase y apparent Michaelis constants
for dNTPs are lO-fold lower than those of DNA polymerase a and DNA
polymerase 8.

Physiological Function of Verte-
brate DNA Polymerases

 

 

In attempts to assign physiological functions to these DNA
polymerases, their levels have been monitored in cells in different
stages of growth. The levels of DNA polymerase activities were
measured when mouse L cells and baby hamster kidney cells were in
stationary phase or lag phase (52,53). In each case, in log phase
the levels of DNA polymerase a were 5- to lZ-fold higher than in
stationary phase. The levels of DNA polymerase B remained unchanged.

Likewise, in regenerating rat liver DNA polymerase a levels increased

15
6— to 7—fold 48 hours after hepatectomy whereas DNA polymerase 8
levels remained constant (54).

The levels of DNA polymerase activities in synchronized HeLa
cells have also been studied. Spadari and weissbach measured DNA
polymerase activities in crude nuclear and cytoplasmic extracts.
They observed a rise in DNA polymerase 7 levels in the early part
of S phase, a steady rise in cytOplasmic a polymerase throughout 8
phase and no change in DNA polymerase 8 levels (55). Chiu and
Baril measured nuclear DNA polymerase activities which had been
partially purified by phosphocellulose chromatography (56). They
observed a 7- to lO-fold increase in DNA polymerase a levels in G1

and early S and a subsequent decline in this activity in late S and

G Nochanges in DNA polymerase 8 levels were observed. No repro-

2.
ducible variation in DNA polymerase 7 was observed. Addition of
cycloheximide to cultures prior to G1 eliminated the rise in a
polymerase levels which suggested that this rise was a result of

de novo protein synthesis.

In another study Bertazzoni et a1. measured DNA polymerase
levels in phytohemagglutinin—stimulated, ultraviolet-irradiated
human lymphocytes (57). DNA polymerase a and 8 activities were
measured in crude extracts or after sucrose gradient centrifugation
of crude extracts. In these cells a wave of DNA replication was
observed which reached maximum levels after five days of stimulation.
Two waves of repair synthesis were Observed which reached maximum.
levels after three days and seven days of stimulation. The levels
of DNA polymerase a paralleled the levels of DNA replication. A

20-fold increase in a polymerase activity was observed after five

days of stimulation. The levels of DNA polymerase 8 increased more

l6
slowly and to a lesser extent and seemed to parallel the second wave
of repair synthesis. After seven days of stimulation, maximum levels
were Obtained which were approXimately 7-fold greater than those of
the unstimulated cells. From these results these investigators sug-
gested a correlation between DNA polymerase a activity and DNA repli-
cation and a correlation between DNA polymerase B activity and repair
synthesis.

Thus, there is evidence from several laboratories which suggests
that DNA polymerase a may be one of the proteins involved in DNA
replication since DNA polymerase a levels change with the prolifera-
tive state of the cell and it can use RNA-primed DNA as a template.
The physiological functions of DNA polymerase B and DNA polymerase Y
are less clear, but DNA polymerase B may be involved in DNA repair

synthesis.

Herpesvirus-Induced DNA Polymerases
Herpesvirus-induced DNA polymerases have been reported for
herpes simplex, Marek's disease virus and cytomegalovirus infections.
The herpes simplex-induced DNA polymerase was the first reported and
has been most extensively studied. The properties of each of these
polymerases will be presented and compared using the herpes shmplex-

induced DNA polymerase as the prototype.

Herpes Simplex-Induced DNA Polymerase

Herpes simplex virus is a general group of herpesviruses which
can be divided into two groups, type 1 and type 2, on the basis of
antigenic and biological differences (58). Man is their only natural
host. Herpes simplex virus-1 is spread primarily by the oral-

respiratory route, while herpes simplex virus-2 appears to be

l7
venereally transmitted. The lesions produced by these viruses occur
in tissues derived from embryonic ectoderm. The skin, oral cavity,
vagina, conjunctiva and nervous system are the most frequent sites
of infection.

Herpes simplex virus undergoes productive infections in vitro
and in active infections in vivo (58). Virus can readily be isolated
from culture and infected tissues. Herpes simplex virus establishes
latent infections in the sensory cells of the trigeminal ganglion of
man and experimental animals.

The first evidence for the existence of an induced DNA polymerase
in HSV infections was obtained when Keir and Gold measured DNA
polymerase activity in extracts of HSV—l-infected and uninfected
baby hamster kidney cells (59). DNA polymerase levels in infected
cells increased two hours after infection, remained at these levels
until eight hours after infection and then declined sharply. The
maximum increase in specific activity of DNA polymerase was about
7-fold at four hours after infection. The majority of this induced
DNA polymerase activity was found in the nuclear fraction of the
infected cells (59-60).

Subsequent characterizations of this induced DNA polymerase
activity were performed using infected cells four hours after infec-
tion since deoxyribonuclease I activity had not yet reached maximum
values at this time. When crude extracts of HSV-l-infected and
uninfected baby hamster kidney cells were compared, the DNA polymerase
activity in infected cells was found to be more heat stable, to
utilize native DNA as a template more effectively, and to have
different Mg2+ requirements than the DNA polymerase activity in

uninfected cells (61). Moreover, the DNA polymerase activity in

18
extracts from HSV-l infected baby hamster kidney cells (BHK21/C13)
or HSV-l infected human epithelioid carcinoma cells (HEp2) was
stimulated 3-fold when 100 mM ammonium sulfate was added to standard
reaction mixtures. The DNA polymerase activity in uninfected extracts
was completely inhibited by this salt concentration (62). Further-
more, antiserum prepared against HSV-infected cells inhibited the
DNA polymerase activity of infected cells but not of uninfected
cells. Thus, a DNA polymerase activity was present in HSV-1 infected
cells which was different catalytically and structurally from the
DNA polymerase activity in uninfected cells. A similar DNA polymerase
activity was also found in HSV-2 infected BHKZl/ClB cells (63).
Weissbach et a1. extended these studies by partially purifying
the HSVbinduced DNA polymerase from infected HeLa cells (64). Crude
extracts of HSVel infected and.mock infected HeLa cells were first
assayed for DNA polymerase activity. As was previously observed in
other cells, HSVel infection of HeLa cells produced a large increase
in DNA polymerase activity. It increased linearly for at least eight
hours after infection. This actiVity was stimulated when 150 mM
potassium sulfate was added to standard reaction mixtures. At four
hours after infection the specific activity of DNA polymerase in
infected cells, when assayed in the presence of 150 mM potassium
sulfate, was 60 times higher than in mock-infected cells. Approxi-
mately 71% of this activity was found in the nuclear fraction of
infected cells. When assays were performed in the absence of
potassium sulfate, no difference in specific activity of host DNA
polymerases was observed between HSV-infected and mock-infected cells.
The HSVbinduced DNA polymerase was purified by DEAE-cellulose

and phosphocellulose chromatography (64). The most highly purified

19

HSV-induced DNA polymerase fraction was purified approximately
1330-fold over the crude cell homogenate fraction. The Hsvbinduced
DNA polymerase was resolved into two actiVities by DEAE-cellulose
chromatography. No differences in structural or catalytic properties
could be found between these two activities, although only one was
completely resolved from HeLa DNA polymerase on. Both peaks of HSV-
induced DNA polymerase activity were resolved from DNA polymerase B,
which does not adsorb to DEAE-cellulose. Phosphocellulose chroma-
tography then resolved the HSVeinduced DNA polymerase from the
induced deoxyribonuclease activity.

The properties of the most highly purified HSV-induced DNA
polymerase fraction were examined (64). It was stimulated 25- to
50-fold by 150 mM potassium sulfate, ammonium sulfate, or potassium
phosphate. HeLa DNA polymerase activities were inhibited.by 90% at
these concentrations. It had a molecular weight of 180,000-200,000
daltons. It was inhibited 95% by 50 pH N—methylmaleimide. In
template-primer studies the HSVBinduced DNA polymerase was determined
to be a DNA-dependent DNA polymerase which could use poly(dC)°oligo(dG)12
at a higher rate than activated DNA. A nuclease activity was associated
with the most highly purified fraction of the enzyme. It solubilized
[3H](dG-dC) or [3H](dA-T) at about 5% of the rate of the polymeriza-
tion of activated DNA in the presence of 150 mM potassium sulfate.
Finally, this partially purified HSV-induced DNA polymerase was com-
pletely inhibited by antiserum prepared against HSVeinfected cells (70).

Thus, there is good evidence which supports the existence of an
induced DNA polymerase in HSV infections. The single property that
readily established the existence of the induced DNA polymerase and

distinguished it from the host DNA polymerases was its stimulation by

20

salt. In the early studies a 3-fold stimulation by 100 mM ammonium
sulfate of HSVbinduced DNA polymerase in crude extracts was observed
(62). When the HSVBinduced DNA polymerase was purified by DEAE-
cellulose and phosphocellulose chromatography, it was stimulated 25-
to 50-fold by 150 mM potassium sulfate or ammonium sulfate (64).

This difference in salt stimulation may be related to the different
degrees of purity of the enzyme. A similar discrepancy exists for
the levels of HSV-induced DNA polymerase found in infected cells.
Weissbach et a1. observed 60 times more DNA polymerase activity in
infected cells than in mock-infected cells (64), while Keir and Gold
noted only seven times more DNA polymerase activity in infected cells
(59). This difference probably reflects the fact that weissbach et
al. assayed DNA polymerase activity in the presence of 150 mM potas-
sium sulfate with activated DNA while Keir and Gold assayed it in the
absence of salt with denatured DNA. In both cases, however, the
HSV-induced DNA polymerase was found predominantly in the nuclear
fraction of infected cells.

The existence of a herpesvirus-induced DNA polymerase had been
demonstrated. However, the origin of this activity was unknown. It
could be viral coded. It could be a derepressed host DNA polymerase
or it could be a modified host DNA polymerase. Studies with
temperature-sensitive mutants of HSV-1 indicate that the HSVhinduced
DNA polymerase is viral coded (65). When human embryonic lung cells
were infected with temperature-sensitive mutants from complementation
groups C and D at the nonpermissive temperature (39°), no DNA polymerase
activity was induced while normal levels of DNA polymerase activity
were induced at the permissive temperature (34°). When cells infected

with those mutants were maintained at 34° and then shifted to 39°,

21
HSVeinduced DNA polymerase activity declined to less than 25% of the
activity present at the time of the shift three hours after the shift.
However, in vitro thermal inactivation studies with crude extracts
from mutant-infected cells and from.wild type-infected cells demon-
strated no difference in temperature sensitivity between the mutant-
induced DNA polymerase and the wild type-induced DNA polymerase.

Marek's Disease Herpesvirus-
Induced DNA Polymerase

 

An induced DNA polymerase in MDHV-infected duck embryo fibro-
blasts has been reported by Boezi et a1. (66). When DNA polymerase
activities from extracts of MDHV-infected DEF were fractionated by‘
phosphocellulose chromatography, a peak of activity was observed
which was not present when DNA polymerase activities from uninfected
DEF were fractionated by phosphocellulose chromatography. This
activity exhibited very different properties from those observed for
the HSVeinduced DNA polymerase. It was completely inhibited by 100
mM ammonium sulfate. It had a sedimentation coefficient of 5.98,
which corresponds to a molecular weight of 100,000 daltons for a
globular protein. It was insensitive to 1 mM p-hydroxymercuribenzoate.
Template primer studies indicated that it was a DNA-dependent DNA
polymerase, but it could only utilize activated DNA and not

poly(dC)-oligo(dG) or poly(dA)-oligo(dT)

12-18 12-18'

Cytomegalovirus-Induced DNA Polymerase

Cytomegalovirus is a herpesvirus so named because of the dis-
tinctly enlarged cells (cytomegaly) containing intranuclear and cyto-
plasmic inclusions produced in infection (67). The human cyto-

megalovirus (HCMV) causes a variety of syndromes, including

22
postperfusion syndrome, cytomegalovirus—mononucleosis and cytomegalic
inclusion disease of infancy. Congenital infection with HCMV may
be fatal or asymptomatic. In the latter case viruria exists, and
in later life extensive abnormalities, particularly neurological
ones, may develop. Patients undergoing immunosuppressive therapy
or extensive blood transfusions appear to be particularly susceptible
to HCMV infections. Evidence suggests that HCMV may establish
latent infections.

In culture HCMV establishes a productive infection in human
fibroblasts. An abortive infection is established in guinea pig
embryo cells. No viral particles are formed, but virus—specific
early antigens are synthesized and cellular DNA synthesis is
stimulated (68). 7

Two laboratories have reported the induction of a DNA polymerase
activity when WI-38 human diploid fibroblast cells are infected with
HCMV. In one case the activity was analyzed in crude extracts
(68). In the second case the activity was partially purified (69).

Hirai et a1. measured DNA polymerase activity in extracts of
HCMV-infected and uninfected WI-38 cells at various times after
infection (68). Induction of DNA polymerase activity occurred
in infected cells at about 20 hours after infection and reached a
maximum stimulation of about 6-fold at 30 hours. The major increase
in DNA polymerase levels occurred in the nuclear fraction of the
infected cells. Levels of HCMV DNA increased at about 48 hours
after infection. The induced DNA polymerase activity was stimulated
3-fold in the presence of 100 mM ammonium sulfate. This salt con-
centration inhibited the WI—38 DNA polymerase activities by 75%.

When infected cells were cultured in the presence of cycloheximide,

23
no induction of DNA polymerase activity was observed, suggesting that
de novo protein synthesis was required for induction.

When DNA polymerase activity was measured in extracts of HCMV-
infected guinea pig cells at 48 hours after infection, a 3— and 6-
fold stimulation of DNA polymerase activity was observed in the nuclear
and cytoplasmic fractions, respectively: however, this activity was
not stimulated in the presence of 100 mM ammonium sulfate (68). This
result suggests that the HCMV-induced DNA polymerase may not be a
necessary enzyme in abortive infections but more sensitive assays are
necessary to establish this point.

Huang has fractionated the DNA polymerase activities in extracts
of HCMV-infected and uninfected WI-38 cells by DEAE-cellulose chroma-
tography followed by phosphocellulose chromatography (69). All levels
of DNA polymerase activities in HCMV-infected cells were substantially
higher than those in uninfected cells. Moreover, a new peak of DNA
polymerase activity in infected cells was found after DEAE-cellulose
chromatography and particularly after phosphocellulose chromatography.
This DNA polymerase activity was stimulated approximately 1.5-fold by
30-60 mM ammonium sulfate. No stimulation of host DNA polymerase
activities occurred in the presence of these salt concentrations.
Sedimentation coefficients of 9.38, 8.25 and 5.35 were found after
glycerol density gradient sedimentation velocity centrifugation of the
HCMV-induced DNA polymerase in the absence of salt. This result sug-
gests that the enzyme was aggregated. The HCMV-induced DNA polymerase
was inhibited 96% by 0.25 mM p-hydroxymercuribenzoate. It did not
appear to be a zinc metalloenzyme. Template-primer studies indicated
that it was a DNA-dependent DNA polymerase; poly(dA)-oligo(dT)

12-18 was

utilized at a higher rate than activated DNA or poly(dC)-oligo(dG)12_18.

24

Thus, a HCMV-induced DNA polymerase activity exists. Its activity
is stimulated in the presence of salt as was also observed for the
HSV-induced DNA polymerase. However, the amount of stimulation
observed in the presence of ammonium sulfate appeared to be substan-
tially lower for the HCMV-induced DNA polymerase than for the HSV-
induced DNA polymerase. After DEAD-cellulose and phosphocellulose
chromatography, the HCMV-induced DNA polymerase was stimulated 1.5-
fold in the presence of 30-60 mM ammonium sulfate, whereas the HSV-
induced DNA polymerase purified in the same manner was stimulated 25—
to 50-fold in the presence of 150 mM ammonium sulfate (69,64).

No genetic evidence is available to determine if the HCMV-induced
DNA polymerase is viral coded as is the case for the HSV-induced DNA
polymerase. Hewever, evidence suggests that de novo protein synthesis
is required for HCMV DNA polymerase induction (68).

The HCMV-induced DNA polymerase as well as the HSV-induced poly-
merase is predominantly a nuclear enzyme. It is interesting to note,
however, that the HCMV-induced DNA polymerase activity was first
Observed 20 hours after infection while the HSV-induced DNA polymerase
activity was first observed two hours after infection (68). Thus,
the amount of time required for these two herpesviruses to establish

productive infections is very different.

Phosphonoacetate and Hegpesvirus Infections
Using a random testing of compounds with a tissue culture screen,
workers at Abbott Laboratories discovered that phosphonoacetate
(Figure l) was an effective inhibitor of the replication of herpes
simplex virus types 1 and 2 (71). This antiviral activity was con-

sistently observed in tissue culture at a concentration of 500 uM.

25

ll
HO-lr-CHz-C
OH

é
\

OH

Figure 1. Structure of phosphonoacetic aci .

26
Since this initial Observation, the workers at Abbott and others have
studied the effects of phosphonoacetate on herpesvirus in tissue
culture, biochemical, and animal studies.

Effect of Phosphonoacetate on Herpesvirus
Infections in Tissue Culture

 

 

Overby et a1. first characterized the inhibitory effect of phos-
phonoacetate on herpesvirus infections in tissue culture (72). Infec-
tion of WI-38 cells with HSV-1 resulted in rounded, clumped, multi-
nucleated cells. However, when HSV-l-infected WI-38 cells were
cultured in the presence of 500 uM phosphonoacetate, cytopathic
effects were not observed. The infected cells could not be distin-
guished from uninfected WI-38 cells. This suggested that phosphono-
acetate prevented the replication of HSV-1.

Studies were then performed to determine at what stage of infec-
tion phosphonoacetate inhibited (72). Phosphonoacetate appeared to
have no effect on adsorption, penetration, or release of virus. When
phosphonoacetate was removed from the culture medium of HSV-l-infected
WI-38 cells, cytopathic effects reappeared. Thus, the viral genomes
were still present and functional in the infected cells. No dif-
ference in RNA and protein synthesis, as measured by [3Hluridine and
[14C1amino acid incorporation, was observed between PA-treated and
untreated infected cells. However, a reduction in DNA synthesis, as
measured by [14C1thymidine incorporation, was observed in PA-treated
infected cultures. This inhibition of DNA synthesis was specific
for viral DNA synthesis as determined by isolation and buoyant
density centrifugation of DNA from infected cultures grown in the
presence and absence of phosphonoacetate. At 12 hours postinfection

two peaks of [14clthymidine, corresponding in density to HSV-l DNA

27
and cellular DNA, were found in CsCl gradients for untreated cells.
Only one peak of [14C]thymidine, corresponding in density to cellular
DNA, was found in CsCl gradients for PA-treated cells.

The effect of phosphonoacetate on uninfected WI-38 cells was
examined (72). Phosphonoacetate was not toxic to contact-inhibited
WI-38 cells. No morphological changes were observed when confluent
WI-38 cells were incubated in the presence of 1 mM phosphonoacetate
for two days. No effect was noted on DNA, RNA, or protein synthesis.
Phosphonoacetate was toxic to growing WI-38 cells, however. When
500 uM phosphonoacetate was present in the culture medium, a 40%
decrease in the number of cells in culture was observed. Partial
inhibition of DNA synthesis was also observed at this concentration
of phosphonoacetate (73). No toxic effects were noted in growing
cells when 50 uM phosphonoacetate was present in the culture medium
(72).

Phosphonoacetate also inhibited the replication of other herpes-
viruses in culture by inhibiting viral DNA synthesis. Those herpes-
viruses studied were: human cytomegalovirus, mouse cytomegalovirus,
herpesvirus saimiri (73), Marek's disease herpesvirus, herpesvirus
of turkeys and owl herpesvirus (74). Thus, phosphonoacetate appears
to be a universal inhibitor of herpesvirus DNA synthesis.
Phosphonoacetate as an Inhibitor of
Herpesvirus-Induced DNA Polymerases

Mao et al. first demonstrated that phosphonoacetate specifically
inhibits herpesvirus-induced DNA polymerases (75). When HSV-l-
induced DNA polymerase or HSV-Z—induced DNA polymerase was assayed

in the presence of 1 uM phosphonoacetate, enzyme activity was

28
inhibited by 50% (75,77). The HCMV-induced DNA polymerase was
inhibited similarly by phosphonoacetate (73).

The sensitivity of other DNA polymerases to phosphonoacetate was
examined. The DNA polymerases from WI-38 cells were relatively
resistant to phosphonoacetate inhibition (73,75). When WI-38 DNA
polymerases, partially purified by phosphocellulose chromatography,
were assayed in the presence of 300 uM phosphonoacetate, only 10-15%
inhibition of enzyme activity resulted (75). DNA polymerase B from
HeLa cells was not inhibited by 500 uM phosphonoacetate. However,
DNA polymerase a and y from HeLa cells were inhibited by 50% when
assayed in the presence of 30 and 400 uM phosphonoacetate, respec-
tively. The vaccinia virus-induced DNA polymerase was inhibited by
50% when assayed in the presence of 30 uM phosphonoacetate (76).
Micrococcus luteus DNA polymerase, hepatitis B virus DNA polymerase
and Rous sarcoma virus reverse transcriptase were not significantly
inhibited by 100 uM phosphonoacetate (77). Thus, phosphonoacetate
is a specific inhibitor of herpesvirus-induced DNA polymerases
because the herpesvirus enzymes are inhibited by phosphonoacetate
concentrations which are at least 30 times lower than those which
inhibit other DNA polymerases.

Phosphonoacetate did not inhibit the herpesvirus-induced DNA
polymerases because of the formation of a PA-DNA complex (73,75).
The specificity of phosphonoacetate for these polymerases precludes
this possibility. Moreover, phosphonoacetate inhibition was not
decreased when DNA concentrations were increased in DNA polymeriza-
tion reaction mixtures. Finally, the presence of phosphonoacetate
in HSV-l-induced DNA polymerization reaction mixtures did not prevent

the formation of an enzyme-DNA complex as determined by glycerol

29
density gradient sedimentation velocity centrifugation of these
reaction mixtures (77). These results suggest that phosphonoacetate

inhibits by direct interaction with herpesvirus-induced DNA polymerases.

Animal Studies with Phosphonoacetate

 

The first studies using phosphonoacetate as an antiherpesvirus
agent in animals, as reported by Shipkowitz et al., involved herpes
simplex virus infections of mice and rabbits (71). Mice were infected
with HSV-2 by topical application to denuded skin. The resulting
herpes dermatitis infection caused death in untreated mice. However,
if phosphonoacetate was administered orally or topically to infected
mice two hours after infection, mortality was significantly reduced.
Rabbits were infected with HSV-l by topical application to the eyes.
The resulting herpes keratitis infection caused corneal lesions in
untreated rabbits. However, if an ointment of phosphonoacetate was
topically applied to the eyes of the infected rabbits two hours after
infection, a reduction in lesions was observed.

The effectiveness of phosphonoacetate in the treatment of herpes
dermatitis in mice and herpes keratitis in rabbits has been confirmed
by others (78,79,80). Moreover, phosphonoacetate suppressed clinical
disease and inhibited herpes simplex virus replication in animals with
established herpesvirus infections.

When chickens were infected with Marek's disease herpesvirus,
phosphonoacetate was not effective in inhibiting the replication of
MDHV or in preventing the development of lymphomas when phosphonoace-
tate was administered intra-abdominally before MDHV inoculation (74).

The initial successes obtained with phosphonoacetate in the

treatment of herpes simplex infections in mice and rabbits led the

30
investigators from Abbott Laboratories to postulate that phosphono-
acetate might be useful in the treatment of herpesvirus infections
in man, such as herpesvirus dermatitis and herpesvirus keratitis (71).
It was also thought that phosphonoacetate might be effective against
herpes encephalitis in man since it greatly reduced mortality of
mice infected with herpesvirus. Moreover, phosphonoacetate might
be useful in preventing sometimes fatal herpesvirus infections in
patients undergoing immunosuppression therapy.

Effect of Phosphonoacetate on a Cell Line
Transformed by Epstein-Barr Virus

 

Epstein-Barr virus is a herpesvirus for which no completely per-
missive culture system exists, i.e., no cell type supports the efficient
replication of EBV. If there is no good source of replicating EBV,
there is no good source of material to use to determine if an EBV-
induced DNA polymerase exists. There are, however, several well char-
acterized human lymphoblastoid cell lines transformed by EBV. These
cell lines have been cultured in the presence of phosphonoacetate in
an attempt to determine the mechanism of EBV DNA replication (81,82).
The results obtained with one cell line, the Rajii cell line, will be
discussed.

The Raji cell line is a transformed lymphoblastoid cell line
derived from a tumor biOpsy from a patient with Burkitt's lymphoma
(83). It is a nonproducer cell line. Raji cells contain approximately
50 EBV genomes per cell but express only a single viral antigen, EBV-
specific nuclear antigen, and produce no virus particles. Replication
of resident EBV genomes is under cellular control mechanisms and

occurs only during early S phase (84). If Raji cells are superinfected

31
with EBV, however, host cell DNA synthesis is completely inhibited
and productive replication of EBV DNA occurs (81).

The effect of phosphonoacetate on the EBV content in Raji cells
before and after superinfection was determined. When Raji cells
were grown in the presence of 1 mM phosphonoacetate for three days
or in the presence of 0.5 mM phosphonoacetate for three weeks, there
was no change in the number of resident EBV genomes as determined
by hybridization techniques (81,82). Thus, the EBV DNA present in
these transformed cells replicates by a mechanism which is insensi-
tive to phosphonoacetate as might be expected since resident EBV DNA
replication is under cellular control.

When Raji cells were superinfected, the amount of EBV DNA
present in these cells increased to such levels that it could be
detected in CsCl gradients if the superinfected cells were labelled
with [3Hlthymidine. When Raji cells were superinfected and then
cultured in the presence of 0.5 mM phosphonoacetate, EBV DNA could
not be detected in CsCl gradients. Thus, productive EBV DNA replica-
tion in the absence of cellular controls is sensitive to phosphono-
acetate. In light of the fact that the known herpesvirus-induced
DNA polymerases are sensitive to phosphonoacetate, the sensitivity
of productive EBV DNA replication to phosphonoacetate suggests that
a herpesvirus-induced DNA polymerase replicates EBV DNA in these
superinfected cells. However, this postulated herpesvirus-induced
DNA polymerase does not appear to replicate EBV DNA in transformed

cells.

MATERIALS AND METHODS

Materials

Phosphocellulose (P-11) and DEAE-cellulose (DE52) were purchased
from Reeve Angel. Whatman no. 40 acid-washed chromatography paper
was from Fisher Scientific Co. Analtech, Inc. was the source of
precoated cellulose thin layer chromatography plates. Nitrocellulose
membrane filters (type B-6) were obtained from Schleicher and Schuell,
Inc. Calf thymus DNA, dithiothreitol, unlabelled nucleotides,
poly (dA) ooligo (dT)

8: poly(rA)-oligo(dT) 8' poly(dC)-oligo(dG)

12—1 12-1 12-18)

and poly(rC)-oligo(dGl were purchased from P-L Biochemicals,

12-18
Inc. Poly d(A-T) and poly(dA) were from Miles Laboratories, Inc.
International Chemical and Nuclear Corp. was the source of [3H]-
deoxynucleoside triphosphates. New England Nuclear was the source
of [32Plsodium pyrophosphate. Deoxythymidine S'phosphomorpholidate,
bovine serum albumin and beef heart lactate dehydrogenase were
purchased from Sigma Chemical Co. Snake venom phosphodiesterase I,
bacterial alkaline phosphatase, and pancreatic DNase I (RNase free)
were obtained from Wbrthington Biochemical Corp. Escherichia coli
DNA polymerase I was purchased from Boehringer Mannheim.

Dowex l (BioRad AG 1-x2, 200-400 mesh) was a gift from Dr.
Fritz Rottman of this department. Nonidet P-40 was a gift from
Dr. Leland Velicer of the Department of Microbiology and Public
Health. Avian myelcblastosis virus reverse transcriptase was a

gift from Dr. J. Beard, Life Science Research Laboratories.

32

33

Phosphonoacetate, disodium salt, was a gift from.Abbott Labora-
tories. Phosphonoacetic acid, phosphonopropionate, the trimethyl
ester of phosphonoacetate, phenylphosphonoacetate, 2-aminophosphono-
acetate, 2-methyl-2-phosphonopropionate and 2-phosphon0propionate
were generously provided by Dr. A. F. Isbell of Texas A&M university.
The monocyclohexylamine salt of phosphonoacetamide, N-methyl phos-
phonoacetamide and N-(phosphonoacetyl)-L~aspartate were gifts from
Dr. George Stark of Stanford University. Acetonyl phosphonate, the
monomethyl ester of acetonyl phosphonate and the monomethyl ester of
acetyl aminophosphate were gifts from Dr. Ronald Kluger of the Uni-
versity of Toronto. Phosphonoacetaldehyde was prepared by Mr. John
Reno of this laboratory. Other reagents were from the usual com-
mercial sources.

[3H]Phosphonoacetic acid (20 Ci/mmol) was prepared by New
England Nuclear using an acid catalyzed exchange reaction developed
by Mr. Steve Saxe of this laboratory. Anhydrous phosphonoacetic
acid (2 mg) was dissolved in carrier-free [3leater (3 ul) and was
stirred gently for 5 hr at 100°. Excess [3H]water was removed, in
vacuo, using water as a solvent. The radioactive material prepared
in this manner was determined to be a homogeneous solution of
phosphonoacetic acid when analyzed by electrophoresis, paper chroma-
tography in solvent system I (Table 4), thin layer cellulose chroma-
tography, DEAE-cellulose (HCO3-) chromatography and Dowex 1 (Cl-)
chromatography. No radioactive material vaporized when a solution
of [3H]phosphonoacetic acid was vaporized to dryness, which indi-
cated no [3H1water remained in the preparation.

Activated calf thymus DNA was prepared by a modification of the

procedure of Schlabach et a1. (85). Calf thymus DNA (approximately

34
200 mg) was dissolved in 10 mM Tris-HCl (pH 8), 50 mM NaCl, 0.5%
sodium dodecyl sulfate (approximately 125 ml) with stirring and was
extracted with one volume of freshly distilled, water-saturated
phenol. After extensive dialysis against three changes (6.1 each)
of 10 mM Tris-HCl (pH 8), 50 mM.NaCl and one change of 10 mM Tris-
HCl (pH 8), 50 mM KCl, the concentration of the DNA solution was

1% =200), A solution of MgCl2

determined spectrophotometrically (E260

was added to a final concentration of 5 mM. Additional buffer was
used if needed so that the final DNA concentration was 1 mg per ml.
Pancreatic DNase I (RNase free) was added to a final concentration
of 0.2 U per ml. Incubation was at 37° for 25 min. The reaction
was terminated by incubation at 65° for 10 min. The DNA solution was
again extracted with one volume of water-saturated phenol, dialyzed

extensively against 10 mM Tris-HCl (pH 8) and stored at -20°.

Preparation and Growth of Cells

 

Cultures of HVT-infected DEF and uninfected DEF were grown in
the laboratory of Dr. Lucy Lee. Cultures of MDHV-infected DEF, unin-
fected CEF and MSB-l cells were grown in the laboratory of Dr.

Keyvan Nazerian.

Duck embryo fibroblasts were prepared from l3-day-old embryos.
Chicken embryo fibroblasts were prepared from ll-day-old embryos.

The embryos were decapitated, washed with a sterile solution of
phosphate-buffered saline, macerated, and trypsin treated as described
by Soloman et a1. (86). The cells were suspended in culture medium
at a concentration of 106 per ml, and 20-ml samples of the suspension
were seeded onto 150 mm-diameter Falcon plastic tissue culture dishes.

The culture medium consisted of 40 volumes of medium 199, 50 volumes

35

of nutrient medium F10, 5 volumes of tryptose phosphate broth, 3
volumes of 2.8% sodium bicarbonate, 4 volumes cf inactivated calf
serum, 100 units of penicillin per m1, 100 ug of streptomycin per
ml, and 10 ug of aureomycin per ml (87). Cultures were incubated
at 37° in a humidified atmosphere containing 5% carbon dioxide.
After 48 hr, the culture medium‘was removed. For the preparation of
uninfected cells, 20 m1 of fresh culture medium was added to each of
the dishes and incubation was continued for another 24 hr. The cells
were then scraped from the tissue-culture dishes, washed with phosphate-
buffered saline, quick frozen and stored at -80°. For the preparation
of infected cells, 20 ml of fresh culture medium containing HVT or
MDHV-infected DEF at a concentration of 2 x 105 per m1 (2 x 104 PFU
per ml) was added to the dishes. After 24 hr of incubation, the cul-
ture medium was replaced with fresh.medium. Cytopathic effect due to
viral infection of the cells was confluent 24 hr later. The infected
cells were then scraped from the tissue culture dishes, washed with
phosphate-buffered saline, quick frozen and stored at -80°.

Cultures of HVT-infected DEF were also grown in roller bottles
(10 x 40 cm) by the same procedure except that DEF were seeded at a
concentration of 4 x 106 per ml in 50 ml culture medium and rotated
at 0.1 rpm at 37° in the absence of carbon dioxide: 4 x 105 per ml
of HVT-infected DEF were used for infection.

A phosphonoacetate-resistant mutant of HVT was isolated by Dr.
Lucy Lee by growing HVT in secondary DEF cultures in medium 199 and
medium F10 supplemented with 4% heat-inactivated calf serum and
increasing concentrations of phosphonoacetate. The concentration of

phosphonoacetate in the medium was increased from 0.3 to 1.6 mM.

36

Each concentration was used for three to four passages of virus.
The virus (HVTPA) from the 30th passage was resistant to 1.6 mM of
phosphonoacetate and was cloned in the absence of phosphonoacetate.

The MSB-l lymphoblastoid cell line was grown in RPMI-1640 medium
(Flow Laboratories) supplemented with 10% bovine fetal calf serum at
41° in a 5% carbon dioxide humidified atmosphere. Cells were seeded
5

in Falcon plastic petri dishes at an initial concentration of 5 x 10

cells per ml and were subcultured every two to three days.

Purification of DNA Polymerases
All procedures were performed at 0-4°. Frozen HVT-infected duck
embryo fibroblasts (20 g wet weight) were suspended in.buffer A (50

mM Tris-HCl [pH 7.5], 25 mM KCl, 5 mM MgCl and 0.25 M sucrose) con-

2.
taining 0.2% nonidet P-40. Cytoplasmic and nuclear extracts were
prepared by a modification of the procedure of Chang et a1. (88). A
cell homogenate was prepared using a Dounce homogenizer and was cen-
trifuged at 1,000 x g for 5 min.

The supernatant fraction was centrifuged at 10,000 x g for 10
min, made 5 mM in 2-mercaptoethanol, and centrifuged at 100,000 x g
for 60 min. The supernatant fraction from the 100,000 x g centrifu-
gation was termed the cytoplasmic extract.

The nuclear pellet from the 1,000 x g centrifugation step was
suspended in buffer A, centrifuged, and then resuspended in buffer A
containing 1% Triton x-lOO (v/v). After 15 min the nuclei were col-
lected by centrifugation and washed three times with buffer A (crude
nuclear fraction). The final nuclear pellet.was suspended in buffer

A containing 5 mM 2-mercaptoethanol and 0.2 M potassium phosphate

(pH 7.5). After gently stirring for 30 min to extract the nuclear

37

DNA polymerase activities, the suspension was centrifuged at 100,000
x g for 60 min. The resulting supernatant fractionAwas termed the
nuclear extract.

The nuclear extract was dialyzed against.buffer B (25 mM Tris-
HCl [pH 8], 100 mM KCl, 5 mM 2-mercaptoethanol, and 20% glycerol
[v/v]) and was applied to a phosphocellulose column. The phospho-
cellulose column (1.4 x 8 cm) had been washed with 5 ml of buffer B
containing 1 mg per m1 of bovine serum albumin to improve recoveries
from the column and then equilibrated with buffer B. After washing
the column with 20 ml of buffer B, the DNA polymerase activity was
eluted with a linear gradient of 100 ml of 0.1 to 1.0 M KCl in buffer
B. Fractions of 2 ml each were collected. A single peak of HVT-
induced DNA polymerase activity eluted at about 0.35 M KCl as
determined by conductivity measurements. The fractions containing
the majority of the HVT-induced DNA polymerase activity were pooled
(phosphocellulose fraction) and dialyzed against buffer C (0.02 M
potassium phosphate [pH 7.51, 0.5 mM dithiothreitol, and 20% [v/v]
glycerol). The dialyzed fraction was applied to a DEAE-cellulose
column (0.6 x 10 cm) which had been washed with 5 ml of buffer C
containing 1 mg per ml of bovine serum albumin and.with 5 m1 of
0.75 M potassium phosphate in buffer C and then equilibrated with
buffer C. After sample application, the column was washed with 20
m1 of buffer C and.was eluted with a linear gradient of 30 ml of
0.02 to 0.4 M potassium phosphate in buffer C. Fractions of 0.5 ml
each were collected. The HVT-induced DNA polymerase activity eluted
as a single peak of activity at about 0.14 M potassium phosphate.
The fractions containing the majority of the DNA polymerase activity

were pooled and stored at -20° after addition of bovine serum albumin,

38
dithiothreitol, and glycerol to give final concentrations of 2 mg per
ml, 0.5 mM and 50% (v/v), respectively. After six weeks at -20°, 75%
of the activity of the HVT-induced DNA polymerase remained.

The MDHV-induced DNA polymerase was extracted from MDHV-infected
DEF and was purified by DEAE-cellulose chromatography as described
above.

Duck embryo fibroblast DNA polymerase a from cytoplasmic extracts
and DNA polymerase B from nuclear extracts were likewise purified
through DEAE-cellulose columns.

When less than 5 g wet weight of cells were used as starting
material, bovine serum albumin was added to nuclear extracts to a
final concentration of 1 mg per ml to stabilize the DNA polymerase

activities.

Assayiof DNA Polymerases

The standard reaction mixture employed for the HVT-induced DNA
polymerase contained, in 200 pl: 50 mM Tris-HCl (pH 8), 2 mM MgC12,
200 mM KCl, 1 mM dithiothreitol, 500 pg per ml of bovine serum
albumin, 200 pg per ml of activated calf thymus DNA, 20 pH [3H1dNTP
(300-400 cpm per pmol), 100 uM each of the other dNTPs, and DNA
polymerase. Incubation was at 37° fer 30 min.

Assay conditions were such that the rate of DNA polymerization
was linear with time and.with the amount of DNA polymerase added.
One unit of DNA polymerase activity was defined as that amount of
activity which directed the incorporation of 1 nmole of [BHIdNTP
into acid-insoluble material in 30 min at 37°.

In experiments in which product inhibition by pyrophosphate

was examined, supplementary MgCl2 was added to reaction mixtures

39
along with pyrophosphate to compensate for the chelation of Mg2+
by pyrophosphate. The concentration of MgCl2 to be added to reac-
tion mixtures in addition to the standard 2 mM MgCl2 concentration
was calculated using the equations described by Moe and Butler (89).
It was determined that equimolar amounts of both pyrophosphate and
MgCl2 were to. be added to the reaction mixtures.

The standard reaction mixture employed for the MDHV-induced DNA
polymerase contained all the same components as the HVT-induced DNA
polymerase assay except that 1 mM MgCl2 was used instead of 2 mM
MgC12.

The standard reaction mixture employed for duck and chicken DNA
polymerase assays contained in 200 pl: 50 mM Tris-HCl (pH 8), 10 mM
MgC12, 1 mM dithiothreitol, 500 pg per m1 of bovine serum albumin,
200 pg per ml of activated calf thymus DNA, 20 pM [3HldNTP (300 to
400 cpm per pmol), 100 pM each of the other three dNTPs, and DNA
polymerase. Incubation was at 37° for 30 min.

The standard reaction mixture employed for E. coli DNA polymerase
I contained all the same components as the duck and chicken DNA
polymerase assays except that 5 mM MgCl2 was used instead of 10 mM
MgC12.

The standard reaction mixture employed for the AMV reverse
transcriptase assay contained in 200 pl: 50 mM Tris-HCl (pH 8),

0.5 mM MnClz, so mM xc1, 1 mM dithiothreitol, 100 um [33mm (80
cpm per pmol), and 0.5 optical density units (at 260 nm) per ml of

poly(rA)-oligo(dT) Incubation was at 37° for 30 min.

12-18°
All DNA polymerase reactions were terminated by addition of 10%
trichloroacetic acid-1% sodium pyrophosphate (2 m1). Bovine serum

albumin (50 p1 of a 5 mg per ml solution) was then added as carrier.

40
After 5 min at 0°, reaction mixtures were centrifuged at 10,000 x g
fer 2 min. The supernatant solution was removed and discarded. The
pellet was suspended in 0.35 ml of 0.5 N NaOH. After readdition of
cold 10% trichloroacetic acid-1% sodium pyrophosphate and after 5
min at 0°, the precipitated material was collected on a nitrocellu-
lose filter. The filter was then washed with three pOrtions of
cold 10% trichloroacetic acid-1% sodium pyrophosphate, dried, and
monitored for radioactivity by liquid scintillation spectrometry.
The scintillation fluid (5 ml) contained 4 g of 2,5-bis[2-(5-t-

butylbenzoxazoyl))thiophene per liter of toluene.

Assay of the dNTP-Pyrophosphate Exchange Reaction

The reaction mixture contained in 200 pl: 50 mM Tris-HCl (pH 8),
1 mM dithiothreitol, 200 mM KCl, 1 mM MgC12, 500 pg per ml bovine
serum albumin, 200 pg per ml activated calf thymus DNA, 0.1 mM each
of the four deoxyribonucleosidetriphosphates, HVTeinduced DNA
polymerase, and various concentrations (0.14-1.1 mM) of [32p]-
labelled sodium pyorphosphate (specific radioactivity of approxi-
mately 100 cpm per pmol). In addition to the 1 mM MgCl2 routinely
added to the reaction mixture, supplementary MgClz, in an amount
equimolar to the sodium pyrophosphate added to the reaction mixture,
was used.

Incubation was at 37° for 30 min. The assay measuring the
conversion of [32Pl-1abelled pyrophosphate to a Norit-adsorbable
form was performed as described by Deutscher and Kornberg (90).

For HVT-induced DNA polymerase, the pyrophosphate exchange reaction
was shown to be dependent on added enzyme, activated calf thymus

DNA, Mg2+ ions, and deoxyribonucleoside triphosphates. When assayed

41
using 1.1 mM [32Pl-labelled sodium pyrophosphate, the reaction was
found to be linear with time for at least 120 minutes and was
directly proportional to the amount of HVT-induced DNA polymerase
added to the reaction mixture. The rate of the dNTP-pyrophosphate
exchange reaction was about 25% of the rate of the DNA polymeriza-

tion reaction.

Analytical Methods

 

Protein concentrations were determined by the method of Lowry
et a1. (91) with bovine serum albumin as the standard.

Lactate dehydrogenase was assayed spectrophotometrically at
340 nm by following the oxidation of NADH in the presence of pyruvate.
The sedimentation coefficient of beef heart lactate dehydrogenase
was taken as 7.48 (92).

The presence of a deoxyribonucleoside triphosphatase activity
in the HVT-induced DNA polymerase preparations was determined by
incubating an excess of HVT-induced DNA polymerase in standard
reaction mixtures at 37° for 30 min. Aliquots (10 pl) were spotted
on Whatman No. 40 acid-washed chromatography paper. Paper chromato—
grams were developed in solvent system I (Table 4) and were then cut
into 2 x 1 cm pieces. These pieces were extracted with water (1 ml)
mixed with Bray's scintillation fluid (10 ml) (93) and monitored
for radioactivity using liquid scintillation spectrometry.

The presence of an inorganic pyrophosphatase activity was
determined by measuring the conversion of [32Plinorganic pyrophosphate
to inorganic phosphate in standard reaction mixtures containing 1 mM
[32P]inorganic pyrophosphate (100 cpm/pmol) by the method of Martin

and Doty (94).

42

The presence of deoxyribonuclease activity in HVT-induced
DNA polymerase preparations was determined by incubating standard
reaction mixtures containing [3Hlpoly d(A-T) (approximately 200
cpm per pmol) and no labelled dNTP or activated DNA with HVT-induced
DNA polymerase and by analyzing for: (1) conversion of acid-
insoluble [3H] material to acid-soluble [3H] material or (2) con-
version of [3H] polymer to [3H]dTMP by descending paper chroma-
tography in isobutyric acid-cone. ammonium hydroxide-water (66:1:33)
(95).

Total phosphorus determinations of samples in solution were
made using the method of Ames and Dubin (96). Samples were ashed
in the presence of 10% Mg(NO3)2 in alcohol and reacted with a 0.36%
ammonium molybdate, 0.86 N sulfuric acid, 1.4% ascorbic acid solu-
tion. All the glassware used in these determinations was rinsed with
concentrated nitric acid and recycled.

Alkaline phosphatase digestions of thymidine nucleotides
employed reaction mixtures containing in 1 ml: 0.3 mM thymidine
nucleotide, 50 mM Tris-HCl (pH 8.5) and 1.25 U bacterial alkaline
phosphatase. Incubation was at 45° for 2 hr. Reaction mixtures were
diluted with 5 mM ammonium bicarbonate and applied to a micro DEAE-
cellulose (HCOB) column (1 x 2.5 cm). Columns were washed with 5 mM
ammonium bicarbonate (6 ml) and 300 mM ammonium bicarbonate (8 m1).
Fractions of 2 ml were collected and absorbance at 267 nm measured.

A thymidine nucleotide was sensitive to alkaline phosphatase if it
did not adsorb to the DEAE-cellulose (HCO3-) column.

Snake venom phosphodiesterase I digestions of thymidine nucleo-

tides employed reaction mixtures containing 10 mM thymidine nucleotide,

30 mM Tris-HCl (pH 8.9), 30 mM magnesium acetate, and 2 U per m1 snake

43

venom phosphodiesterase I. Incubation was at 37° for 30 min.
Digestions were terminated by placing the reaction tubes on ice.
Aliquots of 10 pl were spotted on Whatman No. 40, acid washed
paper. Chromatograms were developed in solvent system I (Table 4).
Reaction products were identified by ultraviolet quenching and a
phosphate spray reagent (97).

Thin layer chromatography on cellulose plates was performed

in ethanol-29% aqueous ammonia-water (6:1:3) (98).

Kinetic Analysis

 

Initial velocity patterns, inhibition patterns, kinetic constants
and reaction mechanisms were defined according to the nomenclature
of Cleland (99,100; Appendices A and C). Each assay was run in
duplicate. The data for the double reciprocal plots were evaluated
using a computer program based on the method of Wilkinson (101).
For evaluation of apparent inhibition constants, replots of the
intercepts and slopes of the double reciprocal plots were analyzed

using linear least-squares analysis.

RESULTS

Marek's Disease Herpesvirus-Induced DNA Polymerase

Identification of an Induced DNA Polymerase
from Marek's Disease Herpesvirus-Infected
Duck Embryo Fibroblasts

 

The first evidence that infection of DEF by MDHV, strain BC-l,
led to the induction of a salt-stimulated DNA polymerase activity
similar to the HSV-induced DNA polymerase activity was obtained
when extracts of infected and uninfected DEF were tested in the MDHV-
induced DNA polymerase assay (+200 mM KCl) and the duck DNA polymerase
assay (-KC1) (Table 1). When nuclear and cytoplasmic extracts of
MDHV-infected DEF were assayed by the MDHV-induced DNA polymerase
assay, the levels of DNA polymerase activity were at least ten times
greater than those in nuclear and cytoplasmic extracts of uninfected
DEF. When nuclear and cytoplasmic extracts of MDHV-infected and
uninfected DEF were assayed by the duck DNA polymerase assay, similar
levels of DNA polymerase activity were found.

The nuclear extract of MDHV-infected DEF was used to partially
purify and characterize the MDHV-induced DNA polymerase since it
contained substantially more MDHV-induced DNA polymerase activity
than duck DNA polymerase activity.

The DNA polymerase activities from a nuclear extract of MDHV-
infected DEF were fractionated by DEAE-cellulose chromatography
(Figure 2, upper panel). The MDHV-induced DNA polymerase activity

44

45

Table 1. Intracellular distributions of DNA polymerase activities in
duck embryo fibroblasts

 

Units per g wet weight cells

 

 

MDHV-Induced DNA Duck DNA Poly-

Sample Polymerase Assay merase Assay
MDHV-infected DEF

nuclear extract 20-80 2-10

cytoplasmic extract 3-30 30-40
DEF

nuclear extract 2 , 5

cyt0plasmic extract 0.3 40

 

Nuclear and cytoplasmic extracts were prepared as described
in Materials and Methods. Both the MDHV-induced DNA polymerase assay
and the duck DNA polymerase assay contained 50 mM Tris-HCl (pH 8),
1 mM dithiothreitol, 500 ug Per m1 bovine serum albumin, 200 pg per
ma activated calf thymus DNA, 20 pM [3H]dNTP, and 100 pM each of the
other dNTPs. In addition, the MDHV-induced DNA polymerase contained
1 mM MgC12 and 200 mM KCl. The duck DNA polymerase assay also con-
tained 10 mM MgCl2 and no KCl (66).

was the only major DNA polymerase activity found. It eluted from

the DEAE-cellulose column as a single peak at about 0.1 M potassium
phosphate. Fractionation of the DNA polymerase activities from a
nuclear extract of uninfected DEF by DEAE-cellulose chromatography
revealed that the MDHV-induced DNA polymerase activity was not present
in the nuclear extract of these cells and that the levels of duck

DNA polymerase activities were approximately 20-fold less than the
level of MDHV-induced DNA polymerase activity (Figure 2, lower panel).
The peak MDHV-induced DNA polymerase fractions were pooled and used

to characterize the enzyme.

46

Figure 2. DEAE-cellulose chromatography of DNA polymerase
activities from nuclear extracts of MDHV-infected duck embryo
fibroblasts (upper panel) and uninfected duck embryo fibroblasts
(lower panel). Nuclear extracts from 2.0 g wet weight of MDHV-
infected DEF and 1.2 g wet weight of uninfected DEF were prepared
and subjected to DEAF-cellulose chromatography as described in
Materials and Methods. Samples (25 pl) of the fractions were
assayed for DNA polymerase activities by the MDHV-induced DNA
polymerase assay (dotted line) and the duck DNA polymerase assay
(solid line). Potassium phosphate concentrations were determined
by conductivity measurements. DNA polymerase activities were
expressed as pmol [3HldTMP incorporated into DNA in 30 min per
g wet weight of cell.

47

.2 76138.: v; m. T8532... v:
3. 2. I. .
0 o o

tttqi — u

 

 

 

,0
4

 

Fraction Number

 
 

 

— —

 

 

_
o m o 8 6 4 2
5 5

22:3 eBEoSooE as»... 2053 3.9538... use“.

Fraction Number

Figure 2

48

Characterization of the MDHV-Induced
DNA Polymerase

 

The general reaction requirements of the MDHV-induced DNA
polymerase activity were examined. The MDHV-induced DNA polymerase
activity required M92+ in activated DNA reaction mixtures (Figure 3,
upper panel). In the absence of MgClz, negligible DNA polymerization
occurred. In the presence of 1 mM MgC12, maximum DNA polymerization
occurred. When Mg2+ was replaced with Mn2+, maximum DNA polymeri-

zation occurred in the presence of 0.05 mM MnCl but this concentra-

2:
tion of MnCl2 was only 33% as effective as 1 mM MgCl2 (Figure 3,
lower panel).

The MDHV-induced DNA polymerase activity required KCl in
activated DNA reaction mixtures (Figure 4). Maximum DNA polymeri-
zation occurred in the presence of 200 to 250 mM KCl. In the presence
of 200 mM KCl, DNA polymerization was 10— to 60-fold greater than
in its absence depending on the enzyme preparation. Stimulation of

DNA polymerization was also observed with other salts. Maximum

stimulation was observed with 200 mM NH Cl, 110 mM (NH4)2SO

4 and

4:
110 mM K2804. However, with these salt concentrations stimulation
was only 0.6 of that with 200 mM KCl (data not shown). Thus, stimu-
lation was more complicated than a simple ionic strength effect.

The MDHV-induced DNA polymerase activity was dependent on the
presence of dithiothreitol in activated DNA reaction mixtures. When
the dithiothreitol concentration was reduced from 1.1 mM to 0.1 mM,
DNA polymerization was reduced by 40%. When 1 mM p-hydroxymercuri-

benzoate was added to reaction mixtures, DNA polymerization was

inhibited by 98% (data not shown).

49

Figure 3. Effect of MgC12 concentration (upper panel) and
MnC12 concentration (lower panel) on the activity of the MDHV-
induced DNA polymerase. Reaction mixtures were prepared for the
MDHV-induced DNA polymerase assay as described in Materials and
Methods except that MgClz or MnC12 was added to the final concen-
trations indicated.

SO

 

 

 

      

-

5 0 5

. _
0
m 7 5 2

2°53 cottoacooc. dszb

 

 

 

 

umBBaBuc. est...

0.2 0.3 0.4 0.5

[Mn Clz] mM

O.|

 

Figure 3

51

Figure 4. Effect of KCl concentration on the activity of the
MDHV-induced DNA polymerase. Reaction mixtures were prepared for
the MDHV-induced DNA polymerase assay as described in Materials

and Methods except that KCl was added to the final concentrations
indicated.

52

 

 

 

 

 

J 1 J l l

o o o o o

9 co o e on
wind pOlDJOdJOOUl dW 1p

200 300 400

IOO

[KCI] mM

Figure 4

53

The MDHV-induced DNA polymerase activity required deoxynucleo—
side triphosphates in activated DNA reaction mixtures. When dATP,
dCTP and dGTP were absent from reaction mixtures, [3HldTMP incor-
poration was only 12% of that in their presence (data not shown).

The MDHV-induced DNA polymerase activity required exogenous
template-primer in reaction mixtures. Template-primer specificities
of the polymerase were examined (Table 2). The MDHV-induced DNA
polymerase did not utilize double-stranded or single-stranded calf

thymus DNA. The oligomer-homopolymers poly(dA)-oligo(dT) d

12-18 an

poly(dC)-oligo(dG) were utilized effectively. No significant

12-18
polymerization was observed with poly(rA)°oligo(dT)12_18, poly(rC)°

oligo(dG) and poly(dA). Thus, the MDHV-induced DNA polymerase

12-18
is a DNA-dependent DNA polymerase and not DNA polymerase y (49),
reverse transcriptase (102,103) or terminal nucleotidyl transferase
(104,105).

The sedimentation coefficient of the MDHV-induced DNA polymerase
was determined by sedimentation velocity centrifugation. The MDHV-
induced DNA polymerase sedimented as a single symmetrical peak in
a 10 to 30% glycerol gradient (Figure 5). The reference protein
lactate dehydrogenase was centrifuged in the same gradient. Based
on the sedimentation coefficient of lactate dehydrogenase, the MDHV-
induced DNA polymerase exhibited a sedimentation coefficient of
7.0 :_0.ZS. For a typical globular protein, a sedimentation coef-
ficient of 7.03 would correspond to a molecular weight of 130,000

daltons.

54

Table 2. Template-primer specificities of MDHV-induced DNA

 

 

polymerase
dNTP 3

Template-primer Substrates [ H]dNMP
Activated calf thymus DNA [3H]dTTP,dGTP,dCTP,dATP 100(100)
Native calf thymus DNA [3H]dTTP,dGTP,dCTP,dATP l
Denatured calf thymus DNA [3H1dTTP,dGTP,dCTP,dATP 3

. 3
Poly(dA)-oligo(dT)12__18 [ H]dTTP 300

. 3
Poly(rA)-ol1go(dT)12_18 I H]dTTP 3

. 3
Poly(dC)-oligo(dG)12_18 [ H]dGTP 80

. 3
Poly(rC)-oligo(dG)12_18 [ H]dGTP 3
Poly(dA) [BHJdGTP 3

 

The composition of the reaction mixtures containing calf
thymus DNA was as described in Materials and Methods. The composi-
tion of the reaction mixtures containing olgio-hcmopolymers was:

50 mM Tris-HCl (pH 8), 0.5 mM MgC12, 150 mM KCl, 1 mM dithiothreitol,
500 pg per ml bovine serum albumin, 20 pM [3H]dTTP (400 cpm per

pmol) or 20 pM [3H]dGTP (300 cpm per pmol), 0.2 optical density
units at 260 nm per m1 of poly(dC)-oligo(dG)12_18 or 0.5 optical
density units at 260 nm per ml of the other template-primers and DNA
polymerase. Incubation was at 37° for 30 min. The picomoles of
[3H]dTMP incorporated into activated DNA are given in parentheses.

55

Figure 5. Glycerol gradient centrifugation of MDHV-induced
DNA polymerase. An aliquot of the DEAF-cellulose fraction of
MDHV-induced DNA polymerase was dialyzed against a buffered solu-
tion containing 50 mM Tris-HCl (pH 7.5), 250 mM KCl and 0.5 mM
dithiothreitol. Samples containing MDHV-induced DNA polymerase
and lactate dehydrogenase (0.2 ml) were layered on 4.8 ml linear
10 to 30% glycerol gradients prepared in the above buffered solu-
ion and containing 0.5 mg per m1 of bovine serum albumin. Gradients
were centrifuged for 16 hr at 4° in a Beckman SW 50.1 rotor. After
centrifugation fractions (0.16 ml) were collected from the bottom
of the centrifuge tube. Samples (25 pl) of the glycerol gradient
fractions were assayed for DNA polymerase activity using the
MDHV-induced DNA polymerase assay. The arrow indicates the peak
position of the reference protein lactate dehydrogenase (LDH).
The recovery of MDHV-induced DNA polymerase activity was approxi-
mately 70%.

56

 

 

0

 

  

11, I l 1 J1

e re a: -
(gourd) peioaodnooul le 1p

11

 

 

3O

25

20

I5

IO

5

Fraction Number

Figure 5

57
Herpesvirus of Turkeys-Induced DNA Polymerase
Purification of the HVT-Induced
DNA Polymerase

When extracts from HVT-infected DEF were examined for an induced
DNA polymerase, an activity was found in amounts similar to that
found in extracts of MDHV-infected DEF. When the DNA polymerase
activities from nuclear extracts of HVT-infected DEF were fractionated
by DEAE-cellulose chromatography, two peaks of HVT-induced DNA
polymerase activity eluted from the column at about 0.08 M and 0.13 M
potassium phosphate. These two activities were present in approxi-
mately equal amounts and were identical with respect to MgCl2 optimum,
KCl optimum, template specificity, sedimentation coefficient and
phosphonoacetate sensitivity (data not shown). This result is dif-
ferent from that obtained for the MDHV-induced DNA polymerase where
a single peak of activity eluted from the DEAE-cellulose column at
about 0.1 M potassium phosphate.

The HVT-induced DNA polymerase was subjected to more extensive
purification. DNA polymerase activities from nuclear extracts of
HVT-infected DEF were fractionated by phosphocellulose chromatography.
The HVTbinduced DNA polymerase eluted from the column as a single
peak of activity at about 0.35 M KCl. The peak fractions of HVT-
induced DNA polymerase activity were pooled, dialyzed and subjected
to DEAE-cellulose chromatography. The HVT-induced DNA polymerase
eluted from the column as a single peak of activity at about 0.14 M
potassium phosphate. A summary of such a purification may be found
in Table 3. The phosphocellulose fraction had a specific activity of

58 units per mg protein and represented a 30-fold purification over the

58

Table 3. Purification of HVT-induced DNA polymerase

 

 

Total Protein Recovery Specific
Fraction (mg) Total Units (%) Activity
Homogenate 920 1580 100 2
Crude nuclear 90 780 50 9
100,000 x g 40 500 30- 12
Nuclear supernatant
Phosphocellulose 5 290 20 58
DEAE-cellulose l 100 6 100

 

HVT-induced DNA polymerase was purified from 20 g (wet weight)
of HVT-infected DEF grown in roller bottles as described in Materials
and Methods.

homogenate fraction; DEAE-cellulose chromatography resulted in an
additional 1.7-fold purification of the HVT-induced DNA polymerase.

No detectable deoxyribonucleotide triphosphatase activity or
inorganic pyrophosphatase activity and little or no deoxyribonuclease
activity was found in the phosphocellulose enzyme fraction when tested
using the standard HVT-induced DNA polymerase assay conditions. This
fraction of HVT-induced DNA polymerase activity was used for many of
the kinetic studies to be reported.

Characterization of the HVT-Induced
DNA Polymerase

 

The phosphocellulose fractions and the DEAE-cellulose fractions
of the HVT-induced DNA polymerase were characterized. In eaCh case,
for the activated DNA polymerization reaction, maximum activity was

obtained in the presence of 2 mM MgCl2 and 200-250 mM KCl. On the

59
average, a lO-fold stimulation of DNA polymerization resulted when
200 mM KCl was added to reaction mixtures. In template-primer
studies the HVT-induced DNA polymerase was able to utilize poly(dA)-
oligo(dT)

and poly(dC)-oligo(dG) effectively when compared

12-18 12-18

with its ability to utilize activated calf thymus DNA. It could not

utilize poly(rA)'oligo(dT) and poly(rC)'oligo(dG) The

12—18 12-18'

sedimentation coefficient of the HVT-induced DNA polymerase was

7.0 :_0.28 as determined by glycerol gradient sedimentation velocity
centrifugation in the presence of 0.25M KCl (data not shown). Thus,
the properties of the HVT-induced DNA polymerase were similar to
those of the MDHV-induced DNA polymerase.

Apparent Michaelis constants for activated DNA and dNTP were
determined for the HVT-induced DNA polymerase from initial velocity
studies (Figure 6; Appendices A and C). With dNTP as variable
substrate and activated DNA as changing fixed substrate, the apparent
Michaelis constant for activated DNA was determined to be 7.3 :_0.2
pg per ml from the replot of intercept against reciprocal activated DNA
concentration. With activated DNA as variable substrate and dNTP as
changing fixed substrate, the apparent Michaelis constant for dNTP
was determined to be 2.1 :_0.5 pH from the replot of intercept

against reciprocal dNTP concentration (data not shown).

Inhibition of HVT-Induced DNA Polymerase by Phosphonoacetate

Mao et a1. (75) have reported that phosphonoacetate is an
effective inhibitor of the herpes simplex-induced DNA polymerase.
Phosphonoacetate was also an effective inhibitor of the DNA polymerases
induced by MDHV and HVT. The addition of l to 3 pM phosphonoacetate

to reaction mixtures resulted in about a 50% decrease in the rate of

60

Figure 6. .Initial velocity pattern for HVT-induced DNA
polymerase with the four dNTPs as the variable substrate and
activated DNA as the changing fixed substrate. Activated DNA
concentrations were (0) 2.5 pg per m1, (0) 5.0 pg per m1, and
(A) 25 pg per ml. Initial velocities were expressed as pmol
of [3H]dCMP incorporated into DNA per 30 min. The specific
radioactivity of [3H]dCTP was 2000 cpm per pmol. Equimolar
concentrations of each of the four dNTPs were present in each
reaction mixture. Replots of the slopes (O) and intercepts
(0) are shown in the left panel.

61

0.0

-

 

123..-?i
«.0 n0 «.0

- m

..0

o mucmflm

 

725311.775 Beets—

 

¢.0 n0 «.0 ..0
- u 4 u

I

I

 

1 000.0

1 00.0

 

 

. ldBOJOlUl

62

the DNA polymerization reaction catalyzed by these enzymes (Figure
16). Either in the presence or absence of phosphonoacetate, the rate
of the DNA polymerization reaction was linear for at least 1 hr.
Phosphonoacetate is a reversible inhibitor of the HVT-induced DNA
polymerase. It could be dialyzed away from the enzyme with full
recovery of enzyme activity.
Phosphonoacetate Inhibition Patterns for
the DNA Polymerization Reaction Catalyzed
pyyHVT—Induced DNA Polymerase

Phosphonoacetate gave linear noncompetitive inhibition with the
four dNTPs as the variable substrate and with activated DNA at a
saturating concentration of 200 pg per m1 (Figure 7; see Appendix A
for explanation of nomenclature). The apparent inhibition constant
(K11) determined from the replot of the vertical intercepts against
phosphonoacetate concentration was 1.4 i_0.6 pM. The apparent inhi-
bition constant (K15) determined from.the replot of the slopes against
phosphonoacetate concentration was 1.0 :_0.2 pM.

With activated DNA as the variable substrate, and the four
dNTPs at 2.5 pM each, phosphonoacetate gave linear noncompetitive
inhibition (Figure 8). A replot of the vertical intercepts yielded
a Kii of 1.5 :_0.2 pM and a replot of the slopes yielded a Kis of
2.5 :_0.6 pM. Phosphonoacetate also gave linear noncompetitive inhi-
bition with activated DNA as the variable substrate and with the
four dNTPs at 100 pM each. K. was determined to be 1.5 :_0.2 pM

1i

and Kis was about 9 :_3 pM. The higher Kis value seen at 100 pM
dNTP than at 2.5 pM dNTP indicated that the phosphonoacetate inhibi-
tion pattern was more nearly uncompetitive at the higher concentra-

tion of dNTP than it was at the lower concentration.

63

Figure 7. Double reciprocal plots with the four dNTPs as
the variable substrate and phosphonoacetate as inhibitor. Acti-
vated DNA concentration was 200 pg per ml. Phosphonoacetate
concentrations were 0 (O), 0.55 ma (0), 1.65 1m (0) and 2.75
ml (A). Initial velocities were expressed as pmol of [3H]dCMP
incorporated into DNA per 30 min. Specific radioactivity of
[3H]dCTP was 2000 cpm per pmol. Equimqu concentrations of
each of the four dNTPs were present in each reaction mixture.
Replots of the slopes (O) and intercepts (O) as a function of
phosphonoacetate concentration are shown in the left panel.

64

h musmflm

..l23._-TEuH_

 

n .o v.0 n.0 N6 _.0
q — q u 1

00.0

0.6

9.0

oNd

 

A .21 v. Tzozcooocofimozn;
n m _

q - -

O
.|\\\\\\IW\\\\\\

no- 0\

o adoIs
S
l
S

“!
o

 

 

o ¢damow|

65

Figure 8. Double reciprocal plots with activated DNA as
the variable substrate and phosphonoacetate as inhibitor. The
four dNTPs were at 2.5 uM each. Phosphonoacetate concentrations
were 0 (0), 0.55 M! (O), 1.1 11M ([3) and 2.2 1.1M (A). Initial
velocities were expressed as pmol of [3H] dCMP incorporated into
DNA per 30 min. Specific radioactivity of [3H]dCTP was 1000
cpm per pmol. Replots of the slopes (O) and intercepts (0)
as a function of phosphonoacetate concentration are shown in the
left panel.

66

m whamwm

L .833 .T 7.20 622:3;

cud 96 2.0 3.0

u m a -

A .21 v . HmzozmooocofimogmH—

 

 
  

D\LDO.O .
.r\\\\\\\JT “W -

\\\\\\\\\L.\\\\JU \\\\\\ AU
0 m\M\IO_.O 5N6! 0.0 ..Ulo
o\u\ > .m \ 26 m
av Au
0 m
C

 

 

 

0
¢\ .. oud

 

67

Phosphonoacetate Inhibition Pattern for the
dNTP-Pyrophosphate Exchange Reaction Catalyzed
byyHVTbInduced DNA Polymerase

Since phosphonoacetate and pyrophosphate have structural features
in common, it was suspected that phosphonoacetate might be inhibiting
the DNA polymerization reaction by interacting with the polymerase at
the pyrophosphate binding site. If so, phosphonoacetate should be a
competitive inhibitor of pyrophosphate in the dNTP-pyrophosphate
exchange reaction. This was the case (Figure 9). The apparent Kis
value fer phosphonoacetate was 1.3 :_0.4 pH. The apparent Km value
fer pyrophosphate was 0.24 i 0.04 mM.
Multiple Inhibition Analysis of Phosphonoacetate
and Pyrophosphate in the DNA Polymerization Reac-
tion Catalyzed by HVT-Induced DNA Polymerase

The results of the dNTP-pyrophosphate exchange reaction indi-
cated that phosphonoacetate interacts with the polymerase at the
pyrophosphate binding site. Additional evidence for this conclusion
was obtained from multiple inhibition analysis of the DNA polymeriza-
tion reaction (106,107). The concentration of phosphonoacetate was
varied in the presence of fixed concentrations of pyrophosphate when
dNTP concentrations were 2.5 uM each and activated DNA concentration
was 200 ug per ml. A plot of % against phosphonoacetate concentra-.
tion resulted in a series of parallel lines (Figure 10). This
result indicated that phosphonoacetate and.pyrophosphate are mutually
exclusive inhibitors and, therefore, bind at the same site on the
DNA polymerase (Appendix D). When dNTP concentrations were 20 uM
eadh and activated DNA concentration was 200 ug/ml, phosphonoacetate

and pyrophosphate were also found to be mutually exclusive inhibitors

of the HVT-induced DNA polymerase (data not shown).

68

Figure 9. Double reciprocal plots of the dNTP—pyrophosphate
exchange reaction with pyrophosphate as the variable substrate
and phosphonoacetate as inhibitor. Activated DNA was at 200
ug per ml and the four dNTPs were at 100 11!! each. Phosphono-
acetate concentrations were 0 (0 ) , 2 11M (0) and 3 (M (D) .
Initial velocities were expressed as pmol [32P]pyrophosphate
converted to Norit-adsorbable form per 30 min.

69

22.5. Encamozqoin;
T .

o

v N

 

.\.

DD

- q

\C

O
0

mu.

m musofim

no.0

 

A .23. TSAoooocofimBmH

 

 

 

n N A
q . u
S 0 No.0 M
.m- -°°.l [3.0 m
a
a O Lmod m.
o o\ o
N00!b . .

 

 

70

Figure 10. Multiple inhibition of the HVT-induced DNA
polymerase by phosphonoacetate and pyrophosphate. Activated
DNA was at 200 ug/ml and the four dNTPs were at 2.5 uM each.
Pyrophosphate concentrations were 0 (0 ) , 0.2 mM ( 0 ) , 0.4 mM
(0) and 0.6 mM (A). Initial velocities were expressed as
pmol [3H]dTMP incorporated into DNA per 30 min. Specific
radioactivity of [3H]dTTP was 2000 cpm per pmol.

71

ca whamflm

.21. TAvoococofimognAH—
N.O QC n.O _ .O

 

 

A A _ _ A _ _

 

 

72

Pyrophosphate Inhibition Patterns for
the DNA Polymerization Reaction Cata-
lyzed by HVT-Induced DNA Polymerase

 

 

 

For comparison to the phosphonoacetate inhibition patterns
and apparent Ki values, inhibition studies using pyrophosphate,
a product inhibitor, were performed. With the four dNTPs as the
variable substrate, pyrophosphate gave linear noncompetitive inhibi-

tion. Kii was 1.3 :_0.2 mM and K was 0.7 :_0.2 mM. With acti-

is
vated DNA as the variable substrate and with the four dNTPs at

2.5 M each, pyrophosphate gave linear noncompetitive inhibition.
Kii was 0.95 :_0.05 mM and Kis was 1.7 i 0.3 mM. Pyrophosphate
gave linear uncompetitive inhibition with activated DNA as the
variable substrate and with the four dNTPs at 20 H" each. Kii
was about 0.9 :_0.1 mM.

Effect of MgClq Concentration on Inhi-

bition of HVT-Induced DNA Polymerase
byiPhosphonoacetate

 

 

 

In the pyrophosphate inhibition studies of the HVT-induced
DNA polymerase, as in parallel studies with E. coli DNA polymerase I,
it is assumed thatMgPPi is the inhibiting species (108). Since
phosphonoacetate binds to the pyrophosphate binding site of the

enzyme, the effect of MgCl concentration on phosphonoacetate inhibi-

2
tion of the HVT-induced DNA polymerase was determined (Figure 11).

As the MgCl concentration in standard reaction mixtures containing

2

1 pM phosphonoacetate increased, the amount of enzyme inhibition
also increased until maximum inhibition was reached at 2 to 5 mM

MgCl This result is consistent with a magnesium-phosphonoacetate

2.
complex as the inhibiting species. It was unnecessary to add sup-

plementary MgCl to reaction mixtures containing phosphonoacetate,

2

73

Figure 11. Effect of MgC12 concentration on inhibition of
HVT-induced DNA.polymerase by phosphonoacetate. Reaction mix-
tures contained in 200 pl: 50 mM Tris-HCl (pH 8), 200 mM KCl,
1 mM dithiothreitol, 500 pg per ml bovine serum albumin, 200
ug per ml activated calf thymus DNA, 20 uM [3H]dCTP (400 cpm
per pmol), 100 uM each of_dATP, dGTP, dTTP, HVT-induced DNA
polymerase, 0 or 1 uM phosphonoacetate and MgClz concentrations
as indicated. For each MgC12 concentration the ratio of
activity in the presence of 1 uM phosphonoacetate to activity
in the absence of phosphonoacetate x 100 was calculated. This
value was subtracted from 100 to give percent inhibition.

74

 

 

 

 

_ A _ b
O 0 O O 0
m 8 6 4 2

8:52... 283..

 

Figure 11

75
since phosphonoacetate was present in*uM concentrations and MgCl2

was present in mM concentrations.
Inhibition by Structural Analogues
of Phosphonoacetate

When tested at a concentration of 200 pM, the following analogues
of phosphonoacetate produced no significant inhibition of either the
polymerization reaction or of the dNTP-pyrophosphate exchange reac-
tion catalyzed by HVT-induced DNA polymerase: methylene diphosphonate,
malonate, phosphoglycolate, sulfoacetate, carbamyl phosphate, phos-
phonoacetaldehyde, phosphonopropionate, amino methyl phosphonate,
a-amino ethyl phosphonoate, trimethyl ester of phosphonoacetate,
c-phenyl phosphonoacetate, 2-amino phosphonoacetate, acetonyl
phosphonate, monomethyl ester of acetonyl phosphonoacetate, mono-
methyl ester of acetyl aminophosphate, thethyl phosphonoacetamide,
and 2-methyl-2-phosphonopropionate. 2-Phosphon0propionate was an
inhibitor of the polymerization reaction and of the pyrophosphate
exchange reaction. As determined in the dNTP-pyrophosphate exchange
reaction, the apparent K13 for 2-phosphonopr0pionate was about 50 pM.
Therefore, 2 phosphonopropionate was about SO-fold less effective as
an inhibitor than phosphonoacetate.

Two other analogues of phosphonoacetate also inhibited the HVT-
induced polymerization reaction. When 30 uM N-(phosphonoacetyl)-L-
aspartate was added to standard reaction mixtures, the DNA polymeri-
zation reaction was inhibited by 50%. When 60 uM phosphonoacetamide,
monocyclohexylamine salt, was added to standard reaction mixtures,
the DNA polymerization reaction was inhibited by 50%. However, thin
layer chromatography of these compounds on cellulose plates indicated

that the Nb(phosphonoacetyl)-L~aspartate preparation was contaminated

76

with phosphonoacetate and that the phosphonoacetamide preparation
contained two unidentifiable components (data not shown). Therefore,
the identity of these inhibiting compounds is unresolved.
The Effect of Phosphonoacetate on the
DNA Polymerization Reaction Catalyzed
byyOther DNA Polymerases

DNA polymerase a, but not B, from uninfected duck embryo fibro-
blasts, was inhibited by phosphonoacetate. The inhibition patterns
with the»a polymerase for the DNA polymerization reaction were similar
to those produced with the HVT-induced polymerase, but the apparent
Ki values were 10 to 20 times greater. DNA polymerase B, when tested
at a phosphonoacetate concentration of 200 uM, was not significantly
inhibited. Likewise, neither E. coli DNA polymerase I nor AMV reverse
transcriptase was significantly inhibited.

Investigation of Phosphonoacetate as an Alternate Product
Inhibitor of the HVT-Induced DNA Polymerization Reaction

Phosphonoacetate was a competitive inhibitor of pyrophosphate
in the dNTP-pyrophosphate exchange reaction. In the polymerization
reaction multiple inhibition studies with phosphonoacetate and pyro-
phosphate indicated that the two were mutually exclusive inhibitors.
Inhibition patterns for phosphonoacetate and pyrophosphate were
similar. Therefore, phosphonoacetate inhibits the HVT-induced DNA
polymerase by binding to the pyrophosphate binding site of the
enzyme. By binding at this site phosphonoacetate could act as a
dead end inhibitor or as an alternate product inhibitor. As a dead
end inhibitor phosphonoacetate would simply bind reversibly to the
rpyrophosphate binding site. As an alternate product inhibitor,

phosphonoacetate would reverse the DNA polymerization reaction via

77
an alternate reaction pathway. In so doing it would generate a
nucleotide analogue of itself which would be an alternate substrate
in the polymerization reaction. In an attempt to provide direct
evidence for phosphonoacetate as an alternate product inhibitor, a
nucleotide analogue of phosphonoacetate, deoxythymidine S'phospho-
phosphonoacetate, was synthesized. If phosphonoacetate were an
alternate product inhibitor, then as an alternate substrate this
compound should: (1) serve as a substrate in the polymerization
reaction, (2) act as a competitive inhibitor of dTTP, and (3) be
generated during phosphonoacetate inhibition. These predictions

were tested.

Synthesis of Deoxythymidine 5'Phosphophosphonoacetate

Deoxythymidine 5'phosphophosphonoacetate was synthesized by
reaction of deoxythymidine S'phosphomorpholidate with phosphonoace-
tate in a modification of the procedure for synthesis of aIBZPladenosine
5'triphosphate (109). Phosphonoacetic acid (1.75 mmnd) was dissolved
in water (1.5 m1): anhydrous pyridine (9 ml) was added followed by
tri-n-octylamine (5.5 mmol). This solution containing the tri-n-
octylanlnonium salt of phosphonoacetate was evaporated to dryness.
The phosphonoacetate was dried by three evaporations with anhydrous
pyridine (5 ml each) and two with anhydrous benzene. Deoxythymidine
S'phosphomorpholidate (.5 mmol) was also dried by evaporations with
anhydrous pyridine and anhydrous benzene. The deoxythymidine
S'phosphomorpholidate was dissolved in anhydrous dimethyl sulfoxide
(7 m1) and was added to the phosphonoacetate. The reaction mixture
was stirred for 1 hr at room temperature under nitrogen, was then

diluted with water (70 ml) and was applied to a DEAE-cellulose

78

(HCO3-) column (2.5 x 60 cm) at 4°. The column was washed with 5 mM
triethylammonium bicarbonate (pH 7.5) until all unadsorbed ultraviolet-
adsorbing material had washed through. The column was eluted with
a 3.5 1 linear gradient of 5 to 350 mM triethylammonium bicarbonate
(pH 7.5). Three peaks of ultraviolet-adsorbing material were found
and were identified as unreacted deoxythymidine 5'phosphomorpholidate
(39%), dTMP (1%) and dTMP-PA (60%), in order of elution. Unreacted
phosphonoacetate eluted in a position between dTMP and dTMP-PA.

Fractions containing dTMP-PA were pooled and lyophilized.
Residual triethylammonium bicarbonate was removed by five evaporations
with methanol (20 ml each). The final residue was dissolved in
methanol (5 ml) and was precipitated as the sodium salt by addition
of sodium iodide (1.2 m1 of a l M solution in acetone) and cold
acetone (30 ml). The precipitate was washed three times with cold
acetone (15 m1) and was dried in vacuo. The resulting powder was
stored at -20°.

If the reaction time was increased, the 60% yield of dTMP—PA
decreased with a proportional increase in the yield dTMP.

This synthesis was performed in collaboration with Barbara
Duhl-Emswiler of the Chemistry Department.
Structural Properties of Deoxythymidine
5'Phosphophosphonoacetate

The deoxythymidine 5'phosphophosphonoacetate preparation was
determined to be homogeneous by paper chromatography in two solvent
systems and by electrophoresis (Table 4). The stability of dTMP-PA
was monitored under several different conditions: (1) incubation in
10 mm Tris-HCl (pH 8) at 37° for six days, (2) incubation in 0.01 N

HCl at room temperature for 24 hr, and (3) incubation in a standard

79
DNA polymerization reaction mixture with an excess of HVT-induced DNA
polymerase at 37° for 4 hr. In each case analysis by paper chroma-
tography in solvent system I indicated that dTMP-PA had not been

degraded.

Table 4. Characterization of deoxythymidine 5'phosphoPhosphonoacetate
by paper chromatography and electrophoresis

 

 

RF Relative Electrophoretic

Compound I II Mdbility
dTMP-PA 0.23 0.28 0.86

dTTP 0.11 0.16 1.0

dTDP 0.19 0.18 0.86

dTMP 0.34 0.23 0.64
dTMP—morpholidate 0.73 0.56 0.36

PA 0.19 - l.ll

 

Descending chromatography with Whatman No. 40 acid washed paper
was used with solvent systems (I) ethyl alcohol-l M ammonium acetate
(pH 7.5) (5:2) and (II) isopropyl alcohol-conc. ammonium hydroxide-
water (7:1:2) (110). Whatman No. 40 acid washed paper was used for
electrophoresis on a water—cooled flat-plate apparatus with 0.05 M
triethylanmonium bicarbonate (pH 7.5), 35 V per cm for 45 min. Com-
pounds were detected by ultraviolet quenching and/or a phosphate
spray reagent (97).

Deoxthymidine 5'phosphophosphonoacetate was determined to have
the structure shown in Figure 12 based on the following: It exhibited
an adsorption maximum for thymine at 267 nm. It had a molecular
weight of 550 :_10 based on the molar extinction coefficient for
thymine (E = 9.6 x 103 at 267 nm). The theoretical molecular weight
for Na3 dTMP-PA‘ZHZO would be 546. It contained 2.0 :_.l phosphorus

atoms per thymine. It was degraded by snake venom phosphodiesterase

80

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81
I into dTMP and PA. The dTMP and PA components of dTMP-PA were also
identified by comparison of proton decoupled 13C nuclear magnetic
resonance spectra of dTMP-PA, dTMP and PA (Figure 13). Alkaline
phosphatase did not degrade dTMP-PA indicating that no terminal
phosphate ester existed in this compound. Evidence for the existence
of a free carboxyl group in dTMP-PA was obtained from the presence
of a carboxyl doublet at 176.4 and 176.9 ppm in the 13C NMR spectrum
and from the presence of a carboxylate anion stretch at 1580 cm-1
in the infrared spectrum.
Deoxythymidine 5 ' Phosphophosphonoacetate

as a Substrate of the HVT-Induced DNA
Polymerization Reaction

 

In order to determine if dTMP-PA could serve as a substrate in
place of dTTP in the activated DNA polymerization reaction, HVT-
induced DNA polymerase was assayed in standard reaction mixtures in
the absence of dTTP, in the presence of increasing concentrations of
dTMP-PA, and in the presence of a saturating amount of dTTP (Table 5).
In the absence of dTTP 12.1 pmol of [3H]dCMP was incorporated. Addi-
tion of increasing concentrations of dTMP-PA to reaction mixtures
did not enhance this incorporation, even when 640 uM dTMP-PA had
been added. In fact, inhibition was observed. Addition of 100 uM
dTTP increased [3H]dCMP incorporation by 3-fold. These results sug-
gest that dTMP-PA cannot replace dTTP as a substrate in the DNA
polymerization reaction. The inhibition that was observed when
increasing concentrations of dTMP-PA were added to reaction mixtures
was probably a result of contaminating phosphonoacetate as explained

below.

82

Figure 13. Proton decoupled 13C nuclear magnetic resonance
spectra of phosphonoacetate (top panel), dTMP (middle panel),
and dTMP-PA (lower panel). Solutions of 0.71 M phosphonoacetate
(pH 8.2), 0.83 M dTMP (pH 8.3), and 0.14 M dTMP-PA (pH 8.6) were
prepared in D20 and were analyzed at 12° using a Brucker WP60
60 MHZ 13C nuclear magnetic resonance spectrometer.

83

 

 

 

 

 

 

 

 

 

 

Figure 13

84
Similar results were also obtained in an activated poly d(A-T)
polymerization reaction. Incorporation of [3H]dAMP in the absence
of dTTP was not enhanced by addition of increasing concentrations of

dTMP-PA to reaction mixtures.

Table 5. Ability of the HVT-induced DNA polymerase to utilize dTMP-PA
in an activated DNA polymerization reaction

 

 

dTMP-PA, uM dTTP, uM pmol [3H]dCMP Incorporated
0 0 12.1
40 0 12.5
80 0 12.0
160 0 11.4
320 0 11.0
640 0 8.7
0 100 37.0

 

All reaction mixtures contained in 200 pl: 20 uM [3H]dCTP
(350 cpm per pmol), 100 uM each dATP, dCTP, 2 mM MgC12, 200 mM KCl,
50 mM Tris-HCl (pH 8), 1 mM dithiothreitol, 500 pg per ml bovine
serum albumin, 200 pg per ml activated calf thymus DNA, HVT-induced
DNA polymerase and the indicated concentrations of dTMP-PA or dTTP.

Deoxythymidine 5'Phosphophosphonoacetate
as an Inhibitor of the HVT-Induced DNA
Polymerization Reaction

 

With dNTP as variable substrate and activated DNA at a concen-
tration of 200 ug per m1, dTMP-PA gave linear noncompetitive inhibi-
tion (Figure 14). Apparent inhibition constants, xii and Kis' were
calculated to be 52 uM and 120 uM, respectively, from intercept and
lepe replots. If dTMP-PA could bind at the dNTP binding site of the
DNA polymerase, it would be a competitive inhibitor of dTTP. This

result was not observed.

85

Figure 14. Double reciprocal plots with the four dNTPs
as variable substrate and dTMP-PA as inhibitor. Activated DNA
was at 200 119 per ml. Deoxythymidine 5'phosphophosphonoacetate
concentrations were: 0 (0 ) , 20 (III (0) and 60 (M (A). Initial
velocities were measured as pmol [3H]d'1‘MP incorporated into DNA
per 30 min. Specific radioactivity of [3H]dTTP was 1000 cpm

per pmol.

86

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87

The noncompetitive inhibition observed for dTMP-PA was probably
due to a small amount of contaminating PA in the dTMP-PA preparation.
Phosphonoacetate gave linear noncompetitive inhibition with dNTP as
the variable substrate and activated DNA at a concentration of 200
ug per ml. Apparent inhibition constants, xii and Kis’ were 1.5 uM
and 1.0 uM, respectively. Therefore, a 1 to 3% molar contamination
of the dTMP-PA preparation by phosphonoacetate would have been suf-
ficient to produce the observed inhibition pattern. When dTMP-PA.was
rechromatographed on a DEAR—cellulose (HC03-) column and the peak
fraction was used in the same kind of inhibition experiment, linear
noncompetitive inhibition was again observed. However, xii and Kis
values had increased from 52 uM and 120 uM to 525 uM and 540 uM,
respectively.

When dTTP was the variable substrate and dATP, dCTP, and dCTP
were each 100 uM and activated DNA was 200 ug per ml, dTMP-PA was
also a noncompetitive inhibitor.

Generation of Deoxythymidine S'Phosphono-
acetate in HVT-Induced DNA Polymerization
Reactions Inhibited by_Phosphonoacetate

The existence of dTMP-PA in polymerization reactions inhibited
by phosphonoacetate was investigated using [Bhlphosphonoacetate of
high specific activity. Reaction mixtures containing 2.7 uM [3H]-
phosphonoacetate (6000 cpm per pmol), 100 uM each of the four dNTPs
and 200 ug per ml activated DNA were incubated in the presence and
absence of HVT-induced DNA polymerase. After incubation the reaction
mixtures were analyzed by Dowex-l chromatography (Figure 15). For
the reaction mixture which contained enzyme, a single peak of radio-

activity corresponding to phosphonoacetate eluted from the column.

88

Figure 15. Analysis of the effect of [BHlphosphonoacetate
on HVT-induced DNA polymerization reactions by Dowex-l (Cl')
chromatography. Reaction mixtures (0.2 m1) contained 2.7 uM
[3H1phosphonoacetate (6000 cpm per pmol), 100 uM each dATP,
dCTP, dGTP, dTTP, 200 pg per m1 activated calf thymus DNA,

50 mM Tris-HCl (pH 8) , 2 mM Mgc12, 200 mM KCl, 1 mM dithio-
threitol, 500 pg per ml bovine serum.albumin and'were incubated
at 37° for 30 min in the presence or absence of HVT-induced
DNA polymerase. Incubations were terminated by addition of
EDTA to a final concentration of 20 mM. Reaction mixtures

were diluted with water (2 ml) and applied to a Dowex-1 (Cl-)
column (0.6 x 10 cm) at 4°. The column was washed extensively
with water and eluted with a 200 m1 linear gradient of 0 to

0.2 M LiCl in 0.01 N HCl. Fractions of 2 ml were collected.
One milliliter of alternate column fractions was added to 8

m1 of a triton—toluene based scintillation fluid (1 1 triton-
X100, 2 l toluene, 16.5 g 2,5diphenyloxazole and 0.3 g
l,4bis[2—(4-methyl-S-phenyloxazolyl)lbenzene) and was monitored
for radioactivity using liquid scintillation spectrometry.
Concentrations of LiCl were determined by conductivity
measurements. The elution position of dTMP-PA.was determined
by Dowex-1 (Cl') chromatography of a reaction mixture con-
taining 4 mM dTMP-PA, 2.7 uM [3H]phosphonoacetate, 2 mM

MgC12, 200 mM KCl and 50 mM Tris-HCl (pH 8). Recoveries from
the Dowex-l (Cl’) column were 80% or better.

89

 

30° + DNA Polymerase ..
"I 0.4
C
"'23 225 r- n _
'2 m 0.3
.5 dTMP-PA ;
E $ '-
5 I50 '- - F:
8 . 0.2 g
g ...-L:
5 7.

 

 

 

 

 

"no 20 4o 60 80 I00

 

 

 

Fraction Number
300 - - DNA Polymerase _
I
'9' -
2 225 " dTMP-PA _ 0 3 e
2 ‘ ' 5
E i ‘ '5"
I50 - "-
g or”; 0 2d“
or" "‘
75
0.0

 

 

    

"I0 20 4o 60 80 I00
Fraction Number

Figure 15

90
No radioactive material eluted from the column in the region where
dTMP-PA eluted. A similar result was obtained for the control
reaction mixture which contained no enzyme. The phosphonoacetate
concentration used in this experiment was one which was used in the
kinetic studies on phosphonoacetate inhibition. The DNA polymeriza-
tion reaction was inhibited by 67%. This analysis could have detected
an exchange reaction whose rate was 3% of the rate of the polymeriza-
tion reaction. Thus, no evidence was obtained which demonstrated
that deoxythymidine 5'phosphoPhosphonoacetate was generated during
inhibition of the HVT-induced DNA polymerase by phosphonoacetate.
Characterization of a Phosphonoacetate-Resistant
HVT-Induced DNA Polymerase

A phosphonoacetate resistant mutant of HVT (HVTfiA) was isolated
by Dr. Lucy Lee (manuscript submitted for publication). The mutant
virus replicates in the presence of 1.6 mM of phosphonoacetate whereas
the replication of the wild type virus was completely inhibited by
0.3 mM. The mutant virus replicates as well as the wild type virus
at 37°, but much less well at 41°. Since phosphonoacetate specifi-
cally inhibits the wild type HVT-induced DNA polymerase, it was of
interest to examine the properties of the PA-resistant HVT-induced
DNA polymerase.

Nuclear extracts from PA-resistant HVT-infected duck embryo
fibroblasts were prepared and fractionated by phosphocellulose chroma-
tography. The viral-induced DNA polymerase, HVTPA-induced DNA
polymerase, eluted from the phosphocellulose column as a single peak
at about 0.35 M KCl. The peak DNA polymerase fractions were pooled

and were used to define the properties of the enzyme.

91
The sensitivity of the HVTPA-induced DNA polymerase to PA was
determined (Figure 16). When added to the standard reaction mixture,
17 uM PA inhibited the HVTPA-induced DNA polymerase by 50%; 1.2 uM
PA inhibited the HVT -induced DNA polymerase by 50%. The apparent

WT
inhibition constant (Ki) for PA of the HVTPA-induced DNA polymerase
was determined and compared with that for the HVTWT-induced DNA
polymerase (Table 6). The apparent inhibition constant for PA for
the HVTPA-induced DNA polymerase was about lO-fold greater than that

for the HVTWT-induced DNA polymerase.

Table 6. Kinetic constants of PA-resistant HVT-induced DNA polymerase
and of wild-type HVT-induced DNA polymerase

 

 

Kinetic constant HVTPA DNA polymerase HVTWT DNA polymerase
Ki PA 18 :_2 uM 1.4 i 0.6 uM
K. 2.5 + 0.5 mM 1.3 + 0.2 mM
1 PPi - -
KDNA 7.2 i 0.2 ug/ml 7.3 :_0.2 ug/ml
' . + . ‘ . .
KdNTP 5 5 __0 6 uM 2 1 1L0 5 uM

 

Ki values for phosphonoacetate (PA) and for pyrophosphate
(PPi) were determined as Kii with dNTP as the variable substrate and
with activated DNA at a concentration of 200 ug per ml. KDNA and
KdNTP values were determined in initial velocity studies.

Evidence has been presented that PA inhibits the herpesvirus-
induced DNA polymerase by binding at the pyrophosphate binding site
of the enzyme. Therefore, an apparent inhibition constant for pyro-

phosphate for the HVT -induced DNA polymerase was determined and

PA
compared with that of the HVTWT-induced DNA polymerase (Table 6).

K1 PPi f°r HUTPA

-induced DNA polymerase was about 2-fold greater

92

Figure 16. Inhibition of the HVTPA—induced DNA polymerase

(O) and the HVTwr—induced DNA polymerase (0) by phosphono-

V represents pmol [ 3H]dTMP incorporated into DNA per
Vi represents

acetate.
The

30 min at 37° in the absence of phosphonoacetate.
dTMP incorporation in the presence of phosphonoacetate.
phosphonoacetate concentration which inhibits the enzyme
activity by 50% is that concentration for which V/Vi is 2.

93

 

 

 

 

 

I 1

IO 20 30

 

PI-DSPHONOACETATE ()1 M)

Figure 16

94
than Ki Ppi for the wild type enzyme, indicating that the mutant
enzyme has a decreased affinity for pyrophosphate.

Apparent Michaelis constants for activated DNA and dCTP were
determined (Table 6). No difference in KDNA for the two enzymes was
seen. The apparent Michaelis constant for dNTP (KdNTP) was about
2.5-fold greater for the HVTPA-induced DNA polymerase than for the

HVTwT-induced DNA polymerase.

The decreased affinity of the HVTPA-induced DNA polymerase for
PA suggested that the structure of the enzyme had changed. A change
in structure might result in a change in the temperature sensitivity
of the enzyme. Therefore, the thermal inactivation of the HVTPA-
induced DNA polymerase and the HVTWT-induced DNA polymerase was.
examined. As shown in Figure 17, the thermal inactivation of both
enzymes followed first order reaction kinetics. The activity of the
HVTPA-induced DNA polymerase decayed with a half life of about 10 min.

The activity of the HVT

WT-induced DNA polymerase decayed with a half

life of about 17 min. The greater temperature sensitivity of the
mutant enzyme as compared with the wild type enzyme was also seen in
crude nuclear extracts.

Although HVTPA-induced DNA polymerase and HVTwT-induced DNA
polymerase showed different sensitivities to PA and heat, other
properties of the two enzymes were similar. Both enzymes eluted
from phosphocellulose at about the same KCl concentration. Both
exhibited similar Optima for MgCl2 and KCl. The sedimentation coef-
ficient for both enzymes was 7.0 :_0.2S as determined by glycerol

gradient sedimentation velocity centrifugation in the presence of

200 mM KCl, 2 mM MgCl2 and 50 mM Tris-HCl (pH 8) (data not shown).

95

Figure 17. Themmal inactivation of the HVTPA-induced DNA
polymerase (0) and the HVT“ DNA polymerase ( 0) at 52°. Aliquots
of pooled phosphocellulose fractions of HVT-induced DNA polymerase
were incubated at 52°. Incubation buffer contained 2 mg per ml bovine
serum albumin, 0.5 mM dithiothreitol, 15 mM Tris-HCl (pH 8),

0.2 M KCl, and 50% glycerol (v/v). At the indicated time intervals,
10 ul aliquots of enzyme were removed to test tubes on ice. After
2 min on ice, DNA polymerase was assayed by addition of warmed

reaction components to the test tubes. Incubation was at 37° for
30 min.

96

 

 

09

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06

im

  

5 . )3? 2
a m o 0.
022329.. >.A..>Fo< 29.55.“.

   

 

0.)

TIME AT 52°(min)

Figure 17

97

Presence of MDHV-Induced DNA Polymerase in
the MSB-l Lymphgblastoid Cell Line

The productive infection of DEF with MDHV results in the induc-
tion of a novel DNA polymerase. Tumors from chickens with Marek's
disease carry the MDHV genome (8; Leinbach, unpublished results).
Therefore, it is of interest to determine if the MDHViinduced DNA
polymerase is present in these transformed lymphoid cells to replicate
the MDHV genome. As a first approach to this problem, the MSB-l
lymphoblastoid cell line was examined for the presence of the MDHV-
induced DNA polymerase.

The MSB-l lymphoblastoid cell line is a continuous transformed
cell line derived from a spleen tumor of a chicken infected with MDHV,
strain BC-l (23). It carries MDHV genomes (26; Leinbach, unpublished
results). In 2-day-old cultures approximately 1 to 2% of the cell
population produces viral antigens and virus particles. In cultures
kept for seven days, 15 to 20% of the cells contain viral antigens
and virus particles. Those cells spontaneously producing viral
antigens and virus particles eventually degenerate and die, whereas
other nonproducer cells actively grow and divide (25).

Nuclear extracts of MSBPl 6-day-old and 2-day-old cultures were
examined by DEAE-cellulose chromatography for DNA polymerase activi-
ties. Because only small quantities of MSB-l cells were available
for this analysis, the MDHV-induced DNA polymerase was determined to
be present in these cells if the following criteria were met: an
activity identified by the MDHVbinduced DNA polymerase assay eluted
from DEAE—cellulose at about 0.1 M potassium phosphate and was inhi-
bited by 50% when l to 3 uM phosphonoacetate was added to the MDHV-

induced DNA polymerase assay.

98

The DNA polymerase activities from a nuclear extract of uninfected
chicken embryo fibroblasts were fractionated by DEAE-cellulose chroma—
tography to demonstrate that the MDHV-induced DNA polymerase activity
was not present in chicken cells (Figure 18). The MDHV-induced DNA
polymerase assay revealed no activity which eluted from the DEAE—
cellulose column at about 0.1 M potassium phosphate.

When DNA polymerase activities from nuclear extracts of 6—day-old
MSB-l cells were examined by DEAE-cellulose chromatography, a major
DNA polymerase activity was found by the MDHV-induced DNA polymerase
assay which eluted from the column at about 0.1 M potassium phosphate
(Figure 19, left panel). This activity (fractions 16-17) was inhibited
by 50% when 3 uM phosphonoacetate was added to the MDHV-induced DNA
polymerase reaction mixture. These results suggest that the MDHVB
induced DNA polymerase is present in these 6—day-old MSB-l cells.

The level of activity of the MDHVhinduced DNA polymerase in the nuclear
extracts of these 6-day-old MSB-l cells is only 2% of the level of
activity of the MDHVbinduced DNA polymerase in the nuclear extracts

of MDHV-infected DEF, however (compare Figure 19 with Figure 2).

When DNA polymerase activities from nuclear extracts of 2-day-old
MSB-l cells were examined by DEAF-cellulose chromatography, only a
manor DNA polymerase activity was found by the MDHV-induced DNA
polymerase assay which eluted from the column at about 0.1 potassium
phosphate (Figure 19, right panel). The peak fraction of this activity,
fraction 16, was tested for sensitivity to phosphonoacetate. When
8 uM phosphonoacetate was added to MDHV-induced DNA polymerase reac-
tion mixtures, 50% inhibition of the DNA polymerase activity was
obtained. This sensitivity to phosphonoacetate was less than expected

for the MDHV-induced DNA polymerase. This could be explained, however,

99

Figure 18. DEAR—cellulose chromatography of DNA polymerase
activities from nuclear extracts of uninfected CEF. Nuclear
extracts from 0.6 g wet weight of uninfected CEF were prepared
and subjected to DEAE-cellulose chromatography as described in
Materials and Methods. Samples (25 ul) of the fractions were
assayed for DNA polymerase activities by the MDHV-induced DNA
polymerase assay (0) and the chicken DNA polymerase assay (0).
Potassium phosphate concentrations (——) were determined.from
conductivity measurements of alternate fractions. DNA polymerase
activities were expressed as pmol [3H]dTMP incorporated into
DNA in 30 min per g wet weight of cells.

100

w [SIDlIdSOIId >4]

‘7. '°. c‘! "t
o o o o

 

 

l I l l

 

 

 

 

 

I l I

f

IO
Fraction Number

 

 

IO N — -
(mud) DSIDJOdJOOU| dW 1p

30

20

Figure 18

101

Figure 19. DEAF—cellulose chromatography of DNA polymerase
activities from nuclear extracts of MSB-l cells. Nuclear extracts
from 0.8 g of 6-day-old MSB-l cells, passages 38 and 39, were pre—
pared and subjected to DEAE-cellulose chromatography as described
in Materials and Methods (left panel). Nuclear extracts from
1.3 g of 2-day-old MSB-l cells, passage 49, were prepared and
subjected to DEAF-cellulose chromatography (right panel). Samples
(25 ul) of the fractions were assayed for DNA polymerase activities
by the MDHV-induced DNA polymerase assay (0) and the chicken DNA
polymerase assay (C)). Potassium phosphate concentrations (-——fl
were determined from conductivity measurements of alternate frac-
tions. DNA polymerase activities were expressed as pmol [3H]dNMP
incorporated into DNA in 30 min per g wet weight of cells.

102

w [anquoqd )4]

0'

8!
0

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0

V.
0

on

mA unseen

.3832 e288“.
ON 0.

.3532 e288...

 

 

 

 

 

 

 

(Iowd) paImodJooul dINNP

103
if fraction 16 contained, in addition, the MDHV-induced DNA polymerase,
a DNA polymerase with a substantially lower sensitivity to phosphono-
acetate than that of the MDHV-induced DNA polymerase. In fact, when
fraction 16 was assayed by the chicken DNA polymerase assay, 50%
inhibition of this DNA polymerase activity was obtained when 40 uM
phosphonoacetate was added to the reaction mixtures. This DNA
polymerase activity probably existed in nuclear extracts from 2—
day-old cultures and not in those from 6-day-old cultures because
the physiology of the two cultures differed greatly. The 2-day-old
cultures contained a greater number of viable cells than did the
6-day-old cultures. Therefore, the MDHVeinduced DNA polymerase is
probably also present in 2-day-old MSB-l cells, although more con-

clusive evidence is needed to firmly establish this point.

DISCUSSION

Characterization of DNA Polymerases of Marek's Disease
Herpesvirus and Herpesvirus of Turkeys

 

 

Infection of duck embryo fibroblasts by MDHV, strain BC-l, led
to the induction of a novel DNA polymerase. Infection of duck embryo
fibroblasts by HVT also led to the induction of a novel DNA polymerase.
The properties of these two enzymes were very similar as might be
expected since the two viruses are closely related. Both exhibited
maximum activity in the presence of 1-2 mM MgCl2 and 200-250 mM
KCl. Both were inhibited by 50% in the presence of 1-3 M phosphono—
acetate. Both were DNA-dependent DNA polymerases and had sedimenta-
tion coefficients in the presence of 0.25 KCl of about 75.

The single major difference between these two enzymes was in
their chromatographic behavior on DEAF-cellulose columns. The MDHV-
induced DNA polymerase eluted as a single peak of activity at about
0.1 M potassium phosphate. The HVT-induced DNA polymerase eluted as
two peaks of activity at about 0.08 M and 0.13 M potassium phosphate.
These two HVT-induced DNA polymerase activities were structurally
and catalytically identical so that their significance remains
unexplained. If the HVT-induced DNA polymerase was chromatographed
first on a phosphocellulose column and then on a DEAF-cellulose
column, only a single DNA polymerase activity was found. This enzyme
fraction was purified approximately 60—fold over the whole cell

homogenate.
104

105

The MDHV- and HVT-induced DNA polymerases could be distinguished
from the DNA polymerases of duck embryo fibroblasts by DEAE-cellulose
chromatography, by'Mg2+ optima, by KCl sensitivity and by phosphono-
acetate sensitivity. DNA polymerase a of DEF eluted from DEAE-
cellulose columns at abouto.14 M potassium phosphate, required 10 mM
MgCl2 for maximum activity, was inhibited by 80% in the presence of
200 mM KCl and was inhibited by 50% in the presence of 16 pM phosphono-
acetate. DNA polymerase B of DEF did not adsorb to DEAF-cellulose,
required 10 mM MgCl2 for maximum activity, was inhibited by 10% in
the presence of 200 mM KCl and was not inhibited by 200 uM phosphono-
acetate (data not shown). All these properties are different from
those of the MDHV— and HVT-induced DNA polymerases as described
above. Moreover, the induced DNA polymerase activities in MDHV-
and HVT-infected DEF were 10-40 times higher than the DNA polymerase
activities in uninfected DEF.

The properties of the DNA polymerases induced by HVT and MDHV,
strain BC-l, are very different from the properties of the DNA
polymerase induced by MDHV, strain GA (66). This latter polymerase
exhibited maximum activity in the presence of 10 mM MgC12, was
totally inhibited by 100 mM (NH4)ZSO4, was insensitive to phosphono-
acetate and had a sedimentation coefficient of 5.28 in the presence
of 0.25 MKCl. It is now apparent, however, that this DNA polymerase
was not a herpesvirus—induced DNA polymerase but a DNA polymerase of
a contaminant of the MDHV-infected cells used to inoculate the DEF.
When aureomycin was routinely added to the tissue culture medium of
cultures of DEF infected with MDHV, strain GA, this DNA polymerase
disappeared. When DNA polymerase activities from nuclear extracts of

these cells were fractionated by phosphocellulose chromatography, and

106

were assayed with DNA polymerase reaction mixtures containing 2 mM
MgCl2 and 200 mM KCl, a DNA polymerase with properties similar to those
of the HVT— and MDHV, strain BC-l-, induced DNA polymerases was faund.

When HVT was grown in DEF in the presence of increasing concen-
trationsof phosphonoacetate, a phosphonoacetate-resistant mutant of
HVT resulted. This mutant replicated as well as the wild type virus
at 37° but not as well as the virus type virus at 41°. The DNA
polymerase induced by the PA-resistant mutant had an apparent inhibi-
tion constant for phosphonoacetate, an apparent inhibition constant
for pyrophosphate and an apparent Michaelis constant for dNTP of
about 10-, 2-, and 2.5-fold, respectively, greater than the wild-
type enzyme. In vitro thermal inactivation studies with the HVT -

PA
and HVTWT-induced DNA polymerases demonstrated that the HVT -

PA
induced DNA polymerase was inactivated 1.7 times faster than the
HVTWT-induced DNA polymerase at 52°. Thus, a mutation in HVT resulted
in an alteration in the structure of the viral-induced DNA polymerase.
These studies suggest that the HVT-induced DNA polymerase is viral
coded and that inhibition by phosphonoacetate occurs by direct inter-
action with the herpesvirus-induced DNA polymerase. Hay and Subak-
Sharpe and Klein and Friedman-Kien have also reported PA-resistant
mutants of HSV (79,111).

The properties of the MDHV; and HVT-induced DNA polymerase were
similar to those of the HSVBinduced DNA polymerase (64,75). All
exhibited maximum activity in the presence of 1-2 mM MgC12. All
were stimulated to similar extents in the presence of salt, although
rmaximum stimulation was observed with different salts. The HSV-

induced DNA polymerase was stimulated 25- to 50-fold in the presence

of 150 mM K2804 and only 0.8 as well in the presence of 250 mM KCl.

107

The MDHV-induced DNA polymerase was stimulated 10- to 60-fold in
the presence of 200 mM KCl and only 0.6 as well in the presence of
110 mM K2S04. All were inhibited about 50% in the presence of 1-3
(M phosphonoacetate. All were high molecular weight DNA-dependent
DNA polymerases. The molecular weight of the HSV-induced DNA
polymerase was about 180,000-200,000 daltons. That of the MDHV-
and HVT-induced DNA polymerases was about 130,000 daltons. Both the
HSV-induced DNA polymerase and the HVT-induced DNA polymerase eluted
from DEAE-cellulose columns in the same region of the gradient as
two peaks of activity. There is evidence that both enzymes are
viral coded (65,111). Therefore, it appears that general structural
and catalytic properties exist for all herpesvirus-induced DNA
polymerases. Nevertheless, the HVT-induced DNA polymerase was not
inhibited by antiserum prepared against HSV-infected cells (data not
shown) indicating that these enzymes are antigenically distinct.

Infection of DEF with MDHV results in a productive infection.
Cellular necrosis occurs and viral antigens and virus particles are
produced, although no cell-free infectious virus is produced. Such
a condition also exists in a small percentage of the cell population
of the MSB-l lymphoblastoid cell line. In both cases MDHV DNA
polymerase is induced. In MSB-l cultures this induction is more
readily apparent in cells which have been cultured for six days than
in cells which had been cultured for two days. This is because the
levels of host DNA polymerase activities are lower in the 6-day-old
cells since fewer cells are viable. These results suggest that in
order to determine if the MDHV DNA polymerase is required to replicate
the resident MDHV genomes in tumor cells, a more specific, sensitive

technique for the detection of the MDHV DNA polymerase is necessary.

108
Immunofluorescence using antibody prepared against the MDHV DNA
polymerase would be such a technique.

The studies with Raji cells grown in the presence of phosphono-
acetate suggest that a herpesvirus-induced DNA polymerase does not
replicate the resident EBV genomes in these transformed cells (81,82).
However, the existence of an EBV-induced DNA polymerase has not yet
been demonstrated directly and direct evidence for or against the
existence of a herpesvirus-induced DNA polymerase in tumor cells is
most important.

Mechanism of Phosphonoacetate Inhibition of
Herpesvirus-Induced DNA Polymerase

The purpose of this study was to elucidate the mechanism by'whiCh_
phosphonoacetate inhibits the HVT-induced DNA polymerase. This prob-
1em was approached using analysis by steady state kinetics. Phosphono-
acetate was a competitive inhibitor of pyrophosphate in the dNTP-
pyrophosphate exchange reaction. Multiple inhibition studies with
phosphonoacetate and pyrophosphate showed that the two were mutually
exclusive inhibitors. Therefore, phosphonoacetate inhibits by binding
to the pyrophosphate binding site of the HVT-induced DNA polymerase.

At this site phosphonoacetate could inhibit as a dead end inhibitor or
as an alternate product inhibitor. As a dead end inhibitor phosphono-
acetate could simply bind to and dissociate from the pyrophosphate
binding site. As an alternate product inhibitor phosphonoacetate

could reverse the DNA polymerization reaction. An attempthas made

to determine which of these two mechanisms best described the mechanism
of inhibition by phosphonoacetate. Rate equations were derived for
each. The predictions which emerged were then tested with the data

obtained from the phosphonoacetate inhibition studies.

109
The alternate product inhibition model is presented in Figure
20. In the presence of phosphonoacetate an alternate reaction path-
way exists in addition to the basic polymerization pathway. Thus,

DNA(n+1) complex. It could

as shown, phosphonoacetate binds to the E
then simply dissociate from the complex or it could remain associated

with the complex and undergo reaction with the nucleotide at the

DNA

,_ .
3 end of the DNA primer chain to form the EdNMP-PA

complex. This
DNA .

complex would in turn yield E and dNMP-PA, a nucleotide analogue
of phosphonoacetate and an alternate substrate for the polymerization
reaction. Thus, phosphonoacetate inhibition occurs because the
EDNA(n+l) complex is diverted by phosphonoacetate from the main
polymerization pathway into an alternate pathway.

The steady state rate equation for this mechanism is given by
equation 1, where KDNA and KDNA' are Michaelis constants for DNA as
d

substrate and product, respectively: K are inhibition

mm 3“ KiDNA'
constants for DNA as substrate and product, respectively (see
Appendix B for method of derivation).

Under the experimental conditions that were used for the phos-
phonoacetate inhibition experiments, the numerator of equation 1 is

approximated by the V (DNA)(dNTP) term. The reciprocal

1(DNA,DNTP)
of equation 1 arranged with DNA as the variable substrate takes the
form.of equation 2. At low dNTP concentrations both the slope and
intercept terms are a function of phosphonoacetate concentration and
the inhibition pattern is noncompetitive. At high dNTP concentra-
tions, however, the slope term is independent of phosphonoacetate
concentration and the inhibition pattern is uncompetitive.

The experimental results are consistent with the predictions of

equation 2. With activated DNA as the variable substrate and with

110

Figure 20. Alternate product inhibition model of phos-
phonoacetate inhibition of herpesvirus-induced DNA polymerase.
The basic reaction mechanism in the absence of phosphonoacetate
is a modified ordered bi bi mechanism (Appendices A and C).

111

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114
a dNTP concentration of 2.5 uM, noncompetitive inhibition was
observed. With a dNTP concentration of 100 uM, the inhibition
pattern was nearly uncompetitive.

If equation 2 is rearranged with dNTP as the variable sub-
strate, the prediction emerges that the phosphonoacetate inhibition
pattern will be noncompetitive regardless of the concentration of
DNA. With dNTP as the variable substrate and with the activated DNA
concentration at 200 ug per ml, the inhibition pattern was none
competitive. Again these results are consistent with equation 2.

The kinetic data obtained from the phosphonoacetate inhibition
studies of the HVT-induced DNA polymerization reaction are not con-
sistent with phosphonoacetate as a dead end inhibitor. If phosphono-.
acetate complexes with the EDNA(n+l) enzyme form as a dead end
inhibitor, the rate equation for this model predicts that with DNA
as variable substrate inhibition by phosphonoacetate would be uncomr
petitive regardless of the concentration of dNTP (see Appendix B
for method of derivation). This same prediction emerges from.the
rate equation which describes phosphonoacetate acting as a dead end
inhibitor with both the EDNA(n+1) and EDNA enzyme forms. ‘If phos-
phonoacetate complexes with both the EDNA(n+l) and E enzyme forms,
the rate equation predicts that with DNA as variable substrate non-
competitive inhibition would be observed regardless of dNTP concen-
tration. Experimentally, when DNA was variable substrate, inhibi-
tion by phosphonoacetate was noncompetitive at 2.5 uM dNTP and
nearly uncompetitive at 100 uM dNTP.

Thus, the phosphonoacetate inhibition studies of the HVT-
induced DNA polymerization reaction favor the alternate product

inhibition model. An attempt was made to provide direct evidence

115
for this model by demonstrating the existence of the alternate reac-
tion pathway predicted by the model. Deoxythymidine 5'phosphophos-
phonoacetate was synthesized and assayed as an alternate substrate
in the polymerization reaction.

As an alternate substrate, dTMP—PA should replace dTTP in HVT-
induced DNA polymerization reaction mixtures. In an activated DNA-
directed reaction incorporation of [3H]dCMP in the presence of dATP
and dGTP was not enhanced by dTMP-PA. As an alternate substrate,
dTMP-PA should be a competitive inhibitor of dTTP. When [3H]dTMP
incorporation was measured with dNTP as variable substrate and
activated DNA at a concentration of 200 pg per ml in.the presence
of changing fixed concentrations of dTMPbPA, a noncompetitive inhibi- .
tion pattern resulted. Thus, dTMP-PA was not demonstrated to be a
substrate for the HVT-induced DNA polymerase or to bind to the dNTP
binding site of the enzyme. I

As an alternate substrate, dTMP-PA should be generated in an
exchange reaction during phosphonoacetate inhibition. When 2.7 uM
[3H1phosphonoacetate was present in standard reaction mixtures, no
exchange of [3H]phosphonoacetate into dTMP-PA was demonstrated by
Dowex-l (Cl-) chromatography of reaction mixtures. Moreover, since
phosphonoacetate was identified as the only radioactive component
in the HVThinduced DNA polymerization reaction, it is unlikely that
the phosphonoacetate analogue with an acyl phosphate linkage was
generated in measurable quantity either. Such an acyl phosphate
linkage might be unstable. However, no [3H]dTMP was generated in
standard reaction mixtures containing 2.5 uM phosphonoacetate during

[3H]dTMP incorporation into activated DNA.

116

Therefore, no direct evidence has been obtained in support of
the alternate product inhibition model as the mechanism of phosphono-
acetate inhibition. Nevertheless, the conclusion from the kinetic.
inhibition studies remains that phosphonoacetate inhibits the
herpesvirus-induced DNA polymerase by binding to the pyrophosphate
binding site of the enzyme. This binding probably involves a
magnesium—phosphonoacetate species.

Phosphonoacetate may be considered as an analogue of pyroPhosphate.
The two exhibited similar inhibition patterns. However, quantitatively
they appear to interact differently with the HVT-induced DNA polymerase.
The apparent inhibition constants for phosphonoacetate were uMu The
apparent inhibition constants for pyrophosphate were mM. The apparent.
affinity of the HVT-induced DNA polymerase for phosphonoacetate is,
therefore, three orders of magnitude greater than the apparent affinity
of the enzyme for pyrophosphate. In addition, pyrophosphate reverses
the polymerization reaction in an exchange reaction whose rate is
about 25% of the rate of the polymerization reaction. Phosphono-
acetate may reverse the polymerization reaction in an exchange reac-
tion whose rate is less than 3% of the rate of the polymerization
reaction.

Therefore, while the kinetic studies on phosphonoacetate inhibi—
tion suggest that phosphonoacetate acts as an alternate product
inhibitor by binding to the pyrophosphate binding site of the HVTb
induced DNA polymerase, the alternate substrate, dNMP-PA, is released
from the enzyme too slowly to be detected. It appears, then, that
phosphonoacetate is an effective inhibitor of the HVT-induced DNA

polymerase because of its tight binding to the enzyme.

117

Mao and Rabishaw (77) published a partial kinetic analysis of
the inhibition of HSV-induced DNA polymerase by phosphonoacetate at
the same time this study was published (112). They obtained results
similar to those reported here. With [3H]dTTP as variable substrate
and with saturating concentrations of dATP, dGTP, dCTP and activated
DNA, inhibition by phosphonoacetate was noncompetitive. With
activated DNA as variable substrate and saturating concentrations
of the four dNTPs, inhibition by phosphonoacetate was uncompetitive.
A study of phosphonoacetate inhibition with activated DNA as variable
substrate and low dNTP concentrations was not reported. The simi-
larity in the results of kinetic inhibition studies with the HVT-
and HSV-induced DNA polymerases suggests that the mechanism of inhi-
bition by phosphonoacetate is the same for all herpesvirus-induced
DNA polymerases.

Phosphonoacetate appears to be a universal inhibitor of the
DNA polymerases induced by the herpesviruses. Therefore, the pyro-
phosphate binding site for this group of DNA polymerases must be
quite similar. The pyrophosphate binding sites of DNA polymerase a
of ducks and HeLa cells and of the vaccinia virus-induced DNA
polymerase (76) appear to be slightly related to this site. The
perphosphate binding sites of DNA polymerase B of ducks and HeLa
cells, of DNA polymerase y of HeLa cells (76), of DNA polymerases
of human WI-38 cells (73,75), of AMV and RSV reverse transcriptases
(77), and of E. coli DNA polymerase I and.M. luteus DNA polymerase
(77), however, appear to be unrelated to this site on the herpesvirus-
induced DNA polymerase.

The studies with the analogues of phosphonoacetate give some

information about the structural requirements for binding at the

118
HVT-induced DNA polymerase pyrophosphate binding site. The results
demonstrate that the carbon chain of the phosphono-compound must be
of specific chain length, that a carboxyl or sulfa-group cannot
substitute for the phosphono-group, that the methylene carbon cannot
have bulky or charged substituents, and that an amino, a methylamino,
an acetaldehyde, an acetonyl, an N-methylacetamide or a phoSphono-

group cannot substitute for the carboxyl group.

APPENDICES

APPENDIX A

NOMENCLATURE USED IN STEADY STATE KINETIC ANALYSES OF
THE HVT-INDUCED DNA POLYMERIZATION REACTION

The following nomenclature as defined by Cleland (99,100) was
used in determining the kinetic mechanism of the HVT-induced DNA

polymerase and the kinetic mechanism for phosphonoacetate inhibition.

Steady State Kinetics - The study of enzyme catalyzed reaction rates
in which reactant concentrations and their Michaelis constants greatly”
exceed enzyme concentration. Under these conditions all enzyme

intermediates are at constant steady state levels.

Initial Velocity Study - Steady state kinetic experiment used to
determine if a kinetic mechanism is sequential or ping pong. The
concentration of one substrate, the variable substrate, is varied

in the presence of fixed concentrations of the second substrate, the
changing fixed substrate, in the absence of products. When a double
reciprocal plot of variable substrate against velocity at different
changing fixed substrate concentrations is made, a family of lines,
an initial velocity pattern, results. Values of the vertical inter-
cepts and of the slopes from the initial velocity pattern are then
replotted against reciprocal changing fixed substrate concentration
in order to identify a kinetic mechanism as sequential or ping pong.
For a sequential mechanism values of both vertical intercepts and
slopes increase as a linear function of the reciprocal changing fixed

119

120
substrate concentration. For a ping pong mechanism slope values
from the initial velocity pattern are independent of reciprocal
changing fixed substrate concentration while vertical intercept
values increase as a linear function of reciprocal Changing fixed
substrate concentration. Kinetic constants for the substrates of

the enzyme reaction and V are also determined from the replots.

Sequential Mechanism - Kinetic mechanism in which all substrates

must add to the enzyme before any products are released.

Ping Pong Mechanism - Kinetic mechanism in which one or more'prdducts

are released from.the enzyme before all the substrates have added.

Ordered bi bi Mechanism - Sequential kinetic mechanism in which the
two substrates add to the enzyme in an obligatory order before the

two products are released from the enzyme in an obligatory order.

Modified Ordered bi bi Mechanism - Ordered bi bi mechanism in which
a polymer is both a substrate and product in an enzyme reaction.
The polymer product cannot be distinguished from the polymer sub-
strate. Consequently, a reaction product is always present during

enzyme reaction (113).

Inhibitor - A compound which slows down the rate of an enzyme catalyzed
reaction by forming complexes with particular forms of the enzyme.
This results in a decrease in the amount of enzyme available for

participation in the normal reaction sequence.

Three types of inhibitors are defined by Cleland (100) and

were considered in this study:

121

Product Inhibitor - A compound which is a product of a particular
enzyme reaction which, when added in sufficient quantity to reaction
mixtures, inhibits by forming reaction complexes for the reverse

enzyme reaction.

Alternate Product Inhibitor — A compound which is a product of a
particular enzyme reaction when an alternate substrate is used. It
inhibits in the same way as a product inhibitor. The enzyme complexes

formed, however, are those of an alternate reaction pathway.

Dead End Inhibitor - A compound which forms reversible enzyme

complexes which cannot participate in the reaction pathway.

Three basic types of inhibition were observed in these studies.
Each type is defined on the basis of the effect the inhibitor has
on the slope and intercept of double reciprocal plots where variable
substrate is plotted against velocity at different fixed inhibitor

concentrations.

Linear Competitive Inhibition - Observed if the slopes of double
reciprocal plots are a linear function of inhibitor concentration

but the intercepts are independent of inhibitor concentration.

Linear Uncompetitive Inhibition - Observed if the intercepts of
double reciprocal plots are a linear function of inhibitor concen-

tration but the slopes are independent of inhibitor concentration.

Linear Noncompetitive Inhibition - Observed if both the intercepts
and slopes of double reciprocal plots increase as a linear function

of inhibitor concentration.

122

Inhibition constants may be determined from slope and inter-
cept replots. The inhibition constant from the slope replot, Kis'
is obtained from the X-intercept of the replot. The inhibition
constant from the vertical intercept replot, Kii' is obtained from
the X-intercept of the replot. For complex kinetic mechanisms
these inhibition constants are apparent inhibition constants. They
are a combination of kinetic constants including the inhibition

constant.

APPENDIX B

PROCEDURE FOR THE DERIVATION OF STEADY STATE RATE EQUATIONS

A steady state rate equation for a particular kinetic mechanism
is derived by first diagramming a kinetic mechanism which illustrates
the order of addition of substrates to and release of products from
different enzyme forms and the rate constants involved. This
mechanism is then translated into a rate equation composed of indi-
vidual rate constants and reactant concentrations by the method of
King and Altman (107,114). Finally, this rate equation is transformed
into a rate equation expressed in terms of kinetic constants as
described by Cleland (99). The complete steady state equation is
then expressed in terms of one Michaelis constant and one inhibition
constant for each reactant and two maximum velocity terms.

Steady state rate equations for dead end inhibitors are derived
by multiplying certain terms in the denominator of the full rate
equation by the factor (1 + I/Ki) where Ki is the dissociation
constant of the enzyme—inhibitor complex. The terms multiplied by
this factor are those which represent the enzyme form with which the
inhibitor combines. In certain cases the rate equation for dead end
inhibition cannot be expressed entirely in kinetic constants because
a kinetic constant is often a combination of rate constants representing

different enzyme forms.

123

APPENDIX C

DETERMINATION OF THE KINETIC MECHANISM FOR THE
HVT-INDUCED DNA POLYMERIZATION REACTION

A modified ordered bi bi mechanism was postulated for the HVT—
induced DNA polymerization reaction. In this mechanism DNA would
add to the enzyme as the first substrate, followed by dNTP as the
second substrate. After addition of dNMP to the 3'-end of the
DNA primer chain by phosphodiester bond formation, PPi would be
released from the enzyme as the first product, followed by DNA as
the second product (Figure 20, outside square). The steady state
rate equation for this mechanism is given by equation 4, where KDNA
and K are Michaelis constants for DNA as substrate and product,

DNA'

respectively, and KiDNA and KiDNA' are inhibition constants for DNA
as substrate and product, respectively. The validity of this equa—
tion for the HVT-induced DNA polymerization reaction was tested by
initial velocity studies and pyrophosphate product inhibition studies.
In an initial velocity study, initial velocities in the absence
of pyrophosphate are measured. Therefore, the negative velocity term
in the numerator of equation 4 and all terms containing (PPi) in the
denominator of equation 4 can be eliminated. The reciprocal of this
simplified equation with dNTP as variable substrate and activated
DNA as changing fixed substrate takes the form of equation 5. This

equation predicts that the slope values and the intercept values of

this initial velocity pattern should be linear functions of reciprocal

124

 

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127
DNA concentration. These results were observed (Figure 6). There-
fore, the HVT-induced DNA polymerization reaction is consistent with
a sequential mechanism as would be expected for a modified ordered
bi bi reaction.
Kinetic constants for dNTP and activated DNA were evaluated using
equation 5 and the slope and intercept replots of Figure 6. For

these evaluations it was necessary to assume that K1 was very

DNA'

large; K is defined as K.7/K.8 (Figure 20) so that in this initial

iDNA'
velocity study this assumption would be valid. The values of these
kinetic constants are given in Table 7. The kinetic constants calcu-

lated when dNTP was variable substrate and activated DNA was changing

Table 7. Kinetic constants for HVT-induced DNA polymerase

 

 

Variable Substrate dNTP Activated DNA
Changing Fixed
Kinetic constant Substrate Activated DNA dNTP

 

V i 36.5 pmol per 37.7 pmol per
1(DNA' ) 30 min 30 min
2. 2.1 M
KdN'l‘P 1 "M u
KDNA 6.9 ug per ml 7.2 ug per ml
KiDNA 2.1 no Per ml 2.6 ug Per m1

 

Kinetic constants were evaluated using equation 5 and slope
and intercept replots of initial velocity patterns with variable
substrate and changing fixed substrate as indicated.

fixed substrate (Figure 6) are in good agreement with those calcu-
lated when activated DNA was variable substrate and dNTP was changing

fixed substrate (data not shown).

128
Pyrophosphate product inhibition experiments were used to deter-
mine if the HVT-induced DNA polymerization reaction did fit the full
steady rate equation for the modified ordered bi bi mechanism (equa-
tion 4). Under the experimental conditions that were used, the

numerator of equation 4 is approximated by the term V (DNA)(dNTP).

1 (DNA ,dN'I‘P)
The reciprocal of equation 4 arranged with DNA as the variable substrate
takes the form of equation 6. At low concentrations of dNTP, both the
slope and the intercept terms are a function of pyrophosphate concen-
trations and the inhibition pattern is noncompetitive. At high con-
centrations of dNTP the slope term is independent of perphosphate
concentration and the inhibition pattern is uncompetitive.

These predictions were satisfied experimentally. When dNTP levels.
were 2.5 uM a noncompetitive inhibition pattern was observed. When
dNTP levels were 20 uM an uncompetitive inhibition pattern was
observed (p. 72).

If equation 6 is rearranged with dNTP as variable substrate, the
prediction emerges that the pyrophosphate inhibition pattern would be
noncompetitive regardless of DNA concentration. With dNTP as the
variable substrate and activated DNA at a concentration of 200 pg
per ml, the inhibition pattern was noncompetitive as predicted
(p. 72).

Therefore, the results from initial velocity experiments and
perphosphate inhibition experiments are consistent with a modified
ordered bi bi kinetic mechanism for the HVT-induced DNA polymerization
reaction. Evidence for this same kinetic mechanism has also been

presented for E. coli DNA polymerase I (108).

129

 

 

 

 

 

 

 

 

 

 

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APPENDIX D

' MULTIPLE INHIBITION ANALYSIS TO DETERMINE THAT TWO
INHIBITORS ARE MUTUALLY EXCLUSIVE

Two inhibitors are mutually exclusive if, when the concentra-
tion of inhibitor1 is varied in the presence of changing fixed

concentrations of inhibitorz, a plot of l/v versus inhibitor1
concentration yields a series of parallel lines. In order to under-
stand the validity of this statement, the circumstances under which
a series of parallel lines arises from.plots of a rate equation must
be defined. Then a rate equation which describes phosphonoacetate
and pyrophosphate as both acting at the pyrophosphate binding site
of the DNA polymerase will be inspected.

In general, a steady state kinetic experiment involves inde-
pendent components (variable substrate, variable inhibitor) and
changing fixed components (changing fixed substrate in initial
velocity studies or changing fixed inhibitor in inhibition studies).
When values of l/v are plotted against values of reciprocal variable
substrate concentrations or variable inhibitor concentrations, a
series of lines is obtained as a result of the different changing
fixed component concentrations used in the experiment. These lines
are parallel if the slope term of the equation describing the rela-
tionship between l/v and the independent component does not contain

changing fixed component concentration terms.

130

131

For the HVT-induced DNA polymerization reaction, evidence exists
that phosphonoacetate and pyrophosphate are mutually exclusive
inhibitors (Figure 10). The kinetic model which best describes
this fact is the alternate product inhibition model (Figure 20).
The rate equation which describes the alternate product inhibition
model when both phosphonoacetate and pyrophosphate are present will
be inspected. Because terms for both phosphonoacetate and pyro-
phosphate must be considered in the rate equation, it will be very
complex. Therefore, the rate equation will be written in "coefficient"
form: for each term in the rate equation, the constants are lumped
together and are identified as the coef of the reactant con—
centration factor in that term in the denominator and as num or

1

num2 in the numerator (99).

Using this simplification, the rate equation appropriate for
a l/v against phosphonoacetate concentration plot (Figure 10) is
given by equation 7. The slope term of this equation contains no
(PPi) terms, where pyrophosphate was the changing fixed inhibitor in
this experiment. Therefore, equation 7, which describes the fact
that phosphonoacetate and pyrophosphate both act at the pyrophosphate
binding site of the DNA polymerase, predicts that a l/v against

phosphonoacetate concentration plot will be a series of parallel

lines as was observed.

132

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