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Journal of Virology, April 2001, p. 3965-3970, Vol. 75, No. 8
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
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.
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.
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
ABSTRACT
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TABLE 1.
Propagation of the RPV isolate and prototype RV (RV-13)
in several cell linesa
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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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).
|
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).
|
nu/nu) purchased from CLEA Japan
(Tokyo, Japan) in each group were inoculated subcutaneously with
5 × 105 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 mm2 in size within 28 days p.i. In
contrast, only two of the eight developed tumors, averaging 23.4 mm2, 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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FOOTNOTES |
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* 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.
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) 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.
and 
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).
