Acyclovir-Resistant Mutants of Herpes Simplex Virus Type 1 Express

JOURNAL OF VIROLOGY, Dec. 1981, p. 936-941
Vol. 40, No.3
Acyclovir-Resistant Mutants of Herpes Simplex Virus Type 1
Express Altered DNA Polymerase or Reduced Acyclovir
Phosphorylating Activities
Wellcome Research Laboratories, Research Triangle Park, North Carolina 27709,' and The Sidney Farber
Cancer Institute, Harvard Medical School, Boston, Massachusetts 021152
The biochemical properties of four acyclovir-resistant mutants are described.
Two of these mutants, PAAr5 and BWr, specified nucleotidyl transferase (DNA
polymerase) activities which were less sensitive to inhibition by acyclovir triphosphate than their wild-type counterparts. Another mutant, IUdRr, exhibited
reduced ability to phosphorylate acyclovir. The fourth mutant, ACGr4, both
induced an altered DNA polymerase and failed to phosphorylate appreciable
amounts of acyclovir. BWr, a new acyclovir-resistant mutant derived from the
Patton strain of herpes simplex virus type 1, induced a DNA polymerase resistant
to inhibition by acyclovir triphosphate, but, unlike the polymerases induced by
PAAr5 and ACGr4, still sensitive to phosphonoacetic acid. Resistance of BWr to
acyclovir mapped close to the PAAr locus and was separable from mutations in
the herpes simplex virus thymidine kinase gene by recombination analysis.
The nucleoside analog 9-(2-hydroxyethoxymethyl)guanine (acyclovir, acycloguanosine) is
a specific and effective inhibitor of herpes simplex virus (HSV) replication (8, 25) and demonstrates little cytotoxicity to uninfected cells (25).
There has accumulated a considerable amount
of evidence indicating that acyclovir exerts its
antiviral effect after conversion to acyclovir triphosphate (acyclo-GTP), which inhibits the
viral nucleotidyl transferase (DNA polymerase)
more efficiently than does the host cell a DNA
polymerase (8, 12). Biochemical evidence indicates that HSV thymidine kinase (HSV-TK) is
the enzyme responsible for phosphorylation of
acyclovir to its monophosphate (8, 13). Host-cell
enzymes are apparently responsible for the phosphorylation of acyclovir monophosphate (acyclo-GMP) (11, 21).
Parallel with biochemical studies are the results of genetic experiments which have implicated the HSV-TK and DNA polymerase genes
as loci which, when mutated, can confer resistance to acyclovir in the cell culture (4, 5, 7, 27).
With regard to the TK gene, several HSV mutants lacking TK activity exhibit resistance to
acyclovir (4, 5, 8, 9, 27), and the degree of resistance generally corresponds to the level of TK
activity (4, 5).
With regard to the DNA polymerase gene,
several mutants which are resistant to phosphonoacetic acid (PAA), a recognized marker for
the HSV DNA polymerase gene (2, 3, 16, 17, 2224), are also resistant to acyclovir, yet exhibit
wild-type levels of TK activity (5, 27). Recombination and complementation analyses of one
of these mutants, PAAN5, showed that it defmes
a codominant locus (termed ACGr-PAA) distinct
from the recessive acgr-tk locus and much more
closely linked to the PAA' locus than it is to the
acgr-tk locus. Complementation analysis indicated that another mutant, ACGr4, was a presumptive double mutant containing mutations
at both loci that lead to a highly resistant phenotype (5). Subsequent intertypic and intratypic
marker rescue experiments with other PAA mutants have also demonstrated linkage of acyclovir resistance with the PAAr locus and with
temperature-sensitive mutations within the
HSV DNA polymerase gene (7; D. M. Coen and
P. A. Schaffer, unpublished data; D. Knipe, personal communication).
To confirm the implications that the HSV-TK
and DNA polymerase genes are loci which, when
mutated, can confer resistance to acyclovir in
cell culture, we examined four acyclovir-resistant mutants derived from the KOS and Patton
strains of HSV type 1 (HSV-1). First, the sensitivity of these mutants to inhibition by acyclovir
and PAA was examined (Fig. 1A and B). Both
the PAA-resistant mutant PAAr5 and the presumptive double mutant ACGr4 (5) were less
sensitive to inhibition by acyclovir than was the
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Received 4 June 1981/Accepted 8 August 1981
VOL. 40, 1981
than that for their wild-type counterparts. However, sensitivity of these viruses to PAA was
considerably different from that observed for the
mutants derived from KOS. Both Patton-derived mutants gave dose-response curves with
PAA comparable to that. of the Patton strains.
In fact, wild-type Patton consistently gave
higher ED50 values with PAA than did the two
Cells infected with mutant viruses were then
tested for their ability to phosphorylate acyclovir. Acyclo-GTP levels in cells infected with the
mutants ACGr4 and IUdRF were 0.3 and 4.0% of
the levels found in cells infected with their wildtype counterparts (Table 1). The levels of acyA.
z 40
z 60
0 40
2^t% 1
FIG. 1. Plaque inhibition dose-response curves for acyclovir (A) and PAA (B) in Vero cells, determined by
using wild-type KOS (0) and Patton (0) and acyclovir-resistant PAAr5 ([1), IUdRrr(), BW' (A), and ACGr4
(A) viruses. Plaque reduction assays to determine ED50 values for acyclovir and PAA were performed as
described by Collins and Bauer (6). Virus stocks were prepared as previously described (8).
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wild-type virus KOS (Fig. 1A). Fifty percent
effective dose (ED50) values for PAAr5 and
ACGr4 were 20- and 490-fold greater, respectively, than that for KOS. PAAr5 and ACGr4
also showed much less susceptibility to inhibition by PAA, with ED50 values more than 10and 5-fold greater, respectively, than that obtained for KOS (Fig. 1B). Mutants of the Patton
strain of HSV-1, IUdRr (a mutant characterized
by Smith et al. [28] as being resistant to acyclovir and iododeoxyuridine) and BWT were also
found to be much less susceptible to inhibition
by acyclovir than was the wild-type virus. The
ED50 values for IJdRr and BW' were approximately 100 and 200 times greater, respectively,
mutant, BWr, were less sensitive to inhibition by
acyclo-GTP than their respective wild types. h5o
values for PAAr5, ACGr4, and BWr were approximately 5-, 9-, and 25-fold higher, respectively,
than the Lo values obtained for their wild-type
counterparts (Table 1). These data confirm the
suggestion of Coen and Schaffer (5) that PAAr5
and ACGr4 contain mutations at the DNA polymerase locus conferring acyclovir resistance.
The sensitivities of the DNA polymerases of
PAAr5, ACGr4, and BWr to PAA inhibition were
also determined (Table 1). The DNA polymerase of PAAr5 and ACGr4 were found to be ap-
FIG. 2. Inhibition of wild-type and mutant virus
DNA polymerases by acyclo-GTP. Virus-induced
DNA polymerase was isolated and identified as described previously (11, 29). DNA polymerase assays
were carried out as described by Elion et al. (8) and
Furman et al. (12). The substrates dATP, dCTP, and
dTTP were present at a concentration of 100 pM, and
dGTP was present at a concentration of 5 p.M. Symbols for polymerases: KOS (0), Patton (O), PAAr5
(0), ACGr4 (A), B W (U), and IUdR r (A).
TABLE 1. Summary of the biochemical properties of acyclovir-resistant mutants and their corresponding
wild types'
106 cells)
Polymerase sensitivity (range)
Viral sensitivity ____
__ ED_____o______M
0.23 (0.11-0.42)
1.17 (0.82-1.62)
2.14 (1.47-3.33)
0.50 (0.21-0.86)
3.57 (2.36-4.71)
3.78 (2.43-5.07)
0.15 (0.09-0.24)
1.93 (0.43-4.43)
3.71 (3.36-4.08)
0.97 (0.55-1.50)
0.23 (0.04-0.89)
0.50 (0.01-1.93)
Experimental details may be found in the legends to Fig. 1 and 2.
b Concentrations of substrates for the acyclo-GTP inhibition assay are described in the legend to Fig. 2.
values were calculated by using the Probit computer program, which places more weight on those points near
the I50 point (10). For the PAA inhibition assay, the concentration of all four deoxynucleoside triphosphates was
The ED0o values were determined by Probit analysis (10).
No inhibition at these concentrations.
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clo-GTP in cells infected with the resistant mutants PAAr5 and BWr were comparable to the
leveLs in cells infected with wild-type viruses.
Moreover, the total amount of phosphorylated
acyclovir (all forms: mono-, di-, and triphosphates) was considerably higher in cells infected
with wild-type viruses, BWr, or PAAr5 than in
cells infected with ACGr4 and IUdRr (data not
shown). Similarly, extracts prepared from cells
infected with IUdRr and ACGr4 contained much
less acyclovir-phosphorylating activity and
HSV-TK activity than did extracts from cells
infected with wild-type virus, whereas BW0 and
PAAr5 induced acyclovir-phosphorylating and
HSV-TK activities comparable to those of their
wild-type counterparts (P. Keller, personal communication). Thus, the acyclovir resistance of
BWr and PAAr5 cannot be attributed to failure
of these mutants to phosphorylate acyclovir.
The lack of acyclovir phosphorylation (TK
expression) by IUdRr probably explains the
cross-resistance to acyclovir and IUdR observed
for this virus (28).
The inhibitory effect of acyclo-GTP on the
DNA polymerase of mutant and wild-type viruses was examined by using [3H]dTTP incorporation as a measure of enzyme activity. Enzyme inhibition curves (Fig. 2) demonstrated
that DNA polymerases of the viruses could be
separated into two classes, a sensitive class and
a resistant class, on the basis of their sensitivities
to inhibition by acyclo-GTP. The sensitive class,
having I50 (50% inhibition) values of about 0.2
,uM acyclo-GTP (Table 1), was comprised of
both wild-type strains and the TK-deficient mutant derived from Patton (IUdRr). The DNA
polymerases induced by the KOS-derived mutants, PAAr5 and ACGr4, and the Patton-derived
VOL. 40, 1981
Genetic experiments previously identified
PAAr5 as a mutant whose resistance to acyclovir
was separable by recombination from the acyclovir resistance mutations in acgr-tk mutants
and closely linked to the PAA resistance locus
(5). To determine whether the mutant BWr behaved similarly in recombination tests, we performed crosses between BW' and the acg'-tk
mutant, ACGr35, which is partially acyclovir
resistant owing to a mutation which reduces TK
activity to about 15% of wild-type levels (5). The
ability to measure recombination between these
two viruses depended upon the fact that neither
plated efficiently in 400 1LM acyclovir (Table 2).
However, when BW' and ACGr35 were crossed,
3.3% of the resulting progeny were resistant to
400 ,uM acyclovir (Table 2). These data imply a
recombination frequency of 6.6%, which is much
greater than any found between mutants within
the same complementation group which map in
the unique sequences of the HSV-1 genome (R.
A. F. Dixon and P. A. Schaffer, unpublished
data). Similar results (not shown) were obtained
when BWT was crossed with the conditionally
resistant acgr_tk mutant, KG-ill, which exhibits
thermolabile TK activity (4).
To determine whether the acyclovir resistance
of BWr was linked to the PAA resistance locus,
we crossed BWr with PAAr5, and the progeny
were examined for their plating efficiency in
both PAA at 1.4 mM and acyclovir at 100 tiM.
Each parent used in the crosses was relatively
resistant to one of these drugs but quite sensitive
to the other or to the combination of both drugs
at these concentrations (Table 2). A recombinant of these two viruses would be expected to
plate efficiently in both drugs. However, only
0.08% of the progeny were resistant to both
drugs, implying a recombination frequency of
only 0.16% (Table 2). In contrast, in a parallel
experiment, when the acgr_tk mutant, ACGr35,
TABLE 2. Recombination of B W, ACGr35, and PAAr5a
acyclovir (100
Nou drIn
dru g
1.1 X
2.6 X
2.4 X
3.3 X
3.2 x
7.2 x
<5.0 X 102
1.6 x 105
1.0 X 103
1.1 x 106
<5.0 X 102
1.0 X 104
5.0 x 102
In acyclovir
(400 jiM)
<4.6 X 10-5
6.2 x 10-3
4.0 X 10-5
3.3 x 10-2
In acyclovir
<4.6 X 10-5
3.8 x 10-4
RF- RF- P+
Ad (%)
2.0 x 10-5
BWT x ACGr35
2.5 X 104
8.0 x 10-4
7.5 x 104
1.0 x 10-2
PAAr5 x ACGr35
a Recombination analysis was performed essentially as described by Schaffer et al. (26), except that Vero cells
were used instead of HEL cells, and recombination was performed at 37°C. Duplicate tube cultures of Vero cells
containing approximately 2 x 105 cells per culture were infected either with pairs of mutants, each at a calculated
multiplicity of 2.5 plaque-forming units (PFU) per cell in a total volume of 0.2 ml, or with single parental virus
controls at a multiplicity of 5 PFU per cell in 0.2 ml. Simultaneous assays of inoculum suspensions were
performed to confirm calculated input multiplicities; if the actual multiplicity varied more than twofold from
the calculated multiplicity, results of tests with these mutants were excluded.
b EOP, Efficiency of plating. EOP = (PFU per milliliter in presence of drug)/(PFU per milliliter in absence
of drug).
'RF - A, Recombination frequency. RF - A = [(PFU per milliliter in presence of acyclovir)/(PFU per
milliliter in absence of acyclovir)] x 2 x 100%.
d RF - P + A, Recombination frequency. RF -P + A = [(PFU per milliliter in presence of PAA and
acyclovir)/(PFU per milliliter in absence of PAA and acyclovir)] x 2 x 100%c.
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proximately seven times more resistant to inhibition by PAA than was wild-type KOS DNA
polymerase. In contrast, BWr DNA polymerase
was found to be no more resistant to inhibition
by PAA than was the DNA polymerase of its
parental virus, strain Patton. The DNA polymerase induced by IUdR' showed not only wildtype sensitivity to acyclo-GTP but also wildtype sensitivity to PAA (Table 1). The apparent
Km values for the four natural deoxynucleoside
triphosphates ranged from 1 to 4 ,uM (unpublished data). All viral DNA polymerase preparations exhibited a fourfold stimulation of activity in the presence of 50 mM ammonium sulfate,
whereas cellular a DNA polymerase activity was
reduced by 50%, indicating that the polymerase
preparations were virus specific (20, 29). In addition, the DNA polymerases induced by KOS,
Patton, IUdRr, and BWr were 50-fold more sensitive than the a cellular DNA polymerase of
HeLa S-3 cells to inhibition by PAA at a concentration of 5 ,uM, thus confirming their viral
We thank C. Lubbers, P. A. Temple, L. B. Sandner, and P.
T. Gelep for excellent technical assistance, J. A. Fyfe for
valuable discussion, G. B. Elion for critical reading of the
manuscript and for continuous support and interest during
this work, and K. 0. Smith for so graciously providing us with
his mutants.
This study was supported in part by Public Health Service
research grant CA20260 and program project grant CA21082
from the National Cancer Institute. D.M.C. was the recipient
of postdoctoral fellowship AI 05817 from the National Institutes of Health.
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and PAA'5 were crossed and the progeny were
analyzed under identical conditions, the recombination frequency was more than 12-fold higher
(Table 2).
Thus, both genetic and biochemical evidence
support the notion that PAAF5, ACGr4, and BWT
contained mutations in their DNA polymerase
genes which conferred resistance to acyclovir.
The results of this study indicate that mutations
can occur in the DNA polymerase gene that will
confer resistance to both acyclovir and PAA or
to acyclovir but not PAA. The latter result
would be expected if the HSV DNA polymerase
conforms to the model proposed by Kornberg
(18) for other DNA polymerases; i.e., DNA polymerase has an active center that is composed
of multiple sites, each with a different function.
Therefore, a mutation which affects the protein
at a site other than the pyrophosphate exchange
site (the presumptive site of PAA inhibition
[19]) will not necessarily affect the pyrophosphate exchange site (resistance to PAA). Nevertheless, the simplest explanation for the data
obtained for the mutants PAAr5 and ACGT4 is
that a single mutation can affect more than one
The new mutant described here, BWr, which
was acyclovir resistant but PAA sensitive, defines yet another phenotype within the DNA
polymerase locus and separates the domain of
the DNA polymerase molecule which specifies
acyclovir sensitivity from the domain which
specifies PAA sensitivity. Thus, mutants associated with the HSV DNA polymerase locus can
be temperature resistant, drug resistant, or both
(1, 14, 15, 17, 23), the degree of resistance to
both PAA and acyclovir varying. A detailed
understanding of the molecular basis for the
wide range of phenotypes within the HSV DNA
polymerase locus awaits further fine-structure
mapping and additional biochemical studies of
its gene product(s).
(This work was presented in part at the 5th
Cold Spring Harbor Workshop on Herpes Viruses, Cold Spring Harbor, N.Y., on 31 August
VOL. 40, 1981
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