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Journal of Virology, April 2001, p. 3965-3970, Vol. 75, No. 8
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.8.3965-3970.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Propagation of Rat Parvovirus in Thymic Lymphoma
Cell Line C58(NT)D and Subsequent Appearance of a Resistant Cell Clone
after Lytic Infection
Yutaka
Ueno,
Tanenobu
Harada,
Hiroyoshi
Iseki,
Takayuki
Ohshima,
Fumihiro
Sugiyama, and
Ken-ichi
Yagami*
Institute of Basic Medical Sciences and
Laboratory Animal Research Center, University of Tsukuba, Tsukuba,
Ibaraki 305-8575, Japan
Received 21 September 2000/Accepted 17 January 2001
 |
ABSTRACT |
Rat parvovirus (RPV) is nonpathogenic in rats but causes persistent
lymphocytotropic infection. We found that RPV was propagated in rat
thymic lymphoma cell line C58(NT)D and induced apoptosis. Interestingly, a resistant subclone, C58(NT)D/R, from surviving cells
after lytic infection had differentiated phenotypic modifications, such
as increased cell adherence, resistance to apoptosis, and suppressed tumorigenicity.
| |
TEXT |
Recent molecular studies on
parvoviral pathogenicity suggest that the viral nonstructural (NS)
protein in which coded genes are highly homologous among parvoviruses
correlates with cytotoxicity (2, 8, 17). The productive
and cytotoxic activity of the NS protein is modulated by cellular
factors that may vary with the host cell type, particularly in
oncogene-transformed cells (2, 13). We have shown that a
transcriptional coactivator, CREB binding protein, is required for
NS1-mediated viral and cellular transcription in parvovirus-infected
cells, resulting in cell proliferation and differentiation to achieve
its lytic cycle (16).
Newly recognized rodent parvoviruses, also called orphan parvoviruses,
are widespread among laboratory mice and rats (21, 23,
24), and viruses isolated have been classified as mouse parvovirus and rat parvovirus (RPV) (1, 5, 6). Although mouse parvovirus grows well in a murine T-cell clone (L3), causing a
cytopathic effect (CPE) (10), effective in vitro RPV
propagation has not been established. We found that RPV involved lytic
infection in the rat thymic lymphoma cell line C58(NT)D and developed
in vitro propagation for RPV. Interestingly, virus-resistant cell clones isolated from subcultures of surviving cells acquired
differentiated phenotypes such as reduced tumorigenicity and
sensitivity to apoptosis.
Viral propagation in cell lines.
The RPV strain was isolated
from rats infected spontaneously at our facility (23) and
passaged three times in specific-pathogen-free newborn Sprague-Dawley
rats. The virus stock was prepared from infected spleens at 7 days
postinoculation (p.i.) We initially inoculated a supernatant of 5%
infected spleen homogenate into cell lines and primary cell cultures of
rats and hamsters to find cells permitting virus propagation. C6 (rat
glioma), BRL-3A (Buffalo rat liver), RBL-2H3 (rat basophilic leukemia),
BHK-21 (Syrian hamster kidney), and Y3-Ag1.2.3 (rat myeloma) cells were
obtained from the Riken Cell Bank, Tsukuba, Japan, and C58(NT)D (rat
thymic lymphoma) cells were purchased from the American Type Culture Collection, Manassas, Va. Infected cells were observed for appearance of the viral CPE and examined for presence of the viral antigen by
immunofluorescent antibody (IFA) and hemagglutination (HA) ability
assays. The isolated virus was propagated only in C58(NT)D cells, not
in other cells tested (Table 1).
Parvoviral DNA was also detected only in infected C58(NT)D cells (data
not shown). In contrast, prototype RV-13 (7) was
propagated, with titers in C58(NT)D cells lower than those in other
cells. Propagation of the isolate in C58(NT)D cells and a difference in
cell tropism was thus clearly demonstrated between the isolate and
prototype RV-13 viruses.
To identify the propagated virus in C58(NT)D cells, we conducted HA
ability, HA inhibition, and IFA assays. The propagated
virus
agglutinated mouse and rat erythrocytes at HA titers of
2
7
to 2
9 at 4°C, but not guinea pig erythrocytes. Prototype
RV, in contrast,
agglutinated erythrocytes of the rat and guinea pig
but not those
of the mouse (data not shown). The propagated virus was
serologically
reacted with antisera prepared from experimentally
infected rats
with RV or RPV by IFA, but only reacted to anti-RPV serum
by HA
inhibition (data not shown). Thus, the propagated virus was
identical
to the original isolate strain of RPV(
23). In
the present study,
the isolate strain, designated RPV/UT, was
propagated only in
the thymic lymphoma cell line, i.e. C58(NT)D cells
(
3), and
involved persistent infection, confirming the
lymphotropism of
this virus in in vitro
experiments.
Viral cytotoxicity and apoptosis induction to C58(NT)D cells.
To preliminarily assess viral cytotoxicity and apoptosis induction,
C58(NT)D cells were infected with the RPV/UT virus at a theoretical
multiplicity of infection of 2, and cell viability was measured by
trypan blue staining. Infected C58(NT)D cells arrested cell growth for
2 days p.i., involved CPE, and reduced the cell number to less than
10% of that of mock-infected cells at 4 days p.i. (Fig. 1A, C, and
D). Viral infectivity of the infected cell culture reached 105.5 50% tissue culture infective
doses/100 µ1 at 2 days p.i. (Fig. 1B). Oligonucleosomal DNA ladders
were clearly visible in infected C58(NT)D cells at 3 days p.i. (Fig.
1E). To confirm that apoptosis was induced in RPV-infected C58(NT)D
cells, we examined the expression pattern of the Bcl-2 gene, which
regulates cell survival against apoptosis in lymphocyte development and
selection. Significant Bcl-2 downregulation was demonstrated in
infected C58(NT)D cells at 3 days p.i. by Western blot analysis (Fig.
1F). These findings indicate that RPV/UT induces apoptosis in infected
C58(NT)D cells.
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FIG. 1.
Cytotoxicity and apoptosis induction of RPV in C58(NT)D
cells. (A) Kinetics of cell survival in the RPV-infected ( ) and the
mock-infected ( ) C58(NT)D cells. (B) Infectivity titers of the
infected C58(NT)D cells (multiplicity of infection, 2). CPE in the
infected cells (C) and the mock-infected control (D) at 4 days p.i. (E)
DNA fragmentation in the RPV-infected cells (*) and mock-infected
cells (unmarked lanes). M, marker. (F) Western blot analysis showing
Bcl-2 and  -actin as a control in the infected and the mock-infected
cells.
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|
Certain autonomous parvoviruses were assumed to induce apoptosis in
infected cells (
4,
11,
12,
15,
18,
26). Our
previous study
(
15) demonstrated caspase-3-dependent apoptosis
in H-1
virus-infected C6 cells. It was also reported that H-1
virus-induced
apoptosis in U937 cells was mediated by downregulation
of c-Myc
oncoprotein overexpression (
18). In the present study,
we
showed that RPV/UT mediates downregulation of Bcl-2 expression
and
induces apoptosis in infected C58(NT)D cells. The Bcl-2 family
acts as
an upstream checkpoint of caspase 3 and mitochondrial
dysfunction in
the apoptosis pathway and has specific roles in
T-cell development and
selection (
14,
25). Our data suggest
that RPV, like H-1
parvovirus, induces apoptosis mediated by the
activation of the caspase
3 signaling
pathway.
Isolation of virus-resistant cell clone C58(NT)D/R.
The CPE of
the virus led to massive cell death, with surviving cells numbering
less than 10% of mock-infected cells. Surviving cells proliferated
continuously, accompanied by phenotypic modification such as increased
cell adherence through subsequent serial passages at 3- to 4-day
intervals. Changes in cell proliferation, viral infectivity of cultured
fluids, and the viral antigen ratio during serial passages are
summarized in Fig. 2A to F. Proliferation of surviving cells was reduced for comparison with mock-infected cells
and recovered at the fifth passage. Cells positive to the viral antigen
by IFA assay progressively decreased at the 4th (Fig. 2C), 8th (Fig.
2D), and 10th (Fig. 2E) passages, and finally disappeared at the 12th
(Fig. 2F) and subsequent passages. The cell adherence of all surviving
cells was enhanced more than that of parental C58(NT)D cells (Fig. 2G
and H). The appearance of resistant subclones was repeatedly confirmed,
and 10 resistant subclones were derived from surviving cells isolated
by limiting dilution. All resistant subclones had the same
morphological properties and resistance to RPV infection (data not
shown). Neither the viral antigen nor viral DNA was detected in them by
IFA assay or PCR, indicating that surviving cells completely eliminated the virus. One of the subclones, C58(NT)D/R, was observed on the cell
surface structure by scanning electron microscopy (SEM). Samples were
fixed with 2% glutaraldehyde and 1% osmium tetraoxide, dehydrated,
and critical-point dried with carbon dioxide. They were coated with
platinum and examined using SEM (JSM-6320F microscope; JEOL, Tokyo,
Japan). Numerous elongated microvilli covering the surface of
C58(NT)D/R cells were also observed (Fig. 2I and J).
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FIG. 2.
Persistent infection of RPV in C58(NT)D cells and an
appearance of resistant cells. (A) Cell growth ratio of the
RPV-infected C58(NT)D cells () compared to uninfected cells () at
serial cell passages. (B) Infective titers of cell fluids at the
indicated cell passages. Cells with a positive reaction to the viral
antigen by IFA assay were progressively decreased at the 4th (C), 8th
(D), and 10th (E) passages, and finally disappeared at the 12th passage
(F) or later. The cells at the 12th passage (H) showed enhanced cell
adherence compared to the parental C58(NT)D cells (G). One of the
resistant cell clones, C58(NT)D/R (J), was observed by SEM and compared
to the C58(NT)D cells (I). Bar = 1 µm.
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|
Phenotypic modification on C58(NT)D/R.
We studied the
susceptibility to apoptosis of irradiated C58(NT)D and C58(NT)D/R cells
using a cell death detection kit enzyme-linked, immunosorbent assay
(Boehringer, Mannheim, Germany). X-ray irradiation at 6 or 8 Gy was
done at 150 V and 20 mA, using a device (model MBR-1520R from Hitachi
Medical, Japan). Interestingly, C58(NT)D/R cells involved definitive
resistance to apoptosis induced by X-ray irradiation, while parental
C58(NT)D cells showed enhanced DNA fragmentation, indicating apoptosis
at 24 h postirradiation (Fig. 3).
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FIG. 3.
Resistance of C58(NT)D/R cells to irradiation-induced
apoptosis. C58(NT)D/R cells and parental C58(NT)D cells were
irradiated with 6 Gy ( and ) or 8 Gy ( and  ) of X rays, and
DNA fragmentation was assayed as an indicator of apoptotic change by
using the cell death detection kit enzyme-linked immunosorbent assay
(Boeringer).
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|
Since parvoviruses inhibit tumorigenesis mediated with oncoviruses and
chemical carcinogens (
19,
20), we compared tumorigenicity
between C58(NT)D and C58(NT)D/R cells. Eight female nude mice
(5 weeks
old, BALB/c
nu/nu) purchased from CLEA Japan
(Tokyo, Japan) in each group were
inoculated subcutaneously with
5 × 10
5 C58(NT)D and C58(NT)D/R cells, respectively.
Mice were housed
in an isolator controlled at 23 ± 2°C and 55% ± 10 % relative
humidity, and given free access to autoclaved food
(NMF; Oriental
Yeast, Tokyo, Japan) and water. They were inspected
daily, and
maximum and minimum diameters of tumor mass were measured
over
28 days. Seven of the eight injected with C58(NT)D cells developed
tumors, averaging 68.8 mm
2 in size within 28 days p.i. In
contrast, only two of the eight
developed tumors, averaging 23.4 mm
2, in the C58(NT)D/R-injected group within 28 days p.i.
(Fig.
4),
indicating that the resistant
C58(NT)D/R subclone possesses more
suppressed tumorigenicity than the
original C58(NT)D clone.
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FIG. 4.
Tumorigenicity of C58(NT)D and C58(NT)D/R cells in nude
mice. (A) Tumor incidence in nude mice inoculated with C58(NT)D (thick
line) and C58(NT)D/R (thin line) is indicated (n = 8).
(B) Tumor size was calculated by averaging the diameters of the tumor
areas in the tumor-bearing nude mice inoculated with C58(NT)D and
C58(NT)D/R and is indicated as mean ± standard error (error bars).
Photographs of examples of tumors in C58(NT)D (C)- and C58(NT)D/R
(D)-inoculated mice are shown.
|
|
Resistant subclones and their phenotypic modification were reported to
occur in the K562 human leukemia cell line (
22) and
the
U937 human promonocytic cell line (
9) infected with the
H-1 virus. A cell clone, KS, resistant to lytic infection with
the H-1
virus, shows a suppressed malignant phenotype and expressed
wild-type
p53, undetectable in parental K562 cells (
22). Resistant
cell clone RU from H-1 virus-infected U937 cells significantly
decreased tumorigenicity and the accumulation of c-Myc and exhibits
monocytic differentiation in cell surface antigens and nonspecific
esterase activity (
9). Our findings with C58(NT)D/R, such
as
enhanced cell adherence, microvillus increase and elongation on
the
cell surface, resistance to apoptosis, and suppressed tumorigenicity,
also suggest that the resistant clone altered the differentiated
or
activated state of lymphoma cells. The appearance of resistant
clones
from RPV-infected C58(NT)D cells is considered to involve
the same
mechanism acting on H-1 virus-infected K562 and U937
cells.
In conclusion, we clarified that RPV was propagated in the rat thymic
lymphoma cell line C58(NT)D and induced apoptosis and
isolated
subclones resistant to RPV infection involving differentiated
phenotypes. This resistant subclone is expected to contribute
much to
the understanding of apoptotic and anticancer mechanisms
mediated by
parvovirus
infection.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Laboratory
Animal Research Center, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan. Phone: (81) 298-53-3386. Fax: (81)
298-53-3380. E-mail:
kenyagam{at}sakura.cc.tsukuba.ac.jp.
| |
REFERENCES |
| 1.
|
Ball-Goodrich, L. J.,
S. E. Leland,
E. A. Johnson,
F. X. Paturzo, and R. O. Jacoby.
1998.
Rat parvovirus type 1: the prototype for a new rodent parvovirus serogroup.
J. Virol.
72:3289-3299[Abstract/Free Full Text].
|
| 2.
|
Caillet-Fauquet, P.,
M. Perros,
A. Brandenburger,
P. Spegelaere, and J. Rommelaere.
1990.
Programmed killing of human cells by means of an inducible clone of parvoviral genes encoding non-structural proteins.
EMBO J.
9:2989-2995[Medline].
|
| 3.
|
Geering, G.,
L. Old, and E. Boyse.
1966.
Antigens of leukemias induced by naturally occurring murine leukemia virus: their relation to the antigens of Gross virus and other murine leukemia viruses.
J. Exp. Med.
124:753-772[Abstract].
|
| 4.
|
Ikeda, Y.,
J. Shinozuka,
T. Miyazawa,
K. Kurosawa,
Y. Izumiya,
Y. Nishimura,
K. Nakamura,
J. Cai,
K. Fujita,
K. Doi, and T. Mikami.
1998.
Apoptosis in feline panleukopenia virus-infected lymphocytes.
J. Virol.
72:6932-6936[Abstract/Free Full Text].
|
| 5.
|
Jacoby, R. O.,
E. A. Johnson,
L. Ball-Goodrich,
A. L. Smith, and M. D. McKisic.
1995.
Characterization of mouse parvovirus infection by in situ hybridization.
J. Virol.
69:3915-3919[Abstract].
|
| 6.
|
Jacoby, R. O.,
L. J. Ball-Goodrich,
D. G. Besselsen,
M. D. McKisic,
L. K. Riley, and A. L. Smith.
1996.
Rodent parvovirus infections.
Lab. Anim. Sci.
46:370-380[Medline].
|
| 7.
|
Kilham, L., and L. J. Olivier.
1959.
A latent virus of rats isolated in tissue culture.
Virology
7:428-437[CrossRef][Medline].
|
| 8.
|
Leruez-Ville, M.,
I. Vassias,
C. Pallier,
A. Cecille,
U. Hazan, and F. Morinet.
1997.
Establishment of a cell line expressing human parvovirus B19 non-structural protein from an inducible promoter.
J. Gen. Virol.
78:215-219[Abstract].
|
| 9.
|
Lopez-Guerrero, J. A.,
B. Rayet,
M. Tuynder,
J. Rommelaere, and C. Dinsart.
1997.
Constitutive activation of U937 promonocytic cell clones selected for their resistance to parvovirus H-1 infection.
Blood
89:1642-1653[Abstract/Free Full Text].
|
| 10.
|
McKisic, M. D.,
D. W. Lancki,
G. Otto,
P. Padrid,
S. Snook,
D. C. Cronin II,
P. D. Lohmar,
T. Wong, and F. W. Fitch.
1993.
Identification and propagation of a putative immunosuppressive orphan parvovirus in cloned T cells.
J. Immunol.
150:419-428[Abstract].
|
| 11.
|
Moffatt, S.,
N. Yaegashi,
K. Tada,
N. Tanaka, and K. Sugamura.
1998.
Human parvovirus B19 nonstructural (NS1) protein induces apoptosis in erythroid lineage cells.
J. Virol.
72:3018-3028[Abstract/Free Full Text].
|
| 12.
|
Morey, A. L.,
D. J. Ferguson, and K. A. Fleming.
1993.
Ultrastructural features of fetal erythroid precursors infected with parvovirus B19 in vitro: evidence of cell death by apoptosis.
J. Pathol.
169:213-220[CrossRef][Medline].
|
| 13.
|
Mousset, S.,
Y. Ouadrhiri,
P. Caillet-Fauquet, and J. Rommelaere.
1994.
The cytotoxicity of the autonomous parvovirus minute virus of mice nonstructural proteins in FR3T3 rat cells depends on oncogene expression.
J. Virol.
68:6446-6453[Abstract/Free Full Text].
|
| 14.
|
Nakayama, K.,
K. Nakayama,
L. B. Dustin, and D. Y. Loh.
1995.
T-B cell interaction inhibits spontaneous apoptosis of mature lymphocytes in Bcl-2-deficient mice.
J. Exp. Med.
182:1101-1109[Abstract/Free Full Text].
|
| 15.
|
Ohshima, T.,
M. Iwama,
Y. Ueno,
F. Sugiyama,
T. Nakajima,
A. Fukamizu, and K. Yagami.
1998.
Induction of apoptosis in vitro and in vivo by H-1 parvovirus infection.
J. Gen. Virol.
79:3067-3071[Abstract].
|
| 16.
|
Ohshima, T.,
E. Yoshida,
T. Nakajima,
K. Yagami, and A. Fukamizu.
2001.
Effects of interaction between parvovirus minute virus of mice NS1 and coactivator CBP on NS1-and p53-transactivation.
Int. J. Mol. Med.
7:49-54[Medline].
|
| 17.
|
Ozawa, K.,
J. Ayub,
S. Kajigaya,
T. Shimada, and N. Young.
1988.
The gene encoding the nonstructural protein of B19 (human) parvovirus may be lethal in transfected cells.
J. Virol.
62:2884-2889[Abstract/Free Full Text].
|
| 18.
|
Rayet, B.,
J. A. Lopez-Guerrero,
J. Rommelaere, and C. Dinsart.
1998.
Induction of programmed cell death by parvovirus H-1 in U937 cells: connection with the tumor necrosis factor alpha signalling pathway.
J. Virol.
72:8893-8903[Abstract/Free Full Text].
|
| 19.
|
Rommelaere, J., and P. Tattersall.
1990.
Oncosuppression by parvoviruses, p. 41-58.
In
P. Tijssen (ed.), Handbook of parvoviruses, vol. II. CRC Press, Boca Raton, Fla.
|
| 20.
|
Rommelaere, J., and J. J. Cornelis.
1991.
Antineoplastic activity of parvoviruses.
J. Virol. Methods
33:233-251[CrossRef][Medline].
|
| 21.
|
Smith, A. L.,
R. O. Jacoby,
E. A. Johnson,
F. Paturzo, and P. N. Bhatt.
1993.
In vivo studies with an "orphan" parvovirus of mice.
Lab. Anim. Sci.
43:1751-1782.
|
| 22.
|
Telerman, A.,
M. Tuynder,
T. Dupressoir,
B. Robaye,
F. Sigaux,
E. Shaulian,
M. Oren,
J. Rommelaere, and R. Amson.
1993.
A model for tumor suppression using H-1 parvovirus.
Proc. Natl. Acad. Sci. USA
90:8702870-8702876.
|
| 23.
|
Ueno, Y.,
F. Sugiyama, and K. Yagami.
1996.
Detection and in vivo transmission of rat orphan parvovirus (ROPV).
Lab. Anim.
30:114-119[Abstract/Free Full Text].
|
| 24.
|
Ueno, Y.,
F. Sugiyama,
Y. Sugiyama,
K. Ohsawa,
H. Sato, and K. Yagami.
1997.
Epidemiological characterization of newly recognized rat parvovirus, "rat orphan parvovirus".
J. Vet. Med. Sci.
59:265-269[CrossRef][Medline].
|
| 25.
|
Williams, O.,
T. Norton,
M. Halligey,
D. Kioussis, and H. J. M. Brady.
1998.
The action of Bax and bcl-2 on T cell selection.
J. Exp. Med.
188:1125-1133[Abstract/Free Full Text].
|
| 26.
|
Yaegashi, N.,
T. Niinuma,
H. Chisaka,
S. Uehara,
S. Moffatt,
K. Tada,
M. Iwabuchi,
Y. Matsunaga,
M. Nakayama,
C. Yutani,
Y. Osamura,
E. Hirayama,
K. Okamura,
K. Sugamura, and A. Yajima.
1999.
Parvovirus B19 infection induces apoptosis of erythroid cells in vitro and in vivo.
J. Infect.
39:68-76[CrossRef][Medline].
|
Journal of Virology, April 2001, p. 3965-3970, Vol. 75, No. 8
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.8.3965-3970.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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