Microbiol. I mmunol. Vol. 23 (1), 1-15, 1979
Spontaneous Production of a C-Type RNA Virus Cell Line Derived from Rat Glioma Koichi
IGARASHI,t
Reiko
SASADA, and
†Biological
Yasuhiko
Yukio
Chemical
Yoshio
KOZAI,
SUGINO
Research Laboratories,Central
Takeda
NIIYAMA,
in a
Research
Industries,
(Received for publication,
Ltd.,
Division,
Osaka
March 8, 1978)
Abstract The spontaneous production of a rat C-type RNA virus (ACV) in a cultured cell line (AC cells) established from a chemically induced rat glioma was studied. The characteristics of ACV were : morphology typical of C-type RNA virus; buoyant density of 1.15 g/ml in a sucrose density gradient; RNA directed DNA polymerase activity; viral core with a density of 1.28 to 1.30 g/ml ; 70S RNA with dimer structure; and structural protein composed of mainly four polypeptides. Kinetical analysis of DNA-DNA hybridization revealed that DNA sequences homologous to DNA transcripts of RNA of ACV were present in rat cells. RNA directed DNA polymerase of ACV partially cross-reacted with antiserum to the polymerase of Rauscher murine leukemia virus. These data suggest that ACV is an endogenous C-type RNA virus of rat origin.
Recent studies have indicated that C-type RNA viruses are found in normal and tumor cells of many vertebrates. In the rat, several lines of experimental evidence for the presence of C-type viruses were obtained. That is, demonstrable C-type viruses of suspected rat origin were observed in rat hepatomas (21, 26, 43), rat mammary tumors (8, 12, 15), and normal or transformed rat cell lines (9, 13, 17, 29, 33-35, 38). Other investigations (2, 27, 28) have shown that nonproductive rat cells transformed with murine or avian sarcoma virus released endogenous C-type virus as a rat leukemia pseudotype when treated with halogen derivatives of uridine. Moreover, endogenous rat C-type virus with properties of helper virus was also isolated from stocks of murine sarcoma virus which had been passaged in rat (1, 39). However, most of the rat viruses hitherto reported have not been fully characterized. This communication virus virus
describes
the spontaneous
in a cell line established from a rat glioma, is an endogenous rat C-type virus. MATERIALS
Cells and viruses. from
Sprague-Dawley
The rat
origin glioma
AND
and
1
and
presents
of C-type evidence
RNA
that
this
METHODS
properties
induced
high production
of AC cells, a cell line
transplacentally
by
established
ethylnitrosourea,
2
K. IGARASHI
have
been
described
a C-type was
virus
used
(RLV), by and was
a
R17 was
line,
line (25) obtained by
was
R17
10%
liver
cell
were
fetal
199
hr
cells
at
cultured
at
near
for
centrifugation
at
The
of
tion
was
precipitated serum
filter
(Whatman
Beckman
virus
son
for
of
et
al
or
was assay
by
addition as
acetate,
dithiothreitol,
0.4
oligo(dT)io,
washed
P-L
DNA
and
dried,
England
Nuclear)
reduced
sucrose
rpm
for
20
hr
concentration of
of
each
and
(TCA)
using
fracthe
100
through
rest ti.g
of
a glass-fiber
radioactivity
mm
virus
counted
by
in
the
reaction 0.005m
polymerase the
a
mixture
(0.1
magnesium
Pure
activity
method
of Aaron-
ml)
contained
acetate,
Chemical
Ind.,
0.001
Ltd.),
polyadenylate•oligodeoxythymidylate or
particles
cell
(3.5
DNA
measured
(Wako
triphosphate 3H-deoxyguanine
from
directed
was The
Inc.)
mm
template, 0,01
the
by sucrose
linear
portion
filtered
clarified
a 60%
activity,
acid
RNA
KCl,
X-100 of
and
purified
to
polymerase
and
was
the A
were Radio-
concentrated
25,000
refractometry.
.
The
15-60%
and
at (14).
diameter)
onto
rotor,
was
in
al
gradient
harvested
a
50.1
DNA
P-L
New
were
mg/ml
fluid et
mCi/mmol,
sucrose
collected
precipitate
0.06m
units/ml
3H-thymidine Co.) or 0.1
added
1.2
culture
was
onto
SW
template
Triton
0.1 mm Chemical
without
acid,
density
mm
fluid
trichloroacetic
modification. 7.8),
plate, Pure
ed
(100
20%
were
polymerase.
exogenous
Biochemicals,
and
sucrose
(54
layered L3,
The
guanylate(poly(rCm)•oligo(dG)12_18,
concentrated
of puls
counter.
0.1% A260
folic
Duesberg
supernatant
through
by
cold
carrier.
slight (pH
min
the
directed
of
with
100 ƒÊg/ml (60%)
from of
on
culture
in
was
RNA
and
medium of
method
dishes
The
determined of
directed
with
hr.
fractions
and
cultures
supplemented
5A
1-4C-uridine
model
kidney
(HEL) All
serum
harvested
the
plastic
virus
90
monkey cells
medium
centrifugation
material
without
Tris-HCl
manganese
for
scintillation
RNA
(3)
24 the
rpm
GF/C),
with
0.04m
for
and virus
was established (RSV), B-77,
Laboratories.
6•@ƒÊg/ml
virus, to
5 ƒÊCi/mlof
and
the
FCS,
The
Falcon
(Spinco
albumin
liquid
Assay in
of
fraction
used
bovine
two
lung
bovine
ACV
(25).
isopycnic
centrifugation,
each
was
in
centrifuged
After
sucrose
and
25,000
20%
virus.
green
McCoy's
Kanamycin
according
concentrated
and
4 C).
of
10 min,
at
gradient
with
purified
presence
in
line virus
African
or
as leukemia
embryonic Flow
produce
herein
murine
essential
propagated
Amersham)
rpm
cushion.
minimum
of
an
from
Laboratories)
purification
confluency
the
obtained
Flow
to
was used. This Rous sarcoma
Human
Eagle's
were
was
Centre
3,000
were
incorporation
in
chemical
line
spontaneously
Rauscher
cells,
Hakura.
cells
is referred
prepare
CV-1
A.
100 ƒÊg/ml
and
intervals,
14C-Uridine AC
Owada. Dr.
supplemented
and
Preparation 8-16
is producing RLV Dr. A. Ishimoto.
cells
(20%)
bactopeptone
To
AC
virus
that from
(FCS,
R17
The
This
study.
in
serum
(24).
origin.
this
grown
calf
medium
elsewhere rat
M. by
Kanamycin.
of
Dr.
gifted
chimpanzee
except
detail
throughout
supplied
cell
in
of suspected
ET AL
culture
poly
(poly(rA)
2'-O-methylcytidylate•oligodeoxy-
Biochemicals,
Inc.)
(dTTP, 50-500 triphosphate (1-20
t,tg
medium.-
concentrations Ci/mmol)
M
0.005m
viral
When of
and
of
0,01%,
as
protein), the
3H-dTTP
exogenous
tem-
mCi/mmol, Daiichi (dGTP, 50 mCi/mmol,
assay and
respectively,
which was Triton and
were
performX-100 0,1
mm
PRODUCTION
OF RAT
C-TYPE
3
VIRUS
deoxyribonucleotide triphosphates (dATP, dGTP and dCTP) were added to the reaction mixture. Incubation was carried out at 37 C for 60 min. DNA synthesis continued linearly for at least 90 min under the conditions employed. The reaction was stopped by addition of cold 10% TCA containing 10mm sodium pyrophosphate and 10 mm monobasic sodium phosphate, and the DNA in the precipitate was collected on a glass-fiber filter. After 5 washings with cold TCA, the radioactivity of the precipitate was determined. Inhibition of RNA directedDNA polymeraseof ACV by antiserum to the Polymeraseof RLV.
Viruses
were
equilibrium directed
DNA
as
of
protein
viral
by
and
Triton
for
of
activity
of
the
the
(a
gift
0 C
60
was
serum
at
for
culture
Y.
at
measured
Ikawa)
After
virus
purified
to
im-
Ltd.) 1 p_g
with 10
10
ti,1 of
incubation,
poly(rCm)
RNA
containing
with
the
by
the
Whatman
disrupted
incubated
C.
using
was
of
were
purified to
(DE22,
(10 ƒÊl)
were 0
and
antiserum
cellulose
albumin
and
min
fluids
goat
Dr.
Dilutions
bovine
15 min
from
a DEAE
(16).
of
IgG
sample
tissue The
through
Fahey
10 ti,g
X-100
concentrations
RLV
fraction
described
spent
centrifugation. of
G(IgG)
column
from
gradient
polymerase
munoglobulin
0.1%
concentrated
sucrose
various
the
•oligo(dG)
as
ti.1 of
enzyme
exogenous
template. Preparation
of
extracted
and
Tsuchida
et
The RNA
18
hr
and
sequences to
1.4
mg
using by
,Vleasurement
(6).
probe C. the
with
ACV
of rat
of
DNAs
sources
of
as
were
described
by
DNA
since
2 mm
per
Tm
et
ml
al
(midpoint
cellular
remaining
at
EDTA, 65
four
presence
proceed
about
of 20
sonically
C (7).
was of
the
specific
activity
of
3H-
to
70S
by DNA
of
most
RNA
of
ACV
excess
of
3H-DNA.
using
an at
performed
24,000
extent
in
contain
heating
was
SDS,
The
DNA activity
treated
nuclease
C
specific
times
denatured
0.05%
37
(EDTA),
seemed 70%
cellular
of
at
deoxyribonucleotides
obtained
and
S1
cpm
hybrid
of
ARTEK 100
C
in
0.01
Kogyo
for M
3H-DNA
formation
(Seikagaku
was
was
Co.)
(4)
(41). of
DNA heated
the
the
with
to
single-strand-specific
endogenous
the
3H-labeled
The from
were 7S
probe
the
in to
The
(40).
hybridization
3H-DNA
allowed
(SDS).
of
DNAs
by
ACV
M ethylenediaminetetraacetate
thus
about
prepared
was
calculated
RNA,
m NaCl,
were of
as
after
was
Green
transcripts
size
Tsuchida
Aliquots amount
Cellular
various
purified
sulfate and
All
a mean
of cellular by
described
65
in
0.75
0.01
incorporation
RNase
7.4),
of
DNA,
hybridization.
(pH
probe
reaction
dodecyl
The
Hybridization
measured
DNA.
of
using
The
Tsuchida
cpmhig
present
to
Tris-HCl
and
by
Pancreatic
DNA-DNA
at
sodium
equimolar
dismembrator 10 min.
viral lines
DNA
addition
reaction.
resistant
DNA
by
0.5%
assuming enzyme
cell
reaction
D(6).
stopped
of the
as
viral
as described was 2 .0 •~ 107
dTTP,
3H-labeled and
polymerase
actinomycin
and
M NaCl
and
and
tissues
(42).
DNA of
extracted 3H-DNA
this
DNA from
3H-thymidine-labeled
100 ƒÊg/ml
0.1
al
directed
for
cellular
purified
melting was
for hybrid
curve).
carried
5 min was
out in
0.75
determined
The to m
hybridization
a
Cot
of 8 •~
NaCl
at
by
digestion
of 103
various
ACV
mol •~
temperatures with
3H-
sec/liter
S1 nuclease
4
K. IGARASHI
ET AL
Isolationof the viral coreof ACV. The viral core was obtained by centrifugation of 3H-labeled virus through a layer of 1.0% Nonidet P-40 (Shell Chemical Co.) on top of a 10 to 70% linear sucrose gradient according to the method of Stromberg (37). Isolation and velocity sedimentation of viral RNA. Purified virus, labeled with 60 of 3H-uridine for 16 hr, in NET buffer (0.1 M NaCl, 0.001 M EDTA, 0.01 M
μCi/ml
Tris-HC1, pH 7.2) was lysed by addition of 1.0% SDS and 0.05% diethylpyrocarbonate (Nakarai Chemicals, Ltd.), and incubated at 25 C for 15 min. The RNA was extracted with water-saturated phenol and chloroform-isoamyl alcohol (24: 1) (41) and precipitated overnight with ethanol at -20 C. Viral RNA, collected by centrifugation and dissolved in NET buffer, was mixed with ribosomal RNA prepared from rat liver and centrifuged in a 10-30% sucrose gradient in NET buffer containing 0.5% SDS for 2 hr at 40,000 rpm in a Spinco SW 50.1 rotor at 20 C. After centrifugation, fractions were collected from the bottom and analyzed for the optical density (A,260)and the radioactivity in the acid insoluble fraction. For analysis of 30-40S RNA, the fractions containing viral 60-70S RNA were pooled, precipitated with ethanol and centrifuged. Then, RNA was dissolved with NET buffer, heated at 70 C for 3 min and centrifuged. The sedimentation coefficient of viral RNA was estimated from the positions of 28S and 18S ribosomal RNAs added as the reference standards in the identical gradient. Acrylamidegel electrophoresis. Viral material was precipitated with acetone and electrophoresed in 13% polyacrylamide gels containing 0.1 % SDS by a modification (32) of the procedure of Maizel (31). Human gamma globulin fraction, bovine albumin, ovalbumin, and beef pancreas chymotrypsinogen A (Schwarz/ Mann Co.) were used as molecular weight standards. Electron microscopy. The AC cells, in the exponential phase of growth, pelletted by centrifugation and purified virus were fixed in 2.5% glutaraldehyde and 1% osmium tetroxide and embedded in epon. Thin sections prepared were stained with uranyl acetate and lead citrate (32). Purified virus was also negatively stained. with 1% uranyl acetate on a glow-discharged carbon coated grid (22). The spreading of ACV RNA, prepared as described by Kung et al (30), was performed with urea-formamide solvent as the spreading solution and distilled water as the hypophase by a drop spreading technique (30). The samples were examined in a JEM100B electron microscope. RESULTS
Virus Detection A combination of the following three methods was employed to detect the endogenous C-type RNA virus, i.e., incorporation of 14C-uridine into viral RNA, assay of RNA directed DNA polymerase and examination by electron microscopy. When fluid of cultures of AC cells labeled with "C-uridine was concentrated and examined by sucrose density gradient fractionation, one major peak of radioactivity was detected at a density of 1.15 g/ml, as shown in Fig. 1. Simultaneous
PRODUCTION
Fig.
1.
Isopycnic
Densities mined
(A) by
RAT
centrifugation were
DNA
from The
is expressed C. Symbols
directed
of
calculated
refractometry.
polymerase min at 37
●,RNA
OF
C-TYPE
the
medium
sucrose
activity
5
VIRUS
of
AC
culture.
concentrations of
RNA
deter-
directed
as 3H-dTTP incorporated (pmol) are; •›, 14C-uridine incorporation
polymerase
activity
measured
DNA for
using
60 and
poly
(rA) • oligo(dT) as exogenous template.
Table
1.
Properties
Complete tion
was carried
Table
2.
of the RNA
reaction
mixture
out
at 37 C for
Template
specificity
directed
is described
DNA
polymerase
in Materials
and
activity
Methods.
of ACV
Incuba-
60 min.
of RNA
directed
DNA
polymerase
of ACV
6
K.
IGARASHI
ET Al
A
B
Fig.
2.
Thin sucrose
Electron section
micrographs of
density
ACV. •~38,000. cells.
C
ative
Budding staining
of 1.15
g/ml.
(B)
Thin
section
purified
(A)
fractions
of
ACV. of
of ACV.
a pellet
117,000. ACV.
at
the
Mature of (C)
AC Neg-
), 187,000.
measurement of RNA directed DNA polymerase activity of these fractions with a synthetic template, poly(rA)•oligo(dT), showed that the enzyme activity was present only around the 1.15 g/ml density region (Fig. 1). Therefore, the peak at a density of 1.15 g/ml seemed to show the presence of suspected C-type virus (ACV). The virus banded at a density of 1.15 g/ml in sucrose density gradients was collected in quantities and the properties of the DNA polymerase were examined. As shown in Table 1, this DNA synthesizing reaction with exogenous templates did not proceed when the synthetic template or detergent was omitted from the reaction mixture. It was also found that the polymerase was able to utilize not only poly(rA)•oligo(dT) but also poly(rCm)•oligo(dG) (18, 19), which were known to be active as the specific exogenous template for RNA directed DNA polymerase of C-type viruses. Poly (dA)•oligo(dT) did not act as a template in this polymerase reaction (Table 2). Moreover, this DNA polymerase could utilize the endogenous template when four deoxyribonucleotide triphosphates (dATP, dGTP, dCTP, dTTP) were present in the reaction mixture (Data not shown). This endogenous
PRODUCTION
Fig.
3. and
OF RAT
Hybridization cellular
The
details
Materials
of
DNAs of and
centration
of
cellular
(10) tion
and corrected to of 0.18 (1 1 ).
tracted
ster an
seconds)
DNA
in
from
were
rat
C7, DNA liver; African
DNA a human
from
[1], DNA green
in
mol
mouse from
monkey
lung
cation cellular
liver; •›,
DNA DNA
thymus;
kidney
A,
cell
line
liver
cell
line; •œ
cell
line
(HEL).
in
is the and
(A260
liver; •¥, calf
described
liter-1 as
ACV
mammals.
(Co
a monovalent Symbols: •¡,
a chimpanzee embryonic
values
calculated
(SD-JCL)
from
from
various are
Cot
7
VIRUS
probe
from
hybridization
Methods.
time
cells;
3H-DNA
extracted the
C-TYPE
con-
t is the
ml-1)12 •~
hr
concentraDNA exfrom from
AC ham-
DNA
from
(CV-1); •¢, DNA
from
enzyme activity was inhibited by addition of RNase and the product of the endogenous polymerase reaction was DNase sensitive, suggesting that the endogenous template was RNA and the product of the reaction was DNA (Data not shown). These results strongly indicate that the active DNA polymerase present in the suspected C-type virus is indeed the C-type virus associated RNA directed DNA polymerase, but not any other known DNA polymerase(s) such as DNA polymerase γ(19).
Electron microscopic examination of the peak fractions around a sucrose density of 1.15 g/ml revealed many typical C-type particles (Fig. 2A and C). Moreover, budding C-type virus was also observed when AC cells at the 61th subculture were examined (Fig. 2B). These particles could not be distinguished morphologically from RLV, a typical C-type RNA virus. All these results testify that the AC cells produce a typical C-type RNA virus in culture spontaneously. This virus was named ACV.
8
K. IGARASHI
Fig.
4.
Effect
polymerase Conditions Results
of of are
are
antiserum RLV
ET AL
to on
described
expressed
in as
RNA
the
Materials of
immune
in
the
absence
of
sources
of
viral
RNA
directed
: •›,
ACV; •œ,
RLV
and •£,
control
DNA of
and
%
activity
were
directed
polymerase
ACV.
Methods. polymerase
serum. DNA
The
polymerase
RSV.
Origin of ACV Two lines of evidence clearly demonstrated that ACV was a rat endogenous virus : (i) homology analysis of nucleic acids, and (ii) immunological specificity of RNA directed DNA polymerase. Conclusive evidence that ACV is not an exogenous virus which infected and propagated in AC cells but is an endogenous C-type virus of rat origin was obtained by the hybridization study. Figure 3 shows a DNA-DNA hybridization experiment performed using a 3H-DNA transcript of ACV and DNAs prepared from cells of various mammalian species. As expected, the highest and comparable extent of homology was found between ACV DNA probe and the DNAs extracted from AC cells and from rat tissue. Detectable but very low homologies were observed with hamster and mouse cellular DNA. In contrast, other animals tested (human, chimpanzee, African green monkey and bovine) did not contain virogene sequences related to ACV. To confirm that the homology observed between ACV DNA probe and AC cell DNA or rat cellular DNA was a result of the presence of highly related sequences in both DNAs and not a result of others, for example, the basepair mismatching the thermal stability of the hybrid was examined. The hybrids formed between ACV DNA probe and the DNA extracted from AC cells and rat liver had Tm of 85 C and 84 C, respectively. Under the same conditions, the Tm value of the hybrid formed between RLV DNA probe and mouse DNA was 82 C. These results clearly show the presence of related virogene sequences derived from ACV in both DNAs of AC cells and of rat tissue, suggesting that ACV is indeed an endogenous rat virus.
PRODUCTION
OF RAT
C-TYPE
VIRUS
9
A
B
Fig. 5. Velocity sedimentation of viral RNA on sucrose gradients. (A) RNA was extracted from ACV labeled with 'H-uridine and analyzed in sucrose gradients. (B) 70S RNA prepared was heated at 70 C for 3 min and analyzed as in (A). Arrows represent the positions of marker 28S and 18S ribosomal RNA.
When the RNA directed DNA polymerase of ACV was preincubated with antibodies against RLV polymerase, the ACV enzyme was partially inhibited, as shown in Fig. 4. Under the same experimental conditions, the greater inhibition of RNA directed DNA polymerase of RLV virions was observed. In contrast, RSV enzyme was not inhibited at all at the highest antiserum concentration used (Fig. 4). These results suggest that the enzyme of ACV virions is more closely related to that of RLV than to RSV enzyme and consistent with the data of DNA-DNA hybridization experiment. Propertiesof Structural Componentsof ACV (i) Viral core. When ACV labeled with 3H-uridine was centrifuged through a layer of Nonidet P-40 on top of a linear sucrose gradient, most of the radioactivity was found at the density of 1.28 to 1.30 g/ml with a single peak (Data not shown),
10
K. TGARASHI
Fig.
6.
Electron
technique. •~
tnicrograph 58,000.
of ACV Arrow
shows
ET AL
RNA
spread
a dimer
by
the
urea-formamidc
linkage.
showing the buoyant density of ACV core. The control tube in which the virus was centrifuged without a surfactant layer had a peak with radioactivity at a density of 1.15 g/ml (Data not shown). The density of ACV core component well coincides with that determined for preparations of the cores of RIX (36), avian leuko-sarcoma viruses (5) and avian myeloblastosis virus (37). (ii) Viral high molecularRNA. The sedimentation profiles of RNA extracted from ACV were studied in a sucrose density gradient. As shown in Fig. 5A, the predominant species of RNA extracted from ACV had a sedimentation coefficient of about 70S relative to markers of ribosomal 18 and 28S RNA prepared from rat liver and the remainder RNA was less than 10S. When the 70S RNA prepared from the above sucrose gradient was heat-treated and analyzed, it was dissociated to subunit RNA with a single 35S species (Fig. 5B). By electron microscopy,, the 70S RNA extracted from ACV was observed to be an extended single strand with a dimer linkage, a characteristic secondary structure, at the central portion of the molecule (Fig. 6). The contour length of the RNA molecule was about 7.1 p.m which corresponds to a molecular weight of about 7.0 / 109daltons. These results suggest that ACV contains high molecular weight 70S RNA consisting of two half-size subunit RNAs.
PRODUCTION
OF
RAT
Fig.
C- TYPE
7.
SDS with
Migration acrylamide Coomassie
11
VIRUS
of ACV gels. blue.
polypeptides Gels
were
on stained
(iii) Viral structural protein. The polypeptidc pattern of ACV in SDS polyacrylamide gels revealed that the virions consisted of four major polypeptides, designated as 1, 2, 3 and 4 according to the order of decreasing electrophoretic mobility (Fig. 7). In addition to these bands, several fine bands, which have lower electrophoretic mobilities than the major components, are present. The molecular weight of each polypeptide was determined from the positions of known marker proteins coelectrophoresed. Polypeptides 1, 2, 3 and 4 in SDS gels have molecular weights of 12,400, 14,000, 15,500 and 31,500 daltons, respectively. The molecular weights of the remaining minor components are from about 40,000 to 100,000 daltons. Although serological analyses to determine the specificity of individual polypeptides have not yet been clone, the polypeptides 1, 2, 3 and 4 of ACV seem to correspond to polypeptides P1, P2, P3 and P4 of MLV (20), respectively. DISCUSSION
Recent reports (2, 27, 28) have indicated that latent endogenous rat C-type
12
K.
IGARASHI
ET AL
virus was induced by treatment with chemical agents, suggesting that the complete C-type virus genome is present in rat cells in an unexpressed form. Sometimes this virus genome is partially expressed. Hino et al (21) showed that the groupspecific antigen of rat C-type viruses was present in about 35-800, of transplantable rat tumor lines. In addition, full expression of rat C-type virus genome, i.e. spontaneous release of C-type RNA virus from rat cells in vitro, was also reported (9, 13, 17, 21, 29, 33, 35, 38, 43). Evidence presented in this communication shows that the genetic information of endogenous rat C-type RNA virus is expressed completely in AC cells resulting in the spontaneous production of ACV. All the characteristics of ACV examined, that is, the morphology and the physicochemical properties buoyant density, RNA directed DNA polymerase activity, viral core, high molecular weight RNA and structural polypeptides-fulfil the criteria for C-type RNA virus. These properties are similar to those of rat C-type viruses reported by Rasheed et al (33) and Svec and Michalides (3e). The slightly low density (d=1.15) of ACV in sucrose gradients appears to be a common characteristic of rat C-type viruses as suggested by Sarma et al (35). DNA-DNA hybridization revealed the presence of the DNA sequences homologous to the DNA transcripts of ACV in rat cells. In addition, immunological studies using antiserum to RNA directed DNA polymerase of RLV indicated the derivation of ACV from the animal closely related to mouse. Therefore, as expected, it was concluded that ACV is indeed a typical C-type virus of rat origin. The reasons for the apparent release of C-type virus in AC cells are obscure. However, it should be noted that AC cells are derived from chemically induced rat tumor. Although little is known about the mechanisms of chemical carcinogenesis, chemical carcinogen may activate or derepress the latent RNA virus genome (34). Huebner and Todaro (23) suggested that such an activation or a derepression of C-type virus genome caused the transformation of cells. But, whether ACV is the causative agent of the malignant behavior of AC cells is unknown at the present moment. Until the 6th passage of AC cells after our reception, the spontaneous release of ACV, as measured by the polymerase assay and 3H-uridine incorporation test, was almost negligible. But, thereafter, a definite increase of the yield of this virus was observed and at the 36th passage, a large amount of virus was spontaneously produced. The increase of overall production of ACV seems to depend on the increased number of virus producing cells in culture. On the other hand, another explanation that the virus production was increased by the increased rate of virus production per cell can not be excluded. In this connection, a recent study (29) suggested that the clonal selection for virus productive cell occurred in a rat tissue culture line. Previously, we observed that the morphological change in AC cells associated with a remarkable retardation of cellular growth was induced in vitro by exposure to agents such as dibutyryl adenosine cyclic 3' : 5'-monophosphate (db-cAMP) (24). Then, the virus productions were compared quantitatively between morphologically differentiated and undifferentiated cells. The media of AC cells
PRODUCTION
OF
RAT
C-TYPE
VIRUS
13
cultured from 0 to 24 hr and 24 to 48 hr with or without 1 mm db-cAMP were harvested, concentrated and the activity of RNA directed DNA polymerase was measured. The results showed that a comparable amount of virus was produced in morphologically differentiated and immature AC cells. This indicates that the organization or disorganization of microtubular system regulated by the intracellular levels of cAMP (24) seems to have little effect on the production of C-type virus. In preliminary studies, it was found that ACV was infections to rat cells but not to mouse, bovine or human cells in vitro. Therefore, ACV may be an ecotropic rat virus. Further test of infectivity is being developed and the pathogenicity of ACV in vitroand in vivois being studied. Previously, Hino et al (21) reported that Ctype virus produced from a rat ascites hepatoma shared common antigen with other C-type viruses produced from other rat tumor lines. In this connection, the immunological studies of ACV and its relationship to other rat C-type virus might be of interest. Weare indebtedto Dr. Y. Ikawafor providingthe antiserumagainstRNA directedDNA polymeraseof RLV, Dr. M. Owadafor supplyingRSV (B77)and Drs.A. Ishimotoand A. Hakura for supplyingthe celllinesused. WealsothankDr. N. Tsuchidafor valuablesuggestions on hybridization experiment. Excellenttechnicalassistancewasprovidedby Mr. M. Koyama. REFERENCES
1)
Aaronson, S.A. 1971. Isolation of a rat-tropic helper virus from M-MSV(O) stocks. Virology 44: 29-36. 2) Aaronson, S.A. 1971. Chemical induction of focus-forming virus from non-producer cells transformed by murine sarcoma virus. Proc. Natl. Acad. Sci. U.S.A. 68: 3069-3072. 3) Aaronson, S.A., Todaro, C. J., and Scolnick, E.M. 1971. Induction of murine C-type viruses from clonal lines of virus-free BALB/3T3 cells. Science 174: 157-159. 4) Ando, T. 1966. A nuclease specific for heat-denatured DNA isolated from a product of Aspergillus oryzae. Biochim. Biophys. Acta 114 : 158-168. 5) Bader, J.P., Brown, N.P., and Bader, A.V. 1970. Characteristics of cores of avian leuko-sarcoma viruses. Virology 41: 718-728. 6) Benveniste, R.E., and Scolnick, E.M. 1973. RNA in mammalian sarcoma virus transformed nonproducer cells homologous to murine leukemia virus RNA. Virology 51: 370-382. 7) Benveniste, R.E., Heinemann, R., Wilson. G.L., Callahan, R., and Todaro, G. J. 1974. Detection of baboon type C viral sequences in various primate tissues by molecular hybridization. J. Virol. 14: 56-67. 8) Bergs, V.V., Bergs, M., and Chopra, H.C. 1970. A virus (RMTDV) derived from chemically induced rat mammary tumors. I. Isolation and general characteristics. J. Natl. Cancer Inst. 44: 913-922. 9) Bergs, V.V., Pearson, G., Chopra, H.C., and Turner, W. 1972. Spontaneous appearance of cytopathology and rat C-type virus (WF-1) in a rat embryo cell line. Int. J. Cancer 10: 165-173. 10) Britten, R.J., and Kohne, D.E. 1968. Repeated sequences in DNA. Science 161: 529-540. 11) Britten, R.J., and Smith, J. 1970. A bovine genome. Carnegie Inst. Wash. Year B. 68 : 378-386. 12) Chopra, H.C., Bogden, A.E., Zelljadt, I., and Jensen, E.M. 1970. Virus particles in a transplantable rat mammary tumor of spontaneous origin. Eur. J. Cancer 6: 287-290. 13) Cremer, N.E., Taylor, D.O., Oshiro, L.S., and Teitz, Y. 1970. Transformation and virus production in normal rat thymus cells and those infected with Moloney leukemia virus. J. Natl. Cancer Inst. 45: 37-48. 14) Duesberg, P.H., Robinson, H.L., Robinson, W.S., Huebner, R.J., and Turner, H.C. 1968. Proteins of Rous sarcoma virus. Virology 36: 73-86.
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Engle, G.C., Shirahama, S., and Dutcher, R.M. 1969. Preliminary report on virus-like particles in a transplantable rat mammary carcinoma. Cancer Res. 29: 603-609. Fahey, J.L. 1967. Chromatographic separation of immunoglobulins, p. 321-332. In Williams, C.A., and Chase, M.W. (eds), Methods in immunology and immunochemistry, Vol. 1, Academic Press Inc., New York. Gazzolo, L., Simkouic, D., and Martin-Berthelon, M.C. 1971. The presence of C-type RNA virus particles in a rat embryo cell line spontaneously transformed in tissue culture. J. Gen. Virol. 12: 303-311. Gerard, G.F., Rottman, F., and Green, M. 1974. Poly(2'-O-methylcytidylate) oligodeoxyguanylate as a template for the ribonucleic acid directed deoxyribonucleic acid polymerase in ribonucleic acid tumor virus particles and a specific probe for the ribonucleic acid directed enzyme in transformed murine cells. Biochemistry 13: 1632-1641. Gerard, G.F. 1975. Poly(2'-O-methylcytidylate) . oligodeoxyguanylate, a template-primer specific for reverse transcriptase, is not utilized by HeLa cell DNA polymerases. Biochem. Biophys. Res. Comm. 63: 706-711. Green, R.W., Bolognesi, D.P., Schafer, W., Pister, L., Hunsmann, G., and De Noronha, F. 1973. Polypeptides of mammalian oncornaviruses. I. Isolation and serological analysis of polypeptides from murine and feline C-type viruses. Virology 56: 565-579. Hino, S., Yoshida, K., Enomoto, J., Oboshi, S., and Yamamoto, T. 1974. Endogenous rat C-type virus in transplantable rat tumors. Japan. J. Exp. Med. 44: 179-189. Horne, R.W., and Ronchetti, I.P. 1974. A negative staining-carbon film technique for studying viruses in the electron microscope. I. Preparative procedures for examing icosahedral and filamentous viruses. J. Ultrastructure Res. 47: 361-383. Huebner, R.J., and Todaro, G. J. 1969. Oncogenes of RNA tumor viruses as determinants of cancer. Proc. Natl. Acad. Sci. U.S.A. 64: 1087-1094. Igarashi, K., Ikeyama, S., Takeuchi, M., and Sugino, Y. 1978. Morphological changes in rat glioma cells caused by adenosine cyclic 3': 5'-monophosphate. Cell structure and function. Jpn. Soc. Cell Biol. 3: 103-112. Ishimoto, A., Ito, Y., and Maeda, M. 1971. Studies on the susceptibility of C57 BL/6 mice to Rauscher virus. Multiplication of Rauscher virus in C57 BL/6 cells in vivo and in vitro. J. Natl. Cancer Inst. 47: 1299-1308. Karasaki, S. 1969. Virus particles associated with the transplantable Novikoff hepatoma. Cancer Res. 29: 1313-1315. Klement, V., Nicolson, M.O., and Huebner, R.J. 1971. Rescue of the genome of focus forming virus from rat nonproductive lines by 5'-bromodeoxyuridine. Nature New Biol. 234: 12-14. Klement, V., Nicolson, M.O., Gilden, R.V., Oroszlan, S., Sarma, P.S., Rongey, R.W., and Gardner. M.B. 1972. Rat C-type virus induced in rat sarcoma cells by 5-bromodeoxyuridine. Nature New Biol. 238: 234-237. Klement, V., Nicolson, M.O., Nelson-Rees, W., Gilden, R.V., Oroszlan, S., Rongey, R.W., and Gardner, M.B. 1973. Spontaneous production of a C-type RNA virus in rat tissue culture lines. Int. J. Cancer 12: 654-666. Kung, H.-J., Bailey, J.M., Davidson, N., Nicolson, M.O., and McAllister, R.M. 1975. Structure, subunit composition, and molecular weight of RD-114 RNA. J. Virol. 16: 397-411. Maize!, J.V., Jr. 1971. Polyacrylamide gel electrophoresis of viral proteins, p. 179-246. In Maramorosh, K., and Koprowski, H.(eds), Methods in virology, Vol. 5. Academic Press Inc., New York. Niiyama, Y., Igarashi, K., Tsukamoto, K., Kurokawa, T., and Sugino, Y. 1975. Biochemical studies on bovine adenovirus type 3. I. Purification and properties. J. Virol. 16: 621-633. Rasheed, S., Bruszewski, J., Rongey, R.W., Roy-Burman, P., Charman, H.P., and Gardner, M.B. 1976. Spontaneous release of endogenous ecotropic type C virus from rat embryo cultures. J. Virol. 18: 799-803. Rhim, J.S., Dub., F.G., Cho, H.Y., Elder, E., and Vernon, M.L. 1973. Activation of a type-C RNA virus from tumors induced by rat kidney cells transformed by a chemical carcinogen. J. Natl. Cancer Inst. 50: 255-261. Sarma, P.S., Kunchorn, P.D., Vernon, M.L., Gilden, R.V., and Berg, V. 1973. Wister-Furth rat C-type virus. Biologic and antigenic characterization. Proc. Soc. Exp. Biol. 142: 461-465.
PRODUCTION
36)
OF
RAT
C-TYPE
VIRUS
15
Shibley, G.P., Carlteon, F.J., Wright, B.S., Schidlousky, G., Monroe, J.H., and Magyasi, S.A. 1969. Comparison of the biologic and biophysical properties of the progeny of intact and etherextracted Rauscher leukemia viruses. Cancer Res. 29: 905-911. 37) Stromberg, K. 1972. Surface-active agents for isolation of the core component of avian myeloblastosis virus. J. Virol. 9: 684-697. 38) svec, J., and Michalides , R. 1977. Biochemical properties of endogenous rat C-type viruses. Neoplasma 24: 601-614. 39) Ting, R.C. 1968. Biological and serological properties of viral particles from a nonproducer rat neoplasm induced by murine sarcoma virus (Moloney). J. Virol. 2: 865-868. 40) Tsuchida, N., and Green, M. 1974. Intracellular and virion 35S RNA species of murine sarcoma and leukemia viruses. Virology 59: 258-265. 41) Tsuchida, N., Long, C., and Hatanaka, M. 1974. Viral RNA of murine sarcoma virus produced by a hamster-mouse somatic cell hybrid. Virology 60: 200-205. 42) Tsuchida, N., Gilden, R.V., Hatanaka, M., Freeman, A.E., and Huebner, R. J. 1975. Type-C virus-specific nucleic acid sequences in cultured rat cells. Int. J. Cancer 15: 109-115. 43) Weinstein, I.B., Gebert, R., Stadler, U.C., Orestein, J.M., and Axel, R. 1972. Type C virus from cell cultures of chemically induced rat hepatomas. Science 178: 1098-1100.
Spontaneous Production of a C-Type RNA Virus Cell Line Derived from Rat Glioma Koichi
IGARASHI,t
Reiko
SASADA, and
†Biological
Yasuhiko
Yukio
Chemical
Yoshio
KOZAI,
SUGINO
Research Laboratories,Central
Takeda
NIIYAMA,
in a
Research
Industries,
(Received for publication,
Ltd.,
Division,
Osaka
March 8, 1978)
Abstract The spontaneous production of a rat C-type RNA virus (ACV) in a cultured cell line (AC cells) established from a chemically induced rat glioma was studied. The characteristics of ACV were : morphology typical of C-type RNA virus; buoyant density of 1.15 g/ml in a sucrose density gradient; RNA directed DNA polymerase activity; viral core with a density of 1.28 to 1.30 g/ml ; 70S RNA with dimer structure; and structural protein composed of mainly four polypeptides. Kinetical analysis of DNA-DNA hybridization revealed that DNA sequences homologous to DNA transcripts of RNA of ACV were present in rat cells. RNA directed DNA polymerase of ACV partially cross-reacted with antiserum to the polymerase of Rauscher murine leukemia virus. These data suggest that ACV is an endogenous C-type RNA virus of rat origin.
Recent studies have indicated that C-type RNA viruses are found in normal and tumor cells of many vertebrates. In the rat, several lines of experimental evidence for the presence of C-type viruses were obtained. That is, demonstrable C-type viruses of suspected rat origin were observed in rat hepatomas (21, 26, 43), rat mammary tumors (8, 12, 15), and normal or transformed rat cell lines (9, 13, 17, 29, 33-35, 38). Other investigations (2, 27, 28) have shown that nonproductive rat cells transformed with murine or avian sarcoma virus released endogenous C-type virus as a rat leukemia pseudotype when treated with halogen derivatives of uridine. Moreover, endogenous rat C-type virus with properties of helper virus was also isolated from stocks of murine sarcoma virus which had been passaged in rat (1, 39). However, most of the rat viruses hitherto reported have not been fully characterized. This communication virus virus
describes
the spontaneous
in a cell line established from a rat glioma, is an endogenous rat C-type virus. MATERIALS
Cells and viruses. from
Sprague-Dawley
The rat
origin glioma
AND
and
1
and
presents
of C-type evidence
RNA
that
this
METHODS
properties
induced
high production
of AC cells, a cell line
transplacentally
by
established
ethylnitrosourea,
2
K. IGARASHI
have
been
described
a C-type was
virus
used
(RLV), by and was
a
R17 was
line,
line (25) obtained by
was
R17
10%
liver
cell
were
fetal
199
hr
cells
at
cultured
at
near
for
centrifugation
at
The
of
tion
was
precipitated serum
filter
(Whatman
Beckman
virus
son
for
of
et
al
or
was assay
by
addition as
acetate,
dithiothreitol,
0.4
oligo(dT)io,
washed
P-L
DNA
and
dried,
England
Nuclear)
reduced
sucrose
rpm
for
20
hr
concentration of
of
each
and
(TCA)
using
fracthe
100
through
rest ti.g
of
a glass-fiber
radioactivity
mm
virus
counted
by
in
the
reaction 0.005m
polymerase the
a
mixture
(0.1
magnesium
Pure
activity
method
of Aaron-
ml)
contained
acetate,
Chemical
Ind.,
0.001
Ltd.),
polyadenylate•oligodeoxythymidylate or
particles
cell
(3.5
DNA
measured
(Wako
triphosphate 3H-deoxyguanine
from
directed
was The
Inc.)
mm
template, 0,01
the
by sucrose
linear
portion
filtered
clarified
a 60%
activity,
acid
RNA
KCl,
X-100 of
and
purified
to
polymerase
and
was
the A
were Radio-
concentrated
25,000
refractometry.
.
The
15-60%
and
at (14).
diameter)
onto
rotor,
was
in
al
gradient
harvested
a
50.1
DNA
P-L
New
were
mg/ml
fluid et
mCi/mmol,
sucrose
collected
precipitate
0.06m
units/ml
3H-thymidine Co.) or 0.1
added
1.2
culture
was
onto
SW
template
Triton
0.1 mm Chemical
without
acid,
density
mm
fluid
trichloroacetic
modification. 7.8),
plate, Pure
ed
(100
20%
were
polymerase.
exogenous
Biochemicals,
and
sucrose
(54
layered L3,
The
guanylate(poly(rCm)•oligo(dG)12_18,
concentrated
of puls
counter.
0.1% A260
folic
Duesberg
supernatant
through
by
cold
carrier.
slight (pH
min
the
directed
of
with
100 ƒÊg/ml (60%)
from of
on
culture
in
was
RNA
and
medium of
method
dishes
The
determined of
directed
with
hr.
fractions
and
cultures
supplemented
5A
1-4C-uridine
model
kidney
(HEL) All
serum
harvested
the
plastic
virus
90
monkey cells
medium
centrifugation
material
without
Tris-HCl
manganese
for
scintillation
RNA
(3)
24 the
rpm
GF/C),
with
0.04m
for
and virus
was established (RSV), B-77,
Laboratories.
6•@ƒÊg/ml
virus, to
5 ƒÊCi/mlof
and
the
FCS,
The
Falcon
(Spinco
albumin
liquid
Assay in
of
fraction
used
bovine
two
lung
bovine
ACV
(25).
isopycnic
centrifugation,
each
was
in
centrifuged
After
sucrose
and
25,000
20%
virus.
green
McCoy's
Kanamycin
according
concentrated
and
4 C).
of
10 min,
at
gradient
with
purified
presence
in
line virus
African
or
as leukemia
embryonic Flow
produce
herein
murine
essential
propagated
Amersham)
rpm
cushion.
minimum
of
an
from
Laboratories)
purification
confluency
the
obtained
Flow
to
was used. This Rous sarcoma
Human
Eagle's
were
was
Centre
3,000
were
incorporation
in
chemical
line
spontaneously
Rauscher
cells,
Hakura.
cells
is referred
prepare
CV-1
A.
100 ƒÊg/ml
and
intervals,
14C-Uridine AC
Owada. Dr.
supplemented
and
Preparation 8-16
is producing RLV Dr. A. Ishimoto.
cells
(20%)
bactopeptone
To
AC
virus
that from
(FCS,
R17
The
This
study.
in
serum
(24).
origin.
this
grown
calf
medium
elsewhere rat
M. by
Kanamycin.
of
Dr.
gifted
chimpanzee
except
detail
throughout
supplied
cell
in
of suspected
ET AL
culture
poly
(poly(rA)
2'-O-methylcytidylate•oligodeoxy-
Biochemicals,
Inc.)
(dTTP, 50-500 triphosphate (1-20
t,tg
medium.-
concentrations Ci/mmol)
M
0.005m
viral
When of
and
of
0,01%,
as
protein), the
3H-dTTP
exogenous
tem-
mCi/mmol, Daiichi (dGTP, 50 mCi/mmol,
assay and
respectively,
which was Triton and
were
performX-100 0,1
mm
PRODUCTION
OF RAT
C-TYPE
3
VIRUS
deoxyribonucleotide triphosphates (dATP, dGTP and dCTP) were added to the reaction mixture. Incubation was carried out at 37 C for 60 min. DNA synthesis continued linearly for at least 90 min under the conditions employed. The reaction was stopped by addition of cold 10% TCA containing 10mm sodium pyrophosphate and 10 mm monobasic sodium phosphate, and the DNA in the precipitate was collected on a glass-fiber filter. After 5 washings with cold TCA, the radioactivity of the precipitate was determined. Inhibition of RNA directedDNA polymeraseof ACV by antiserum to the Polymeraseof RLV.
Viruses
were
equilibrium directed
DNA
as
of
protein
viral
by
and
Triton
for
of
activity
of
the
the
(a
gift
0 C
60
was
serum
at
for
culture
Y.
at
measured
Ikawa)
After
virus
purified
to
im-
Ltd.) 1 p_g
with 10
10
ti,1 of
incubation,
poly(rCm)
RNA
containing
with
the
by
the
Whatman
disrupted
incubated
C.
using
was
of
were
purified to
(DE22,
(10 ƒÊl)
were 0
and
antiserum
cellulose
albumin
and
min
fluids
goat
Dr.
Dilutions
bovine
15 min
from
a DEAE
(16).
of
IgG
sample
tissue The
through
Fahey
10 ti,g
X-100
concentrations
RLV
fraction
described
spent
centrifugation. of
G(IgG)
column
from
gradient
polymerase
munoglobulin
0.1%
concentrated
sucrose
various
the
•oligo(dG)
as
ti.1 of
enzyme
exogenous
template. Preparation
of
extracted
and
Tsuchida
et
The RNA
18
hr
and
sequences to
1.4
mg
using by
,Vleasurement
(6).
probe C. the
with
ACV
of rat
of
DNAs
sources
of
as
were
described
by
DNA
since
2 mm
per
Tm
et
ml
al
(midpoint
cellular
remaining
at
EDTA, 65
four
presence
proceed
about
of 20
sonically
C (7).
was of
the
specific
activity
of
3H-
to
70S
by DNA
of
most
RNA
of
ACV
excess
of
3H-DNA.
using
an at
performed
24,000
extent
in
contain
heating
was
SDS,
The
DNA activity
treated
nuclease
C
specific
times
denatured
0.05%
37
(EDTA),
seemed 70%
cellular
of
at
deoxyribonucleotides
obtained
and
S1
cpm
hybrid
of
ARTEK 100
C
in
0.01
Kogyo
for M
3H-DNA
formation
(Seikagaku
was
was
Co.)
(4)
(41). of
DNA heated
the
the
with
to
single-strand-specific
endogenous
the
3H-labeled
The from
were 7S
probe
the
in to
The
(40).
hybridization
3H-DNA
allowed
(SDS).
of
DNAs
by
ACV
M ethylenediaminetetraacetate
thus
about
prepared
was
calculated
RNA,
m NaCl,
were of
as
after
was
Green
transcripts
size
Tsuchida
Aliquots amount
Cellular
various
purified
sulfate and
All
a mean
of cellular by
described
65
in
0.75
0.01
incorporation
RNase
7.4),
of
DNA,
hybridization.
(pH
probe
reaction
dodecyl
The
Hybridization
measured
DNA.
of
using
The
Tsuchida
cpmhig
present
to
Tris-HCl
and
by
Pancreatic
DNA-DNA
at
sodium
equimolar
dismembrator 10 min.
viral lines
DNA
addition
reaction.
resistant
DNA
by
0.5%
assuming enzyme
cell
reaction
D(6).
stopped
of the
as
viral
as described was 2 .0 •~ 107
dTTP,
3H-labeled and
polymerase
actinomycin
and
M NaCl
and
and
tissues
(42).
DNA of
extracted 3H-DNA
this
DNA from
3H-thymidine-labeled
100 ƒÊg/ml
0.1
al
directed
for
cellular
purified
melting was
for hybrid
curve).
carried
5 min was
out in
0.75
determined
The to m
hybridization
a
Cot
of 8 •~
NaCl
at
by
digestion
of 103
various
ACV
mol •~
temperatures with
3H-
sec/liter
S1 nuclease
4
K. IGARASHI
ET AL
Isolationof the viral coreof ACV. The viral core was obtained by centrifugation of 3H-labeled virus through a layer of 1.0% Nonidet P-40 (Shell Chemical Co.) on top of a 10 to 70% linear sucrose gradient according to the method of Stromberg (37). Isolation and velocity sedimentation of viral RNA. Purified virus, labeled with 60 of 3H-uridine for 16 hr, in NET buffer (0.1 M NaCl, 0.001 M EDTA, 0.01 M
μCi/ml
Tris-HC1, pH 7.2) was lysed by addition of 1.0% SDS and 0.05% diethylpyrocarbonate (Nakarai Chemicals, Ltd.), and incubated at 25 C for 15 min. The RNA was extracted with water-saturated phenol and chloroform-isoamyl alcohol (24: 1) (41) and precipitated overnight with ethanol at -20 C. Viral RNA, collected by centrifugation and dissolved in NET buffer, was mixed with ribosomal RNA prepared from rat liver and centrifuged in a 10-30% sucrose gradient in NET buffer containing 0.5% SDS for 2 hr at 40,000 rpm in a Spinco SW 50.1 rotor at 20 C. After centrifugation, fractions were collected from the bottom and analyzed for the optical density (A,260)and the radioactivity in the acid insoluble fraction. For analysis of 30-40S RNA, the fractions containing viral 60-70S RNA were pooled, precipitated with ethanol and centrifuged. Then, RNA was dissolved with NET buffer, heated at 70 C for 3 min and centrifuged. The sedimentation coefficient of viral RNA was estimated from the positions of 28S and 18S ribosomal RNAs added as the reference standards in the identical gradient. Acrylamidegel electrophoresis. Viral material was precipitated with acetone and electrophoresed in 13% polyacrylamide gels containing 0.1 % SDS by a modification (32) of the procedure of Maizel (31). Human gamma globulin fraction, bovine albumin, ovalbumin, and beef pancreas chymotrypsinogen A (Schwarz/ Mann Co.) were used as molecular weight standards. Electron microscopy. The AC cells, in the exponential phase of growth, pelletted by centrifugation and purified virus were fixed in 2.5% glutaraldehyde and 1% osmium tetroxide and embedded in epon. Thin sections prepared were stained with uranyl acetate and lead citrate (32). Purified virus was also negatively stained. with 1% uranyl acetate on a glow-discharged carbon coated grid (22). The spreading of ACV RNA, prepared as described by Kung et al (30), was performed with urea-formamide solvent as the spreading solution and distilled water as the hypophase by a drop spreading technique (30). The samples were examined in a JEM100B electron microscope. RESULTS
Virus Detection A combination of the following three methods was employed to detect the endogenous C-type RNA virus, i.e., incorporation of 14C-uridine into viral RNA, assay of RNA directed DNA polymerase and examination by electron microscopy. When fluid of cultures of AC cells labeled with "C-uridine was concentrated and examined by sucrose density gradient fractionation, one major peak of radioactivity was detected at a density of 1.15 g/ml, as shown in Fig. 1. Simultaneous
PRODUCTION
Fig.
1.
Isopycnic
Densities mined
(A) by
RAT
centrifugation were
DNA
from The
is expressed C. Symbols
directed
of
calculated
refractometry.
polymerase min at 37
●,RNA
OF
C-TYPE
the
medium
sucrose
activity
5
VIRUS
of
AC
culture.
concentrations of
RNA
deter-
directed
as 3H-dTTP incorporated (pmol) are; •›, 14C-uridine incorporation
polymerase
activity
measured
DNA for
using
60 and
poly
(rA) • oligo(dT) as exogenous template.
Table
1.
Properties
Complete tion
was carried
Table
2.
of the RNA
reaction
mixture
out
at 37 C for
Template
specificity
directed
is described
DNA
polymerase
in Materials
and
activity
Methods.
of ACV
Incuba-
60 min.
of RNA
directed
DNA
polymerase
of ACV
6
K.
IGARASHI
ET Al
A
B
Fig.
2.
Thin sucrose
Electron section
micrographs of
density
ACV. •~38,000. cells.
C
ative
Budding staining
of 1.15
g/ml.
(B)
Thin
section
purified
(A)
fractions
of
ACV. of
of ACV.
a pellet
117,000. ACV.
at
the
Mature of (C)
AC Neg-
), 187,000.
measurement of RNA directed DNA polymerase activity of these fractions with a synthetic template, poly(rA)•oligo(dT), showed that the enzyme activity was present only around the 1.15 g/ml density region (Fig. 1). Therefore, the peak at a density of 1.15 g/ml seemed to show the presence of suspected C-type virus (ACV). The virus banded at a density of 1.15 g/ml in sucrose density gradients was collected in quantities and the properties of the DNA polymerase were examined. As shown in Table 1, this DNA synthesizing reaction with exogenous templates did not proceed when the synthetic template or detergent was omitted from the reaction mixture. It was also found that the polymerase was able to utilize not only poly(rA)•oligo(dT) but also poly(rCm)•oligo(dG) (18, 19), which were known to be active as the specific exogenous template for RNA directed DNA polymerase of C-type viruses. Poly (dA)•oligo(dT) did not act as a template in this polymerase reaction (Table 2). Moreover, this DNA polymerase could utilize the endogenous template when four deoxyribonucleotide triphosphates (dATP, dGTP, dCTP, dTTP) were present in the reaction mixture (Data not shown). This endogenous
PRODUCTION
Fig.
3. and
OF RAT
Hybridization cellular
The
details
Materials
of
DNAs of and
centration
of
cellular
(10) tion
and corrected to of 0.18 (1 1 ).
tracted
ster an
seconds)
DNA
in
from
were
rat
C7, DNA liver; African
DNA a human
from
[1], DNA green
in
mol
mouse from
monkey
lung
cation cellular
liver; •›,
DNA DNA
thymus;
kidney
A,
cell
line
liver
cell
line; •œ
cell
line
(HEL).
in
is the and
(A260
liver; •¥, calf
described
liter-1 as
ACV
mammals.
(Co
a monovalent Symbols: •¡,
a chimpanzee embryonic
values
calculated
(SD-JCL)
from
from
various are
Cot
7
VIRUS
probe
from
hybridization
Methods.
time
cells;
3H-DNA
extracted the
C-TYPE
con-
t is the
ml-1)12 •~
hr
concentraDNA exfrom from
AC ham-
DNA
from
(CV-1); •¢, DNA
from
enzyme activity was inhibited by addition of RNase and the product of the endogenous polymerase reaction was DNase sensitive, suggesting that the endogenous template was RNA and the product of the reaction was DNA (Data not shown). These results strongly indicate that the active DNA polymerase present in the suspected C-type virus is indeed the C-type virus associated RNA directed DNA polymerase, but not any other known DNA polymerase(s) such as DNA polymerase γ(19).
Electron microscopic examination of the peak fractions around a sucrose density of 1.15 g/ml revealed many typical C-type particles (Fig. 2A and C). Moreover, budding C-type virus was also observed when AC cells at the 61th subculture were examined (Fig. 2B). These particles could not be distinguished morphologically from RLV, a typical C-type RNA virus. All these results testify that the AC cells produce a typical C-type RNA virus in culture spontaneously. This virus was named ACV.
8
K. IGARASHI
Fig.
4.
Effect
polymerase Conditions Results
of of are
are
antiserum RLV
ET AL
to on
described
expressed
in as
RNA
the
Materials of
immune
in
the
absence
of
sources
of
viral
RNA
directed
: •›,
ACV; •œ,
RLV
and •£,
control
DNA of
and
%
activity
were
directed
polymerase
ACV.
Methods. polymerase
serum. DNA
The
polymerase
RSV.
Origin of ACV Two lines of evidence clearly demonstrated that ACV was a rat endogenous virus : (i) homology analysis of nucleic acids, and (ii) immunological specificity of RNA directed DNA polymerase. Conclusive evidence that ACV is not an exogenous virus which infected and propagated in AC cells but is an endogenous C-type virus of rat origin was obtained by the hybridization study. Figure 3 shows a DNA-DNA hybridization experiment performed using a 3H-DNA transcript of ACV and DNAs prepared from cells of various mammalian species. As expected, the highest and comparable extent of homology was found between ACV DNA probe and the DNAs extracted from AC cells and from rat tissue. Detectable but very low homologies were observed with hamster and mouse cellular DNA. In contrast, other animals tested (human, chimpanzee, African green monkey and bovine) did not contain virogene sequences related to ACV. To confirm that the homology observed between ACV DNA probe and AC cell DNA or rat cellular DNA was a result of the presence of highly related sequences in both DNAs and not a result of others, for example, the basepair mismatching the thermal stability of the hybrid was examined. The hybrids formed between ACV DNA probe and the DNA extracted from AC cells and rat liver had Tm of 85 C and 84 C, respectively. Under the same conditions, the Tm value of the hybrid formed between RLV DNA probe and mouse DNA was 82 C. These results clearly show the presence of related virogene sequences derived from ACV in both DNAs of AC cells and of rat tissue, suggesting that ACV is indeed an endogenous rat virus.
PRODUCTION
OF RAT
C-TYPE
VIRUS
9
A
B
Fig. 5. Velocity sedimentation of viral RNA on sucrose gradients. (A) RNA was extracted from ACV labeled with 'H-uridine and analyzed in sucrose gradients. (B) 70S RNA prepared was heated at 70 C for 3 min and analyzed as in (A). Arrows represent the positions of marker 28S and 18S ribosomal RNA.
When the RNA directed DNA polymerase of ACV was preincubated with antibodies against RLV polymerase, the ACV enzyme was partially inhibited, as shown in Fig. 4. Under the same experimental conditions, the greater inhibition of RNA directed DNA polymerase of RLV virions was observed. In contrast, RSV enzyme was not inhibited at all at the highest antiserum concentration used (Fig. 4). These results suggest that the enzyme of ACV virions is more closely related to that of RLV than to RSV enzyme and consistent with the data of DNA-DNA hybridization experiment. Propertiesof Structural Componentsof ACV (i) Viral core. When ACV labeled with 3H-uridine was centrifuged through a layer of Nonidet P-40 on top of a linear sucrose gradient, most of the radioactivity was found at the density of 1.28 to 1.30 g/ml with a single peak (Data not shown),
10
K. TGARASHI
Fig.
6.
Electron
technique. •~
tnicrograph 58,000.
of ACV Arrow
shows
ET AL
RNA
spread
a dimer
by
the
urea-formamidc
linkage.
showing the buoyant density of ACV core. The control tube in which the virus was centrifuged without a surfactant layer had a peak with radioactivity at a density of 1.15 g/ml (Data not shown). The density of ACV core component well coincides with that determined for preparations of the cores of RIX (36), avian leuko-sarcoma viruses (5) and avian myeloblastosis virus (37). (ii) Viral high molecularRNA. The sedimentation profiles of RNA extracted from ACV were studied in a sucrose density gradient. As shown in Fig. 5A, the predominant species of RNA extracted from ACV had a sedimentation coefficient of about 70S relative to markers of ribosomal 18 and 28S RNA prepared from rat liver and the remainder RNA was less than 10S. When the 70S RNA prepared from the above sucrose gradient was heat-treated and analyzed, it was dissociated to subunit RNA with a single 35S species (Fig. 5B). By electron microscopy,, the 70S RNA extracted from ACV was observed to be an extended single strand with a dimer linkage, a characteristic secondary structure, at the central portion of the molecule (Fig. 6). The contour length of the RNA molecule was about 7.1 p.m which corresponds to a molecular weight of about 7.0 / 109daltons. These results suggest that ACV contains high molecular weight 70S RNA consisting of two half-size subunit RNAs.
PRODUCTION
OF
RAT
Fig.
C- TYPE
7.
SDS with
Migration acrylamide Coomassie
11
VIRUS
of ACV gels. blue.
polypeptides Gels
were
on stained
(iii) Viral structural protein. The polypeptidc pattern of ACV in SDS polyacrylamide gels revealed that the virions consisted of four major polypeptides, designated as 1, 2, 3 and 4 according to the order of decreasing electrophoretic mobility (Fig. 7). In addition to these bands, several fine bands, which have lower electrophoretic mobilities than the major components, are present. The molecular weight of each polypeptide was determined from the positions of known marker proteins coelectrophoresed. Polypeptides 1, 2, 3 and 4 in SDS gels have molecular weights of 12,400, 14,000, 15,500 and 31,500 daltons, respectively. The molecular weights of the remaining minor components are from about 40,000 to 100,000 daltons. Although serological analyses to determine the specificity of individual polypeptides have not yet been clone, the polypeptides 1, 2, 3 and 4 of ACV seem to correspond to polypeptides P1, P2, P3 and P4 of MLV (20), respectively. DISCUSSION
Recent reports (2, 27, 28) have indicated that latent endogenous rat C-type
12
K.
IGARASHI
ET AL
virus was induced by treatment with chemical agents, suggesting that the complete C-type virus genome is present in rat cells in an unexpressed form. Sometimes this virus genome is partially expressed. Hino et al (21) showed that the groupspecific antigen of rat C-type viruses was present in about 35-800, of transplantable rat tumor lines. In addition, full expression of rat C-type virus genome, i.e. spontaneous release of C-type RNA virus from rat cells in vitro, was also reported (9, 13, 17, 21, 29, 33, 35, 38, 43). Evidence presented in this communication shows that the genetic information of endogenous rat C-type RNA virus is expressed completely in AC cells resulting in the spontaneous production of ACV. All the characteristics of ACV examined, that is, the morphology and the physicochemical properties buoyant density, RNA directed DNA polymerase activity, viral core, high molecular weight RNA and structural polypeptides-fulfil the criteria for C-type RNA virus. These properties are similar to those of rat C-type viruses reported by Rasheed et al (33) and Svec and Michalides (3e). The slightly low density (d=1.15) of ACV in sucrose gradients appears to be a common characteristic of rat C-type viruses as suggested by Sarma et al (35). DNA-DNA hybridization revealed the presence of the DNA sequences homologous to the DNA transcripts of ACV in rat cells. In addition, immunological studies using antiserum to RNA directed DNA polymerase of RLV indicated the derivation of ACV from the animal closely related to mouse. Therefore, as expected, it was concluded that ACV is indeed a typical C-type virus of rat origin. The reasons for the apparent release of C-type virus in AC cells are obscure. However, it should be noted that AC cells are derived from chemically induced rat tumor. Although little is known about the mechanisms of chemical carcinogenesis, chemical carcinogen may activate or derepress the latent RNA virus genome (34). Huebner and Todaro (23) suggested that such an activation or a derepression of C-type virus genome caused the transformation of cells. But, whether ACV is the causative agent of the malignant behavior of AC cells is unknown at the present moment. Until the 6th passage of AC cells after our reception, the spontaneous release of ACV, as measured by the polymerase assay and 3H-uridine incorporation test, was almost negligible. But, thereafter, a definite increase of the yield of this virus was observed and at the 36th passage, a large amount of virus was spontaneously produced. The increase of overall production of ACV seems to depend on the increased number of virus producing cells in culture. On the other hand, another explanation that the virus production was increased by the increased rate of virus production per cell can not be excluded. In this connection, a recent study (29) suggested that the clonal selection for virus productive cell occurred in a rat tissue culture line. Previously, we observed that the morphological change in AC cells associated with a remarkable retardation of cellular growth was induced in vitro by exposure to agents such as dibutyryl adenosine cyclic 3' : 5'-monophosphate (db-cAMP) (24). Then, the virus productions were compared quantitatively between morphologically differentiated and undifferentiated cells. The media of AC cells
PRODUCTION
OF
RAT
C-TYPE
VIRUS
13
cultured from 0 to 24 hr and 24 to 48 hr with or without 1 mm db-cAMP were harvested, concentrated and the activity of RNA directed DNA polymerase was measured. The results showed that a comparable amount of virus was produced in morphologically differentiated and immature AC cells. This indicates that the organization or disorganization of microtubular system regulated by the intracellular levels of cAMP (24) seems to have little effect on the production of C-type virus. In preliminary studies, it was found that ACV was infections to rat cells but not to mouse, bovine or human cells in vitro. Therefore, ACV may be an ecotropic rat virus. Further test of infectivity is being developed and the pathogenicity of ACV in vitroand in vivois being studied. Previously, Hino et al (21) reported that Ctype virus produced from a rat ascites hepatoma shared common antigen with other C-type viruses produced from other rat tumor lines. In this connection, the immunological studies of ACV and its relationship to other rat C-type virus might be of interest. Weare indebtedto Dr. Y. Ikawafor providingthe antiserumagainstRNA directedDNA polymeraseof RLV, Dr. M. Owadafor supplyingRSV (B77)and Drs.A. Ishimotoand A. Hakura for supplyingthe celllinesused. WealsothankDr. N. Tsuchidafor valuablesuggestions on hybridization experiment. Excellenttechnicalassistancewasprovidedby Mr. M. Koyama. REFERENCES
1)
Aaronson, S.A. 1971. Isolation of a rat-tropic helper virus from M-MSV(O) stocks. Virology 44: 29-36. 2) Aaronson, S.A. 1971. Chemical induction of focus-forming virus from non-producer cells transformed by murine sarcoma virus. Proc. Natl. Acad. Sci. U.S.A. 68: 3069-3072. 3) Aaronson, S.A., Todaro, C. J., and Scolnick, E.M. 1971. Induction of murine C-type viruses from clonal lines of virus-free BALB/3T3 cells. Science 174: 157-159. 4) Ando, T. 1966. A nuclease specific for heat-denatured DNA isolated from a product of Aspergillus oryzae. Biochim. Biophys. Acta 114 : 158-168. 5) Bader, J.P., Brown, N.P., and Bader, A.V. 1970. Characteristics of cores of avian leuko-sarcoma viruses. Virology 41: 718-728. 6) Benveniste, R.E., and Scolnick, E.M. 1973. RNA in mammalian sarcoma virus transformed nonproducer cells homologous to murine leukemia virus RNA. Virology 51: 370-382. 7) Benveniste, R.E., Heinemann, R., Wilson. G.L., Callahan, R., and Todaro, G. J. 1974. Detection of baboon type C viral sequences in various primate tissues by molecular hybridization. J. Virol. 14: 56-67. 8) Bergs, V.V., Bergs, M., and Chopra, H.C. 1970. A virus (RMTDV) derived from chemically induced rat mammary tumors. I. Isolation and general characteristics. J. Natl. Cancer Inst. 44: 913-922. 9) Bergs, V.V., Pearson, G., Chopra, H.C., and Turner, W. 1972. Spontaneous appearance of cytopathology and rat C-type virus (WF-1) in a rat embryo cell line. Int. J. Cancer 10: 165-173. 10) Britten, R.J., and Kohne, D.E. 1968. Repeated sequences in DNA. Science 161: 529-540. 11) Britten, R.J., and Smith, J. 1970. A bovine genome. Carnegie Inst. Wash. Year B. 68 : 378-386. 12) Chopra, H.C., Bogden, A.E., Zelljadt, I., and Jensen, E.M. 1970. Virus particles in a transplantable rat mammary tumor of spontaneous origin. Eur. J. Cancer 6: 287-290. 13) Cremer, N.E., Taylor, D.O., Oshiro, L.S., and Teitz, Y. 1970. Transformation and virus production in normal rat thymus cells and those infected with Moloney leukemia virus. J. Natl. Cancer Inst. 45: 37-48. 14) Duesberg, P.H., Robinson, H.L., Robinson, W.S., Huebner, R.J., and Turner, H.C. 1968. Proteins of Rous sarcoma virus. Virology 36: 73-86.
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IGARASHI
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PRODUCTION
36)
OF
RAT
C-TYPE
VIRUS
15
Shibley, G.P., Carlteon, F.J., Wright, B.S., Schidlousky, G., Monroe, J.H., and Magyasi, S.A. 1969. Comparison of the biologic and biophysical properties of the progeny of intact and etherextracted Rauscher leukemia viruses. Cancer Res. 29: 905-911. 37) Stromberg, K. 1972. Surface-active agents for isolation of the core component of avian myeloblastosis virus. J. Virol. 9: 684-697. 38) svec, J., and Michalides , R. 1977. Biochemical properties of endogenous rat C-type viruses. Neoplasma 24: 601-614. 39) Ting, R.C. 1968. Biological and serological properties of viral particles from a nonproducer rat neoplasm induced by murine sarcoma virus (Moloney). J. Virol. 2: 865-868. 40) Tsuchida, N., and Green, M. 1974. Intracellular and virion 35S RNA species of murine sarcoma and leukemia viruses. Virology 59: 258-265. 41) Tsuchida, N., Long, C., and Hatanaka, M. 1974. Viral RNA of murine sarcoma virus produced by a hamster-mouse somatic cell hybrid. Virology 60: 200-205. 42) Tsuchida, N., Gilden, R.V., Hatanaka, M., Freeman, A.E., and Huebner, R. J. 1975. Type-C virus-specific nucleic acid sequences in cultured rat cells. Int. J. Cancer 15: 109-115. 43) Weinstein, I.B., Gebert, R., Stadler, U.C., Orestein, J.M., and Axel, R. 1972. Type C virus from cell cultures of chemically induced rat hepatomas. Science 178: 1098-1100.
