Download The Role of Axginine in the Replication of Herpes Simplex Virus

J. gen. Virol. (1967), 1, 471-478
471
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The Role of Axginine in the Replication of Herpes Simplex Virus
BY Y. B E C K E R , U. O L S H E V S K Y
AND J U L I A L E V I T T
Department of Virology, Hebrew University-Hadassah Medical School,
Jerusalem, Israel
(Accepted 3 June I967)
SUMMARY
Arginine is essential for the replication of herpes simplex virus. Omission of
arginine from the culture medium did not affect the synthesis of viral D N A
but prevented the formation of virions. Addition of arginine immediately
stimulated protein synthesis which was followed by virus formation.
INTRODUCTION
The need for arginine in the replication of adenovirus was described by Rouse,
Bonifas & Schlesinger (I963), who demonstrated the inhibition of virus synthesis in
the absence and its restoration on the addition of arginine. Tankersley (i964) showed
that arginine was essential for the replication of herpes simplex virus, as did Roizman,
Borman & Rousta (I965) and Sharon (I966), but the mechanism was not explained.
The present communication deals with the role of arginine in the intranuclear replication of herpes simplex virus and its effect on the synthesis of viral DNA, viral coat
proteins and the formation of herpes virions.
METHODS
Virus, cells and medium
The HF strain of herpes simplex virus, plaque-purified, was propagated in the BSC1
line of monkey kidney cells. The cells were grown in milk-dilution bottles using
Eagle's medium (Eagle, I959) supplemented with Io ~o (v/v) calf serum. Infected
cultures which were used as virus stock preparations contained 5 ~ calf serum and
were stored at 4 °. The virus was released from the cells before use by ultrasonic vibration for I rain. in a MSE ultrasonic disintegrator operating at I'3g. Monolayer
cultures were infected at a multiplicity of Io p.f.u./cell. The virus, suspended in 5 ml.
Eagle's medium without serum, was adsorbed to the cells (2 x Io 6 cells per milk-dilution
bottle) for 3 hr. At the end of the adsorption period the virus was removed and IO ml.
fresh Eagle's medium was added. Infectivity titrations of the virus stocks were done
by the plaque assay described by Levitt & Becker 0967). The amount of leucine in
Eagle's medium was reduced to one-hundredth the normal concentration (final concentration o-oo8 raM). As required, arginine was either omitted from the medium or
added at concentrations ranging from o.0042 to 4.2 mM. Puromycin dihydrochloride
(obtained from N.B. Co., U.S.A.), at a concentration of Io #g./ml., was used to inhibit protein synthesis.
Radioactive isotopes
The D N A in the infected and control cultures was labelled either with [2-14C]thymidine or [aH]thymidine (specific activity 35 me/m-mole and 2"7 mc/m-mole,
3o-2
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472
Y. BECKER, U. OLSHEVSKY AND J. LEVITT
respectively). Proteins were labelled with either [14C]leucine or [aH]leucine (specific
activity 165 and 2oo mc/m-mole, respectively) and also with [14C]arginine (specific
activity 15o mc/m-mole). The isotopes were obtained from the Radiochemical Centre,
Amersham, Buckinghamshire, England.
Fractionation of cells
Infected and control cells labelled with radioisotopes were withdrawn at intervals
after infection; the medium was removed and the cells were kept at 4 °. The cells were
scraped with a glass rod into 2 to 4 ml. cold hypotonic buffer (RSB) and disrupted by
means of twenty strokes in a Dounce glass homogenizer (Levitt & Becker, I967). By
this procedure less than 2 ~oo of the radioactive DNA in the nuclei was released into the
cytoplasmic fraction. The nuclei were separated from the cytoplasm by centrifugation
at 8oog for 2 min., resuspended in hypotonic buffer and disrupted by ultrasonic
treatment for I min. Samples of o. I ml. were removed from the different cell fractions,
treated with 2o 70 trichloracetic acid, collected on Millipore filters and counted in a
Packard liquid scintillation counter. The amount of DNase-resistant radioactive DNA
was determined by treatment with Ioo#g. DNase per o.I ml. sample, before the
addition of trichloracetic acid.
Sucrose density gradient centrifugation
Mature herpes virions were isolated from the nuclear fractions by zone centrifugation in sucrose gradients (15 to 3o 70, w/w). The gradients were centrifuged at 15,ooo
rev./min, for 5o min. in the SW z5"I rotor of the Beckman model L-2 ultracentrifuge as
described by Levitt & Becker (1967). Fractions were collected and incubated for
3o rain. at 37 ° with DNase (2 x crystallized; Worthington Co., U.S.A.), 35 #g. per
fraction in the presence of IO-~M-Mg2+, before treatment with trichloracetic acid.
RESULTS
The effect of arginine on the formation of herpes simplex virus
At least o'1o5 mM arginine was required to obtain maximum virus yield (Table I).
This was also demonstrated by using the DNase resistance of mature herpes virions
to measure virus synthesis (Levitt & Becker, 1967). BSC1 cultures were infected with
herpes simplex virus, labelled with tritiated thymidine and incubated in Eagle's
medium lacking arginine. Different concentrations of arginine were added to some of
the cultures 3 hr and to the rest 13 hr after infection. The cultures were harvested
19 hr after infection and the amount of coated viral DNA determined. The amount of
radioactive DNA resistant to DNase increased in the infected cells as the concentration of arginine was increased, indicating that the process of coating of viral DNA
occurred only in the presence of arginine (Fig. I).
An attempt was made to determine whether incomplete herpes virions were synthesized in the nuclei of the infected cells in the absence of arginine. Accordingly, the
cells were infected with herpes simplex virus and incubated in Eagle's medium lacking
arginine (Fig. 2A) or containing o"1o5 mM arginine (Fig. 2B, C). The cultures were
labelled with [aH]thymidine and [14C]leucine (Fig. 2 A, B), or with [SH]thymidine and
[14C]arginine (Fig. z C). Eighteen hr after infection the cultures were harvested and the
cell nuclei separated and analysed on sucrose gradients. In the absence of arginine
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Arginine and replication of herpes virus
473
(Fig. 2 A) no virus band was evident and radioactive proteins were found only in the
soluble fraction at the top of the sucrose gradient. In the presence of arginine however,
mature herpes virions labelled in the coat proteins with either [z4Clleucine or [14C]arginine appeared in the virus band which was characterized by the presence of radioactive D N A resistant to DNase. In addition, two peaks containing radioactive proteins
10,000
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C o n c e n t r a t i o n o f a r g i n i n e (mM)
Fig. I. T h e effect o f d i f f e r e n t c o n c e n t r a t i o n s o f a r g i n i n e o n t h e e x t e n t o f v i r u s f o r m a t i o n
19 hr after infection. •
O, arginine added 3 hr after infection; © . . . . . . ©, arginine
added I3 hr after infection.
Table I. Dependence of virus yieM on the concentration of arginine
Virus yield (p.f.u.) after 18 hr
incubation
F
A r g i n i n e c o n c e n t r a t i o n (raM)
Expt I
Expt z
2-2 × T05
C o m p l e t e m e d i u m (3 h r a f t e r i n f e c t i o n )
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0
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x IO 7
were evident in the region of the gradient between the virus band and the soluble
fraction at the top. These two peaks were shown by electron microscopy to contain
viral capsids and capsomeres (Olshevsky, Levitt & Becker, I967), indicating that
arginine was required for the formation both of mature virions and of the viral coat
proteins. The two upper protein bands were not evident in the cytoplasmic fraction of
infected cells.
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474
Y. B E C K E R , U. O L S H E V S K Y AND J° L E V I T T
The effect of arginine on the synthesis and coating of viral DNA
Infected cells were labelled with [3H]thymidine 3 hr after infection and incubated in
Eagle's medium with or without arginine. Samples were withdrawn at different times
and the amounts of radioactive D N A before and after DNase treatment were determined. Uninfected controls were similarly treated. More D N A was synthesized in
20,000
10,000
5,000
10,000 A
5,000
Protein
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[14C]Feucin/
1,000
o
B
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C
50001- Protein
l" ['4C]arginine .~ft/~
Protein . ~
['4C]leucine7
1,000
500
500
~
[ HIDNA
....
0
5 10 15 20 25 30 35
0
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Fraction
0
5 lO 15 20 25 30
Fraction
Fraction
Fig. 2. Effect of arginine depletion on the synthesis of herpes simplex virus; zone centrifugation in sucrose density gradients. A, without arginine; B, C, with o' I o5 mM arginine. A, B, C
labelled with [3H]thymidine; A, B with [14C]leucine; C with ['C]arginine. © ...... ©,
[3H]DNA resistant to DNase; ®
Q, [a4C]proteins.
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80
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20
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10
0
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6
I
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9
12
15
Time after infection (hr)
0
18
I
6
9
12
15
Time after infection (hr)
18
Fig. 3. The effect of arginine on the synthesis of D N A and herpes virions. •
Q, infected
cells plus o'Io5 m ~ arginine; (3 -.
(3, infected cells minus arginine; ~ ...... [3, uninfected
control. A, before and B, after treatment with I mg./ml. DNase.
infected than in uninfected cells and the synthesis of D N A was not affected by the
absence of arginine. The coating of viral D N A was studied by measuring the increase
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Arginine and replication of herpes virus
475
in the amount of radioactive D N A resistant to DNase treatment at different times
after infection. In the absence of arginine the amount of DNase-resistant D N A was
negligible, indicating that although viral D N A was being synthesized, coating of the
D N A and formation of herpes virions were prevented (Fig. 3 A, B).
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6
9
12
15
18
o
I
I
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t
3
6
9
12
15
18
Time (hr)
Time (hr)
Fig. 4. The effect of arginine depletion on the rate of protein synthesis. A, infected cultures;
B, uninfected controls. •
0, medium containing o-Io5 mt~ arginine; © . . . . . . 0, medium
lacking arginine.
The rate of protein synthesis in the absence of arginine
Cultures were incubated in Eagle's medium with or without arginine and at different
times were labelled for I5 min. with [l~C]leucine and the radioactivity in the protein
was determined. The rate of protein synthesis in infected cells was lower than in uninfected cells (Fig. 4)- The lack of arginine in the medium resulted in reduction of the
rate of protein synthesis both in infected and in uninfected cells. Further analysis
demonstrated the site of arginine incorporation to be localized in the cytoplasm.
Treatment with puromycin abolished, to a great extent, the incorporation of [I~C]arginine into cell proteins.
The effect of addition of arginine on the rate of protein synthesis
To determine whether the protein-synthesizing mechanism in the cytoplasm of
infected cells was affected by the absence of arginine, cultures infected with herpes virus
and incubated for [ 5 hr in the absence of arginine were pulse-labelled for 5 rain. with
[14C]leucine at different times before and after the addition of arginine. The rate of
protein synthesis increased three- to fourfold after the addition of arginine (Fig. 5).
Labelling the cells for z min. at 5 min. intervals demonstrated that the increase in the
rate of protein synthesis occurred within 2 min. of the addition of arginine.
The effect of arginine on the coating of viral DNA
Cultures infected with herpes simplex virus were labelled with [3H]thymidine and
incubated in medium with and without arginine. Twelve hr after infection, arginine
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476
Y. B E C K E R ~ U . O L S H E V S K Y
A N D J. L E V I T T
was added to some of the cultures without arginine. Cultures were harvested at
different times and the amount of DNase-resistant radioactivity determined. DNaseresistant radioactivity after the addition of arginine I2 hr after infection increased in
parallel with the increase in infected cultures kept in complete medium (Fig. 6). This
and other similar experiments showed that radioactive DNA resistant to DNase
appeared within 2 hr of the addition of arginine. Sucrose-gradient analysis of infected
cell nuclei labelled both with [l~C]leucine and [aH]thymidine also showed that radioactive proteins and DNA appeared in the band containing mature virions 2 hr after
the addition of arginine. This agrees with the finding that under normal circumstances viral proteins are transported to the nucleus and viral DNA is coated within
two hr (Olshevsky et aL I967).
i
I
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i
1
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10
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%
.=2
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-=2 6
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90
T i m e (min.)
Fig. 5
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H
120
0
6
Fig. 5. T h e effect o f arginine o n the rate o f protein synthesis. •
O - - - - O, arginine (o'04 raM) a d d e d (arrow).
9
12
15
18
T i m e after infection (hr)
Fig. 6
21
• , arginine n o t added;
Fig. 6. Reversal o f arginine-deficiency effect. •
• , c o m p l e t e m e d i u m ; © . . . . . . O, n o
arginine; • - • , arginine a d d e d I2 h r after infection (arrow).
DISCUSSION
The experiments described in the present paper confirm and extend the initial
finding of Tankersley (I964) that arginine is essential for the synthesis of herpes
simplex virus. Determination of virus yield both by the plaque assay and by the increase in DNase-resistant radioactive DNA demonstrated that virus synthesis was
completely inhibited in the absence of arginine. The extent of virus synthesis was
found to be dependent on the amount of arginine present in the medium of the infected cultures. The effect of arginine deprivation was further investigated by studying
the synthesis of DNA and proteins in infected cells. DNA synthesis was not affected
by the lack of arginine and there was no difference in the increase in the amount of
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Arginine and replication of herpes virus
477
DNA synthesized in cultures incubated in medium with or without arginine. It was
also shown that the decline in the rate of protein synthesis in infected cells continued
in the absence of arginine, though the extent of incorporation decreased in both
infected and control cultures. These experiments demonstrated that the synthesis of
enzymes necessary for the synthesis of viral DNA was not affected in the absence of
arginine. Thus, the inhibition of virus formation does not occur at the level of viral
DNA.
Utilization of arginine for the synthesis of herpes virions was demonstrated by
labelling infected cells with [14C]arginine and analysing the infected nuclei in sucrose
gradients. It was found that similarly to [14C]leucine, [x4C]arginine is incorporated in
the coat proteins of mature herpes virions. This indicates that arginine is essential for
the synthesis of the viral coat proteins and is incorporated into them. In the absence of
arginine, also the two radioactive protein peaks present in the sucrose gradient above
the virus peak do not appear. When arginine was added to deficient medium the
synthesis of proteins was resumed immediately. This indicates that the viral messenger
RNA was present in the cytoplasm, probably in association with the cytoplasmic
ribosomes, but lacked the ability to continue protein synthesis in the absence of
arginine. Addition of the essential amino~acid may be thought to initiate the synthesis
of nascent peptide chains and the viral coat proteins, which are transported from the
site of synthesis in the cytoplasm to the nuclei to form mature virions. Bonifas (I967)
similarly concluded that arginine is needed for the synthesis of coat proteins of
adenoviruses. The possible utilization of arginine in other metabolic processes is being
further investigated.
The results of the present experiments therefore indicate that the genetic information
required for the early enzymes necessary for the synthesis of DNA polymerase
(Keir et aL I966) as well as the inhibitors of cellular nucleic acid synthesis are not
affected in the absence of arginine. Cell damage is evident in the infected cells in the
absence of arginine. Arginine is necessary for the expression of the late viral functions
concerned with the synthesis of viral coat proteins. Thus in the absence of arginine,
the viral DNA molecules remain in the infected nuclei as DNase sensitive DNA. It
may be assumed that if the infected cells contain an enzyme capable of degrading
arginine, synthesis of herpes virions would similarly be prevented. Such a situation is
not known for herpes viruses but the infection of cells with Shope papilloma virus
(Shope, x933) causes the induction of the enzyme arginase which is virus specific
(Rogers & Moore, x963). Under similar conditions Ito & Evans (I96I) found only
naked viral DNA molecules and not virions in the tumour cells. Recently, it was
found that the synthesis of SV4o coat proteins in BSC1 cells was inhibited in the
absence of argjnine but the synthesis of the tumour antigen, which is an early viral
function, was not affected (Goldblum, Ravid & Becker, unpublished).
It is of especial interest that Subak-Sharpe et al. (I966) found evidence of arginyltransfer RNA in herpes infected cells and that this must be specified by the virus
genome. Herpes virus resembles adeno- and papovaviruses in its requirement for
arginine. All three groups replicate in cell nuclei, and are also characterized by their
ability to cause latent infections. The finding that in the absence of arginine, viral
DNA synthesis continues undisturbed, whereas the formation of virions is inhibited,
might explain the latent behaviour of these intranuclear viruses.
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478
Y. BECKER, U. OLSHEVSKY AND J. LEVITT
This work was aided by grant No. Ro5-TWoo3 from the National Institutes of
Health, Bethesda, Md, U.S.A. One of the authors (J.L.) is a Ph.D. student at the
Hebrew University, Jerusalem.
REFERENCES
BONIFAS, V. H. 0967). Time course and specificity of the arginine requirement for adenovirus synthesis. Arch. ges. Virusforsch. 20, 2o.
EAGLE,H. (I959). Amino acid metabolism in mammalian cell cultures. Science, N. Y. x3o, 432.
ITO, Y. & EVANS,C. A. (I96I). Induction of tumours in domestic rabbits with nucleic acid preparations from partially purified Shope papilloma virus and from extracts of the papillomas of domestic
and cottontail rabbits. J. exp. Med. xx4, 485.
KERR,H. M., SUBAK-SHARPE,H., SHEDDEN,W. I. H., WATSON,D. H. & WILDY,P. (I966). Immunological evidencefor a specificDNA polymeraseproduced after infectionby herpes simplexvirus.
Virology 3o, 154.
LEvrrr, J. & BECKER, Y. (1967). The effect of cytosine arabinoside on the replication of herpes
simplex virus. Virology 3x, IZ9.
OLSHEVSKV,U., LEVITT,J. & BECKER,Y. (I967). Studies on the synthesis of herpes simplex virions.
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ROIZMAN, B., BORMAN,C. S. & ROUSTA, M. K. (I965). Macromolecular synthesis in cells infected
with herpes simplex virus. Nature, Lond. 2o6, 1374.
RousE, H. C., BOMFAS,V. H. & SCHLESINGER,R. W. (I963). Dependence of adenovirus replication on
arginine and inhibition of plaque formation by pleuropneumonia-like organisms. Virology 20,
357.
SHARON, N. 0966). Stimulation of herpes simplex virus propagation in WISH cells by arginine.
Proc. 9th Microbiological Congress p. 5o8.
SHOEE, R. E. (I933). Infectious papillomatosis of rabbits. J. exp. Med. 58, 6o7.
SUBAK-SHARPE,H., SHEPHERD,W. M. & HAY, J. (I966). Studies on s-RNA coded by herpes virus.
CoM Spring Hath. Symp. quant. BioL 3 x, 583.
TANKERSLEY,R. W., Juy. (I964). Amino acid requirements of herpes simplex virus in human cells.
J. Bact. 87, 609.
(Received25 May 1967)
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