J. gen. ViroL (I98O), 48, I23-I33
123
Printed in Great Britain
Integration and Transcription of Virus D N A in Herpes Simplex
Virus Transformed Cell Lines
By D O U G L A S
F. M O O R E *
AND D A V I D T. K I N G S B U R Y
Department of Microbiology, College of Medicine, University of California,
Irvine, California 92717, U.S.A.
(Accepted I7 December I979)
SUMMARY
The physical state of the HSV I DNA present in two biochemically transformed
cell lines, a revertant line and a supertransformed cell line, was determined. These
cells all contained fragments of the HSV genome and the transformed and supertransformed cell lines expressed virus thymidine kinase. It was found that the virus
D N A in these cells was maintained in a complex state with approximately half of
the HSV DNA present in a covalently integrated state and the other half in a nonintegrated state. There was no major cell line difference in the distribution of integrated and non-integrated virus DNA. RNA transcripts representing 5 % of the
HSV I genome are present in each of these lines. This is more than is required to
code for the virus thymidine kinase present in the transformed and supertransformed
cell lines and suggests the presence of other virus proteins in these cells.
INTRODUCTION
Murine cells lacking the enzyme thymidine kinase (tk-) have been transformed to a tk +
phenotype by u.v.-irradiated herpes simplex virus types I and 2 (HSV I and HSV 2; Munyon
et al. 197I ; Davis et al. I974). The thymidine kinase expressed in these cells was of virus
origin as shown by electrophoretic mobility, thermolability and serological specificity
(Munyon et al. I972; Davis et al. I974; Thouless et al. I976). The levels of thymidine kinase
in these cells did not increase and decrease with the cell cycle as does the cellular thymidine
kinase (Lin & Munyon, I974) and the virus thymidine kinase was inducible by superinfection with tk- HSV (Lin & Munyon, I974; Kit & Dubbs, I977). A protein synthesized
early after infection by HSV is necessary for this induction to take place (Leiden et al. I976).
Solution hybridization experiments demonstrated that these biochemically transformed
cell lines contain fragments of the HSV genome ranging from 3 0o to 2o o~ and were present
in single and multiple copies (Kraiselburd et al. I975; Davis & Kingsbury, i976; Sugino
et al. I977). Revertants have been selected from biochemically transformed cell line I39 by
treatment with bromodeoxyuridine (BrdUrd) or aH-thymidine, and some of these lines
have been supertransformed by infection with u.v.-irradiated HSV (Chadha et al. J977).
Most revertant cell lines contained no detectable virus DNA; an exception was line t39
BrdUrd-2. The supertransformed lines contained virus DNA fragments representing I4 oJ
o
to 28 % of the genome (Sugino et al. I977)* Present address: Clinical Microbiology Laboratory AL 332, UCLA Hospitals and Clinics, Center for
the Health Sciences, Los Angeles, Calif. 9oo24, U.S.A.
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I2 4
D.F.
MOORE AND D.T.
KINGSBURY
Cell fusion experiments indicated that the virus DNA fragment coding for thymidine
kinase in biochemically transformed human cells is associated with two specific chromosomes (Donner et al. I977). This suggests that the virus DNA may be present in an integrated
state. It is known that HSV D N A can be found associated with the cellular genome after
infection of permissive and non-permissive cells (Biegeleisen & Rush, I976; Padgett et al.
I978).
The experiments presented here focus on two different aspects of the virus D N A present
in biochemically transformed cells. First, the physical state of the virus DNA in two biochemically transformed cell lines, a revertant and a supertransformed cell line, has been
determined. Experiments using the network technique and alkaline sucrose gradients
indicated that the virus DNA in these cell lines was present in a covalently integrated state
as well as a non-integrated state. Second, the fraction of the HSV I genome that is represented by R N A transcripts in these cell lines was determined. D N A - R N A solution hybridizations indicated that approx. 5 % of the virus genome was transcribed in all the cell
lines regardless of the size of virus DNA fragment present in the cell.
METHODS
Cells and viruses. Table I lists the designations and properties of the HSV r-transformed,
revertant and supertransformed cell lines used in this study. The properties of these cells
and the derivation of the parental cell lines have been described elsewhere (Munyon et al.
I97r; Chadha et al. T977). The media used for propagation of each cell type have been
described (Davis & Kingsbury, 1976). The revertant clone, r39 BrdUrd-2, was maintained
in media containing 3o/zg/ml BrdUrd. N H F cells are normal human foreskin fibroblasts
originating in this laboratory. HSV 1 hybridization probes were prepared from virions of
the JH strain of HSV I. This strain shows complete homology to the KOS strain used in
the transformation experiments (Sugino & Kingsbury, 1976).
Extraction of high molecular weight cellular DNA and the preparation of hybridization
probes. High mol. wt. DNA was extracted from cell monolayers using a phenol extraction as
described earlier (Davis & Kingsbury, I976 ) with modifications in the technique to avoid
shearing forces whenever possible. 6H-thymidine labelled HSV DNA for use as a hybridization probe was prepared as described earlier (Davis & Kingsbury, 1976). The HSV r
strain JH probes used in these studies had a specific activity of either 6-6 × I@ or 2"5 × lO6
ct/min//zg. All DNA used in hybridization reactions was sheared at o to 2 °C for 2 min
with a Branson sonifier fitted with a micro tip. This treatment gave fragment lengths of
350 nucleotides as determined by sedimentation in alkaline sucrose gradients.
Extraction of cellular RNA. Cell pellets were resuspended in 5 vol. of o-2 M-NaC1 and
I mM-EDTA and lysed by the addition of SDS to 1%. The lysate was sheared in a Waring
blender and incubated with 50/~g/ml of Proteinase K at 37 °C for I h. This suspension was
extracted with phenol, phenol-chloroform and chloroform, and the RNA was precipitated
by the addition of cold ethanol, storage overnight at - 2o °C and centrifugation at 4ooo g.
The RNA was resuspended in 5o mM-MgCI~ and Io mM-tris, pH 7"5, and incubated for r h
with 5o/~g/ml pancreatic DNase. This was followed by the addition of Proteinase K at
5o/tg/ml and incubation for I h. The solution was again extracted with phenol and chloroform, and then precipitated by the addition of cold ethanol as above. The RNA pellet was
resuspended in r mM-EDTA, the absorbance at 26o and 28o nm measured and the RNA
stored at - 7 o °C. All steps were carried out using sterile solutions and glassware.
Hybridization conditions. All D N A - D N A solution hybridizations were carried out as
described earlier (Sugino et al. ~977) except that the DNA in the reactions was denatured by
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H S V D N A in transformed cells
Iz 5
the addition of N a O H to IOO mM before addition of the sodium phosphate reaction buffer.
In every case the concentration of unlabelled ' d r i v e r ' D N A was adjusted to provide an
excess of cellular D N A to give a ratio of 5o to Ioo cellular genomes for each virus probe
genome.
Hybridizations with cellular R N A were carried out in zoo #1 vol. Approx. z × IO-s/zg
of SH-HSV D N A probe was denatured by the addition of N a O H to IOO mM and incubation
for Io min at room temperature. PB (sodium phosphate buffer containing equal molar
concentrations of monobasic and dibasic sodium phosphate, p H 6.8) was added to o'48 M
final concentration and cellular R N A was added to a concentration of 1.7 to 3"6 rag/m!.
A zero time sample was removed and the reactions were covered with mineral oil and incubated at 7o °C. All time points taken during the reaction were immediately diluted into
o.I2 M-PB containing o ' 2 % SDS. The single and double strands were separated on a
hydroxyapatite column. Single strands were etuted with o.~z M-PB with o . z % SDS and
double strands were eluted with o'3 M-PB. The amount of reassociation was calculated by
determining the percentage of counts in the sample that were in a double-stranded form at
each time point.
The results of the hybridizations were plotted using the equation:
I
fss~t~ - KC0t + I,
where I/fss~,~, the fraction of probe remaining single stranded at time t, equals C0/C
(Britten & Kohne, I968 ). By plotting I/fss against Cot the slope of the line is equal to K.
The increase in reaction rate caused by the presence of virus sequences in transformed cell
D N A results in an increase in the slope of the plot. By comparing the slope of a reaction
containing transformed cell D N A to the slope of a reaction containing a known amount
of HSV D N A (self reaction) the amount of virus D N A present in transformed cell D N A , or
fractions thereof, can be calculated (Gelb et al. I97I ). The curves of the reactions which
contain transformed celt D N A are not linear throughout the reaction because only a
fragment of the virus genome is present in the transformed cell (Davis & Kingsbury, I976;
Sugino et al. I977). Therefore the initial slope, corresponding to the driven part of the
reaction, was used for quantification of the amount of virus D N A present in all cases.
Network analysis. The formation of D N A networks of high mol. wt. cellular D N A was
similar to the method described by Bellett 0975). High mol. wt. D N A was diluted to 1"25
m g / m l and denatured by the addition of N a O H to Ioo raM. PB was added to o'48 M and the
solution reannealed to a Cot of Ioo mol. s/l at 60 °C. The solution was diluted twofold in
0"48 M-PB at 60 °C and centrifuged in a SW5o'I rotor for zo min at 350o0 rev/min at
~o °C. The supernatant was removed, dialysed against distilled water and lyophilized before
suspension in I mM-EDTA and shearing. The network pellet was resuspended directly into
I mM-EDTA and sheared.
The amount of cellular D N A contained in the network and supernatant fractions was
determined by measuring the A260 of the two fractions. The amount of HSV D N A in each
fraction was determined by D N A - D N A solution hybridizations as described above. The
network experiments included a trapping control consisting of a sample of the networking
reaction to which unsheared ZH-HSV D N A was added. After the network procedure, the
supernatant and network of the control reaction were placed in scintillation vials and counted. The percentage of counts found in the network was considered the trapping value and
was used to correct the data as presented in Results.
Alkaline sucrose gradients. Alkaline sucrose gradients (5 to 2o ~y,,), containing o'o3 M-tris,
O'OO5 M-EDTA, l-o M-NaCI and o'3 M-NaOH, were formed in SW27 tubes. Five × IO5 cells
were layered on a gradient followed by ~ ml of lysis buffer containing o. 5 M-NaOH and
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I26
D.F. MOORE AND D.T. KINGSBURY
Table I. Cell lines and their properties
HSV I DNA present*
A
Cell line
Type
•
% genome
139
I7t
139 Brd Urd-2
t39 RI2S3
Transformed, tk+
Transformed, tk +
Revertant, tkSupertransformed, tk +
9
I7
9
28
,
,
Copies/ha;loid genome
Source
2'5"1"
x'5
I'o
3"0
W. Munyon
W. Munyon
K. Chadha
K. Chadha
* Sugino et al. (I977).
"]" Davis & Kingsbury 0976).
o.oI M-EDTA. Control gradients contained cells grown in media containing 0.02 m C i / m l
a2PO4= and unsheared aH-HSV D N A as a trapping control. The gradients were incubated
for I8 h at o °C and centrifuged for 4 h at 27000 rev/min. The control gradients were
collected into 32 fractions and the acid-precipitable radioactivity was counted in a sample
of each. Thirty-three gradients containing unlabelled cells were collected into a top, middle
and bottom fraction and the fractions were pooled, diluted l : I with distilled water and
neutralized with HC1. The top and middle fractions contained very small amounts of D N A ,
I5o/zg of Balb/C mouse D N A was added to each as a carrier. All fractions were sheared
and passed through a large hydroxyapatite column equilibrated to 60 °C to concentrate the
DNA. The column was washed with Io ml of o'oo48 M-PB and the D N A was eluted with
Io ml of o'48 M-PB. The D N A was dialysed against distilled water, lyophilized and the
amount of HSV D N A contained in each fraction was determined by D N A - D N A solution
hybridizations as described above.
RESULTS
Network analysis of integrated virus DNA
The network technique, as described by Varmus et al. (I973) and Bellett 0975), was used
to determine the physical state of the HSV I D N A contained in two biochemically transformed cell lines, a revertant of one of these cell lines and a supertransformed cell line
(Table ~). High tool. wt. D N A from these cells was alkali denatured and reassociated to a
Cot of IOO tool. s/t. At this Cot the repetitive sequences present in the cellular D N A
reassociated forming a network containing 66 to 85 o/o of the D N A present in the reaction.
This network, which should contain any integrated virus D N A , was separated from the
supernatant by centrifugation as described in Methods.
For each network experiment, five quantitative hybridizations with high sp. act. SHHSV D N A were carried out. The network and supernatant fractions from a networking
reaction were hybridized as was an untreated sample of the same DNA. In addition, a self
reaction, containing only 3H-HSV probe and D N A known to be free of HSV sequences,
and a reconstruction reaction, containing a known amount of virus D N A , were run at the
same time.
The reaction kinetics of all four networking experiments presented in Fig. I to 4 indicate
that there was virus D N A present in both the network and supernatant fractions of the
D N A from each cell line. In order to keep the ratio of cellular to probe D N A high enough
to ensure maximum sensitivity variable fractions of the network supernatant and pellet
were used. In Fig. I to 4 the apparent rate of hybridization of the network supernatant is
very high; however, this fraction constitutes the supernatant from the entire networking
reaction uncorrected for the total number of cells from which it was obtained. The hybridization to the networking pellet was done on approx. 25% of the entire sample, so that
i
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H S V DNA in transformed cells
2.2
I27
I
I
I
2
4
6
I
I
I
IS
12
14
2.0
1-5!
1.8
1"4
$
•
•
1-3
~, t-6
1.2
1.4
1.1
1.2
1
2
3
4
5
6
0
7
E Co t X 10 -3
8
10
E Co t x 10 -3
Fig. 2
Fig.
Fig. t. Kinetics o f hybridization of aH-HSV D N A with D N A f r o m a n e t w o r k reaction of 139 cell
D N A . T h e reassociation of >8 x io -a # g of aH-HSV D N A (sp. act. 6.6 x 1@ ct/min//~g) was determ i n e d in the presence of 450 #g o f one of the following: I39 D N A ( • ) , I39 network D N A • ) ,
I39 s u p e r n a t a n t D N A (O), B a l b / C D N A (IS]) a n d B a l b / C D N A plus 5"4 x io -a/zg of H S V D N A
( © ) in a total reaction of 5o0 #1. T h e Cot values have been corrected for salt concentration a n d
represent Cot equivalents (E Cot) in o'I 2 M-PB.
Fig. 2. Kinetics of hybridization o f 3H-HSV D N A from a network reaction of I71 cell D N A . T h e
reassociation o f 1.8 x lo -a # g of 3H-HSV D N A (sp. act. 6.6 x ion ct/min//zg) was determined in the
presence of 45o # g o f one o f the following: 171 D N A ( • ) , 171 network D N A ( • ) , 171 s u p e r n a t a n t
D N A ( $ ) , B a l b / C D N A ([]) a n d B a l b / C D N A plus 5"4x lo-~,u,g of H S V D N A ( ~ ) in a total
reaction of 5o0/zl. T h e Cot values have been corrected for salt concentration a n d represent Cot
equivalents in o" x2 M-PB.
2.2
I
I
I
I
I
I
I
2.0
1,2(
1.8
o
~1"6
~ 1.1(
IlL
1.4
1"2
l
1.0
2
4
6
8
E Cot X 10 -3
Fig. 3
10
12
1'0
Q
2
4
6
8
10
E Co t × 10-3
12
14
Fig. 4
Fig. 3. Kinetics of hybridization of aH-HSV D N A with D N A from a network reaction of I39
BrdUrd-2 cell D N A . T h e reassociation o f 1.8 x l o -z/zg of aH-HSV D N A (sp. act. 6 6 x i o e c t / m i n /
#g) was determined in the presence of 450/~g of one of the following: 139 BrdUrd-2 D N A ( • ) , 139
BrdUrd-2 network D N A (m), 139 BrdUrd-2 s u p e r n a t a n t D N A (O), B a l b / C D N A ([B) a n d B a l b / C
D N A plus 7.2 × io -3 # g of H S V D N A ((5) in a total reaction o f 500 #1. T h e Cot values have been
corrected for salt concentration a n d represent Cot equivalents in o.Iz M-PB.
Fig. 4. Kinetics of hybridization of 3H-HSV D N A with D N A f r o m a network reaction of 139 RI 2S3
cell D N A . T h e reassociation o f ].8 x io -3 # g of ~H-HSV D N A (sp. act. 6.6× IOn c t / m i n / # g ) was
detern,tined in the presence o f 45o # g o f one o f the following: 139 RT2S3 D N A ( • ) , 139 R A I 2 S 3
network D N A (m), I39 R~2S3 s u p e r n a t a n t D N A (O), h u m a n placenta D N A (E3) a n d h u m a n
placenta D N A plus 5"4 x Io -3 # g of H S V D N A ( © ) in a total reaction of 500 #1. T h e Cot values have
been corrected for salt concentration a n d represent Cot equivalents in o.I 2 M-PB.
5
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VIR 4 8
16
~28
D.F.
MOORE AND
D.T.
KINGSBURY
Table 2. Amount of integration of H S V DNA in transformed, revertant and
supertransformed cell lines
Cell type
Biochemically transformed
Revertant
Supertransformed
Cell line
% virus
% total
DNA inte- HSV DNA
grated recovered
(139
] 139 (alkaline sucrose)
50*
43
I4O
96
L 171
139 BrdUrd-2
139 R 1253
62
45
61
1 I0
I 15
120
* The % virus DNA integrated was calculated from the equation of Bellett (I975) which states:
I=
Vn-Cn
1-(Ds -I- Cn)
where I is the fraction of the virus DNA integrated; Vn is the fraction of the virus DNA found in the
network fraction; Ds is the fraction of the cellular DNA in the supernatant; and Cn is the fraction of
trapping in the control network. The total recovery of virus DNA is a comparison of the total amount
recovered in each fraction divided by the amount of virus DNA in an equivalent sample.
several correction factors must be applied. Calculations to quantify the amount of virus
D N A present in an integrated and non-integrated state were done in three parts. First,
the amount of virus D N A present in each hybridization reaction was calculated by comparing the initial rate of the reactions to the initial rate of the self reaction. The reconstruction reaction was used to ensure that the hybridizations and calculations were accurate.
Second, the amount of virus D N A detected in the network and supernatant fractions was
corrected to account for the relative fractional amount of the original D N A present in each
fraction.
In order to be able to compare experiments two other factors must be taken into account
when calculating the amount of integration of virus DNA. These factors are the amount of
trapping of non-integrated sequences into the network and the amount of cellular D N A that
actually enters the network. To correct for these factors an equation derived by Bellett
(I975) was used to calculate the amount of virus D N A integrated into the cellular genome
in each case. In all experiments the amount of trapping was below 5 %. The results of
multiple experiments analysed by this method are contained in Table 2. In all cases approx.
5o% of the virus D N A was associated with the ceIlular D N A and 50 % was present in a
non-integrated state. All network reactions reported were done using alkaline denaturation
of cellular D N A before the networking reaction in contrast to the heat denaturation used
by Varmus et al. (I973) and in part by Bellett (1975). Thus, the association of virus D N A to
cellular sequences must be by covalent linkages.
Alkaline sucrose gradient analysis of integrated virus DNA
To ensure that the results from the network experiments were not due to some undefined
aspect of HSV integration a second technique was used to analyse the integration of HSV
D N A in cell line I39. The alkaline sucrose gradient analysis used is based on the technique
of Varmus et al. 0976). Five × Io 5 139 cells were layered on alkaline sucrose gradients and
lysed by the addition of o'5 M-NaOH followed by incubation overnight at o °C before
centrifugation. This results in a very gentle extraction of the D N A present in the cells
because no shearing forces are involved; hence the cellular D N A remains in a very high
tool. wt. form which can be separated from smaller non-integrated virus sequences. Any
covalently integrated virus D N A should remain attached to the high tool. wt. cellular D N A
during centrifugation and be found associated with this D N A in the bottom fractions of the
gradient.
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H S V DNA in transformed cells
Top
?
Bottom
2~
o
i
X
.=.
E
"7.
Middle
I2 9
1o
I
-50
- 40
Z
IL
~
-30
",
-
>
20
-10
~.__/''r
10
....................
• ...........................
20
i
30
Fraction
Fig. 5. A 5 to 2 0 °/o alkaline sucrose gradient was layered with aH-HSV D N A and 5 x lO6 I39 cells
grown in the presence of 32p. After addition of lysing buffer and incubation at o °C for 18 h, the
gradient was centrifuged for 4 h in a SW27 rotor at 27000 rev/min, collected and the acid-precipitable radioacivity counted in each fraction. - - - , z2P-labelled I39 D N A : - - - , 3H-labelled
HSV DNA. Thirty-three gradients were layered with 5 × IO5 I39 cells, lysed and centrifuged as
above. The top, middle and bottom fractions were collected and pooled, and the D N A concentrated
from each as discussed in Methods. The D N A from each fraction was hybridized with aH-HSV
D N A and the distribution of virus D N A in the gradients is represented by the bars superimposed
on the gradient profile.
The profile of a control gradient containing a2P-labelled I39 cells and 3H-HSV DNA
shows that the high mol. wt. cellular DNA sediments rapidly to the bottom of the gradient
while the added virus D N A remains in the top fractions (Fig. 5)- The 32p peak at the top of
the gradient consisted of acid-insoluble counts. This material which absorbs very little at
26o nm has been observed by others and most likely is composed of phospholipids and
phosphoproteins although it may contain a small amount of mitochondrial DNA. This
control gradient indicates that with the amount of cells lysed on the gradients no trapping
of non-integrated D N A takes place.
Multiple gradients with unlabelled I39 cells were centrifuged and collected into top,
middle and bottom fractions. The DNA was extracted from each of these fractions and the
amount of virus D N A present in each fraction was determined by hybridization to the aHHSV probe. The percentage of the virus D N A present in I39 cells found in each fraction is
represented by the bars superimposed on the profile of a control gradient in Fig. 5. The
hybridizations indicate that 43 ~o of the virus DNA in I39 cells is associated with high tool.
wt. cellular sequences while 32 ~ is non-integrated and remains in the top fractions. The
25 % of the virus DNA which was found in the middle fraction could have been due to
mixing of the gradients during collection, preferential integration of some virus sequences
into small cellular sequences, or rapid sedimentation of a non-integrated supercoiled
circular form of the virus DNA.
These results, which show that 43 % of the virus D N A was integrated in I39 cells, are
very close to the results from the networking experiments. This is an indication that the
two techniques are measuring the same physical parameter and that the network analysis
is as accurate as alkaline sucrose gradients for the determination of the physical state of
virus DNA.
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5-2
130
D.F. MOORE AND D.T. K I N G S B U R Y
15
1
1
15~
I
I
o
Nsl0
•
~s
0
0
f
I"1
tA
Io
I
1
I
I0
I
1
E Cot x 10-3
n
I
10
E Cot X 10-3
Fig. 6
Fig. 7
Fig. 6. Kinetics of hybridization of 3H-HSV DNA with transformed cell RNA. The reassociation
of 7"I x io -3/zg of ~H-HSV DNA (sp. act. 2'5 x i@ ct/min/#g) was determined in the presence of
7IO/zg of t39 RNA (0) or 139 BrdUrd RNA (©) in a reaction volume of 20o #1. The self reaction
has been subtracted from each time point. The Cot values have been corrected for salt concentration
and represent Cot equivalents in o.12 M-PB.
Fig. 7. Kinetics of hybridization of 3H-HSV DNA with transformed cell RNA. The reassociation
of 7"~ x io -~ #g of 3H-HSV DNA (sp. act. z5 x ~o0 ct/min//,~,g) was determined in the presence
of 7Io#g of I7I RNA ([]) or 139 RI2S3 RNA ( I ) in a reaction volume of 2oo#1. The self
reaction has been subtracted from each time point. The Cot values have been corrected for salt
concentration and represent Cot equivalents in o.12 M-PB.
Table 3- Percentage of the HSV I genome transcribed in biochemically transformed,
revertant and supertransformed cell lines
Cell line
% HSV-I genome
transcribed
(range)
% of HSV-I
virus fragment
transcribed
139
5 (z-8)
55
I7I
139 BrdUrd-z
I39 RI2S3
5 (3-9)
4 0-8)
5 (2-II)
29
46
18
Detection of virus RNA transcripts
Total cellular R N A was extracted from 139 , J39 BrdUrd-2, r39 RI2S3 and I7I cells,
and hybridized with a small a m o u n t o f 3 H - H S V D N A as described in Methods. The kinetics
of reaction are shown in Fig. 6 for R N A from 139 and 139 BrdUrd-2 ceils a n d in Fig. 7
for R N A from 17~ and T39 RI2S3 cells. The reactions shown are typical. Because of the
small a m o u n t of reaction the hybridizations were repeated five to eight times to ensure an
accurate estimate of the extent of reaction. In addition several R N A concentrations were
examined in order to obtain a true plateau value. The range of values and the average extent
of reaction for R N A from each cell line is presented in T a b l e 3- In all cases the reassociation
of a self-reaction c o n t a i n i n g N H F R N A was subtracted from the experinaental values. I n
each case, the plateau of the reaction was reached by an R N A Cot of 50o during which time
the probe self-reaction was less than 1%.
The results indicate that each cell line contains approximately the same fraction of the
HSV genome transcribed into RNA. In the case of 139 and ~39 BrdUrd-2, 5 c~,, of the genome
represented as R N A could be the total coding capacity of the virus D N A fragment present in
the cells, assuming transcription of only one strand of the virus D N A . Cell lines I7t and
I39 R12S3 contain larger fragments of the virus genome and could theoretically have
larger a m o u n t s of virus R N A transcribed. The results here indicate that there was a very
similar a m o u n t of the virus genome present as R N A transcripts in every cell line and that
the level of transcription is i n d e p e n d e n t of virus D N A fragment size.
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H S V D N A in t r a n s f o r m e d cells
I3 T
DISCUSSION
The maintenance of virus DNA in an integrated state in the genome of cells transformed
by DNA tumour viruses has become a common phenomenon. Cells transformed by human
and avian adenoviruses contain virus DNA present in a covalently integrated state (Doerfler,
I968; Bellett, i975) as do cells transformed by SV4o (Sambrook et al. I968; Botchan et al.
I976; Ketner & Kelly, I976). Analysis of the physical state of the virus DNA present in
cells transformed by herpesvirus has become a complex problem. Cells transformed by
Epstein-Barr virus (EBV) can contain linear integrated virus sequences or non-integrated
circular forms of the virus DNA. Some cell lines, most notably Raji, seem to contain both
integrated and non-integrated forms of EBV DNA (Adams et al. 1973; Adams & Lindahl,
T975; Lindaht et al. ~976; Adams et al. T977).
Integrated virus DNA is not present in transformed cells alone. Virus DNA can become
integrated during lytic infections with SV4o (Hirai & Defendi, I972), adenovirus (Burger &
Doerfler, ~974; Tyndall et al. I978), and HSV (Biegeleisen & Rush, I976); likewise, HSV I
DNA can become integrated within I6 h after infection of non-permissive XC cells (Padgett
et al. I978 ).
The experiments reported here demonstrate that cells biochemically transformed by
HSV I contain integrated virus D N A . However, it was also demonstrated that, in every case,
non-integrated virus DNA is also present. Approximately half of the virus DNA was in an
integrated state and half in a non-integrated state in every cell line examined. Since all the
procedures to assay integration used in these experiments employed alkali denaturation
conditions, the integration measured must be due to covalently integrated virus sequences.
The two major techniques used in these experiments, network formation and alkaline
sucrose density gradient centrifugation are known to measure the same physical parameter
as a caesium chloride gradient, the other widely used technique for studying virus integration (Tyndall et al. I978). Network formation and alkaline sucrose have the extra advantage of much less non-specific trapping of the virus DNA in the high mol. wt. cellular
DNA. In all cases the cells were subjected equally to both procedures and the amount of
agreement between them is remarkable.
Unlike an earlier study of the integration of an HSV-I derived gene (Pellicer et al. I978)
the cell lines studied here were transformed with u.v. inactivated virus particles and not a
purified and defined sequence of the HSV genome. The lack of knowledge about the exact
fragment present in these cells makes the use of some of the techniques reported earlier
(Pellicer et al. r978) for the study of integration very difficult.
Because of the small amounts of virus DNA present in the cell lines examined a quantitative analysis of the networking experiments, as described by Bellett (I975), was used.
This analysis takes into account the amount of trapping of non-integrated virus sequences
into the network and required a control networking to be carried out with each networking
experiment.
In contrast to SV4o and adenovirus, where a large amount of information concerning
the virus transcripts present in transformed cells is available, little is known of the virus
RNA transcripts present in HSV transformed cells. Reports by Collard et al. (I973) and
Jamieson et aL (1976) have established the presence of virus RNA in HSV transformed cells.
Since cell lines biochemically transformed by HSV invariably contain only a fraction of the
virus genome (Davis & Kingsbury, I976; Sugino et aL 1977) RNA transcripts representing
only a portion of the virus genome are expected.
The experiments reported here indicate that approx. 5 % of the HSV genome was transcribed in every cell line examined. A transcript of this size indicates that the entire fragment
of virus DNA present in I39 and 139 BrdUrd-2 cells is being transcribed assuming transcription of only one strand of DNA. Cell lines i7i and I39 RI2S3 contained larger virus
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I32
D.F.
MOORE AND
D.T.
KINGSBURY
DNA fragments and could have RNA transcripts of a more extensive region of the genome.
The results from these experiments, however, indicate that only a 5 % fragment was transcribed in these cells also.
The limited amount of transcription detected in this case could be due to several factors.
A portion of the transcripts could be present in very low concentrations and not be detectable
or the R N A could be rapidly degraded. It is also possible that there is some type of transcriptional control operating on the virus DNA sequences in the cell.
Thymidine kinase, with a mol. wt. of 44ooo requires 0"85 % of the coding capacity of the
HSVgenome (Wigler et al. I977). The presence of RNA transcripts greatly exceeding this
amount suggests the presence of other virus proteins in biochemicaUy transformed cells.
Initial observations made in this laboratory indicate that 6o to 75 % of the virus RNA
transcripts present in the biochemically transformed cell lines 139 and 139 RI2S3 are
homologous to the immediate early (alpha) RNA transcripts present during a permissive
infection with HSV. Thus it is possible that proteins present early in productive infection
with HSV are also present in transformed cells. It may be the presence of these proteins,
associated primarily with virus replication that leads to what appears to be a dynamic
process within the cell in contrast to the stable integration of the tk gene fragment reported
previously (Pellicer et al. 1978).
The able technical assistance of Valerie H. Williamson is most gratefully acknowledged.
This work was supported by contract number NO I CP5356o from the Virus Cancer Program,
National Cancer Institute.
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