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Introduction
Foamy virus (FV) belongs to the retroviridae family and
can infect a wide range of hosts, including chimpanzees,
monkeys, cats, and cows[1,2]. The virus undergoes reverse
transcription (RT) in the cytoplasm and integrates into the
host genome upon viral infection. Like most retroviruses,
the genomic structure of FV from 5' to 3' includes 5' long
terminal repeat (LTR), gag, pol,
env, and 3' LTR[1]. However, FV presents some unique features that are distinct from other
members of retroviridae[3]. A few regulatory genes,
bel-1 (Tas), bel-2,
bel-3, and bet are located between
env and 3' LTR[4,5]. An internal promoter and
a cis-acting element[6,7] were identified within
env gene sequence and a region from 5' LTR to
pol, respectively. In addition, the replication
strategy and viral genomic contents of FV are also quite different
from most retroviruses, but present marked similarities with
hepadnaviruses[3].
Traditional titration of the infectious FV is usually
achieved by using the cytopathic effect
(CPE)[8]. However, not all the cells that are infected by FV show
CPE[9,10]. Therefore, the method based on CPE may have a limitation
on the titration of FV. Other methods, such as RT-PCR, which
has been reported for the detection of prototype foamy
viruses[11,12], does not give the direct quantitation on viral
loading during infection. Recently, a new method called the green
fluorescent protein (GFP)-based indicator cell line was
established for the titration of prototype foamy virus (PFV)
and feline foamy virus (FeFV)[13,14]. The indicator cell line
was constructed by the stable integration of the GFP gene
under the control of the foamy virus LTR promoter. Viral
titration can be monitored by GFP expression after FV
infection. This method is rapid and accurate in detecting FV
infection and is very useful for clinical diagnosis as well as
basic research.
It has long been noted that both RNA and infectious
DNA genomes are present in extracellular FV
virions[15,16]. Azidothymidine (AZT), a potent inhibitor of reverse
transcri-ptase, has been introduced to the indicator cell line of FV for
the detection of the time point of RT during PFV and FeFV
infections[3]. Studies indicate that the RT of FV is not at the
early phase of infection, but occurs late in the replication
cycle before new virus buds from an infected cell. This is
held true for both human and feline
FV[16_18]. However, the step of RT in bovine foamy virus (BFV) has not been defined.
In this study, we employed the indicator cell line system
to assay the infectivity of BFV in 3 different cells in a
non-pathogenic condition. In addition, the replication strategy
of BFV was evaluated by assaying the effect of AZT on BFV
production.
Materials and methods
Cell culture Fetal bovine lung (FBL) cells, 293T, HeLa,
and the BFV indicator cell line (BICL) cells were preserved
by our laboratory (Laboratory of Molecular Virology, Nankai
University, Tianjin, China). The BICL, 293T, and HeLa cells
were cultured in Dulbecco's modified Eagle's medium
(DMEM) supplemented with 10% heat-inactivated fetal calf
serum (FCS), 105 U/L penicillin, and 0.1 g/L streptomycin.
The FBL cells were cultured in DMEM supplemented with
10% FCS, 105 U/L penicillin, 0.1 g/L streptomycin, and
non-essential amino acids. All the cells were cultured at 37 °C in
5% CO2.
Reagents AZT and Lipofectamine was purchased from
Invitrogen (Carlsbad, CA, USA). DMEM was purchased
from Gibco (Carlsbad, CA, USA). The pre-stained molecular
weight markers were purchased from Bio-Red (Hercules, CA,
USA). Other chemicals used in the Western blotting and in
the β-galactosidase enzyme assay in this study were of
analytical grade and commercially available.
Preparation of virus The strain
BFV3026 was isolated from peripheral lymphoid cells by our
laboratory[19]. The virus was harvested and treated in 3 different ways:
(i) after being infected with
BFV3026, the infected cells were trypsinized and
gathered for the co-culture experiment; (ii)
after being infected with BFV and centrifuged at 4
000×g for 10 min, the cell supernatant was filtered through a 0.22 µm filter
membrane for subsequent use; and (iii) after transfection of FBL,
293T, and HeLa cells with the full-length BFV proviral clone
pCMV (cytomegalovirus) - BFV, the cells were
trypsinized and gathered for the co-culture experiment.
Plasmid construction pcDNA3.1(-) was purchased from
Invitrogen (USA). To generate the infectious BFV molecular
clone pCMV-BFV, we assembled the subgenomic BFV clones
step by step based on the provirus clone pBS (short form of
pbluescript)-BFV. The U3 promoter region of 5' LTR was
replaced by the immediate early gene promoter of human
CMV in pBS-BFV to generate pCMV-BFV.
DNA transfections and β-galactosidase enzyme assay
The cells were plated in 6-well plates
(2×105/well) and cultured at 37 °C for 24 h. After that, the cells were transfected
with 3 μg pCMV-BFV plasmid and 0.5 μg
pSV-β-galactosidase control vector (Promega, Madison, WI,, USA) to each
well according to the manufacturer's instructions
(No 92 Lipofectamine reagent manual, Invitrogen, USA). In the
control well of each kind of cell, 3 μg pcDNA 3.1(-) and 0.5
μg pSV-β-galactosidase control vector were added. The
transfected cells were tested for the transfection efficiency
following the manufacturer's instructions (Technical Bulletin
No 097, Promega, USA) and equal aliquot cells were added
into the BICL cells to detect BFV infection.
GFP activation assay by BFV The BICL cells were plated
at a density of 2×105 per well in 6-well plates in DMEM
medium. On the following day, the medium was removed
and the cells were incubated with infected cells through the
co-culture method or incubated with the supernatant of
infected cells. Forty-eight hours after co-culturing, the
infected cells were observed and counted for GFP
expression under an inverted fluorescence microscope (Olympus
IX-71, Shinjuku-ku, Tokyo, Japan). Subsequently, the total
BICL cells were trypsinized and counted with a
hemocyto-meter. The percentage of GFP-positive cells was calculated
by the ratio of GFP positive cells against the total cultured
cells.
SDS-PAGE and Western immunoblotting The infected
cells were mixed with 5×sample buffer containing 0.6 mol/L
Tris-HCl (pH 6.8), 25% glycerol, 2% SDS, and 0.1%
bromophenol blue, and incubated for 5 min at 100 °C. 50 µL of each
sample was loaded and electrophoresed for 80 min at the
constant voltage of 100 V. After electrophoresis,
polypeptides and markers separated on each gel were
electrotrans-ferred onto a 0.2 µm nitrocellulose membrane overnight at 30
V under chilled conditions. Each membrane was blocked
with a 1% skim milk solution by submerging the membrane in
the solution for 30 min at a room temperature.
For the Western immunoblot analysis, the nitrocellulose
membrane was incubated with an antibody against BTas (Tas
of BFV) at a dilution of 1:5000 in 20 mmol/L Tris-buffered
saline solution with 0.05% Tween 20 (TBST, pH 7.5) for 90
min at an ambient temperature with slow rocking, and then
rinsed at least 3 times with TBST. Then the membrane was
incubated with goat anti-mouse IgG or IgM labeled with
horseradish peroxidase (Santa Cruz biotechnology, Santa
Cruz, CA, USA) for 60 min at an ambient temperature. After
the developing and fixing process, the membrane was
exposed to Kodak XAR-5 film (KODAK (CHINA) LIMITED,
Beijing, China). β-actin was detected as a loading control;
the β-actin antibody was purchased from Sigma (St Louis,
MO, USA).
The result was photographed by AlphaImager 2200
(Alpha Innotech, San Leandro, CA, USA) and analyzed by
ImageJ software (Research Services Branch, National
Institute of Mental Health, Bethesda, Maryland, USA). The
integrated density of each band represented the protein
expression levels.
AZT inhibition experiments The cell lysate prepared
from BFV-infected FBL cells were used to infect
2×105 FBL, 293T, and HeLa cells through the co-culture method at a
ratio of 1:10. The infected cells were co-cultured with the
indicator cells at 48 h post infection. Then the BICL cells
were tested by FV-activated GFP assay. The final
concentration of AZT was 25 µmol/L.
Results
Evaluation of BFV infectivity in non-cytopathic
conditions by BICL BFV preferentially propagates in bovine
fibroblastic cells such as FBL. We found that no significant
CPE was induced in human cell lines such as HeLa and 293T
cells after incubation with excessive
BFV3026. However, the infectivity of BFV on these cells was unclear.
In order to clarify this issue, a BICL system was used for
the detection of viral infectivity. This system is very
sensitive since the infectivity of BFV can be detected by
monitoring GFP expression even in a non-cytopathic state. The
infectivity of BFV against FBL, HeLa, and 293T cells was
evaluated by an indicator cell line through a co-culture
approach. We infected the above cell lines at low MOI
(multiplicity of infection) to ensure that all cells infected did
not induce CPE within 48 h post infection. Then the infected
cells were co-cultured with 2×105 BICL cells at a ratio of 1:10.
The activation of the GFP expression could be induced in
BICL after being co-cultured with the BFV-infected cells
(Figure 1). The GFP-positive cells were counted. Here we
defined the GFP-positive percentage as 1. The ratio of these
3 cell lines was 1.00: 0.19: 0.17.
Moreover, we detected the BFV infection on the
expression level. FBL, HeLa, and 293T cells were infected at the
same MOI. Forty-eight hours after the infection, these
infected cells were lysed, and the expression of BTas was
detected by Western immunoblotting (Figure 2). The
integrative density ratio of BFV-infected FBL, HeLa, and 293T cells
was 1.00:0.22:0.15 (Table 2).
Using BICL to detect the infectivity of plasmid
pCMV-BFV in mammalian cells pCMV-BFV was the infectious
clone of BFV driven by the human CMV immediate early
gene promoter instead of its U3 promoter region of 5' LTR
(Figure 3A). FBL cells were first transfected with
pCMV-BFV and incubated for 4 d. The transfected FBL cells were
co-cultured with BICL cells at a ratio of 1:10. The induction
of the GFP expression could be clearly observed 2 d later
(Figure 3B), indicating that pCMV-BFV can successfully
generate infectious BFV viruses in the cells. Similarly, in
non-cytopathic cell lines such as HeLa and 293T, we also
detected the GFP expression (Figure 4). However, the
infectivity produced by pCMV-BFV in HeLa and 293T cells
(1.62%±0.40% and 1.33%±0.47%, respectively) was lower
than that in FBL cells (2.52%±0.78%; Table 3). Their initial
was 1.00: 0.53: 0.64. After transfection efficiency, the
β-gal report system was employed to calibrate this result. The
ratio after calibration was 1.00: 0.21: 0.14 (Table 3).
Inhibitory effect of AZT on BFV replication
FBL, 293T, and HeLa cells were first infected with
BFV3026 at a ratio of
1:10. Then these cells were either treated with 25 µmol/L
AZT or left untreated for 48 h. The titers of
BFV3026 in these cells were determined on BICL indicator cells which had been
pretreated with AZT for 24 h or left untreated. When the
virus was produced and assayed in the absence of AZT, the
virus replicated very well in all 3 cells and led to high titers of
infectious virus 48 h after infection (Table 4, positive control,
experiment I). When AZT was present during virus
production and in the BICL indicator cells, almost few virus
replications were detected (Table 4, negative control, experiment
II), indicating that AZT effectively inhibited the RT. BFV
titers decreased when AZT was absent from virus
production and present in the viral titration assay (experiment III in
Table 4). Remarkably, the viral titer was down to the
negative control level when AZT was absent from the production
of BFV and present during the titration assay ( Table 4,
experiment IV).
Discussion
Quantitative detection of viral infection is important for
biological studies of FV infection. In this study, we
demonstrate that the titration of BFV can be achieved by using a
GFP-based indicator cell line system. This system is very
useful and sensitive in the detection of BFV replication in
non-cytopathic conditions. In addition, we have
successfully used this indicator cell line system to show that the RT
of BFV also occurs at the late phase during BFV infection.
Although FV can strongly induce CPE in a broad range
of infected cells in vitro, the viruses usually do not cause
clinical syndromes in natural hosts[2]. FV often cause
apathogenic or persistent FV infection in certain cell types
such as primary monocyte, leukemia, and epithelial cancer
cell lines[14,20,21]. Thus, traditional CPE-based FV titration is
greatly restricted in these non-cytopathic cells. As an
sensitive assay, RT-PCR has been used for the identification of
FV infection in cultured cells, experimental animals, or
natural hosts[12,17,22]. However, because of the hard-to-handle
standard test, it is difficult for RT-PCR to quantitate viral
infection, thus it may not be used widely for viral titration. In
1993, Yu et al constructed an indicator cell line system and
used this system to detect prototype FV infection by
employing beta-galactosidase gene as an
indicator[23]. Later studies demonstrated that other reporter genes, such as
luciferase, could also be used for HFV
titration[12,24]. However, both systems need either fixation with a special chemical
substrate or the destruction of infected cells by lysis buffer.
Therefore, it could not be directly applied to viral detection
in living cells. Recently, a GFP-based indicator cell assay
has been introduced for the titer detection of FeFV infection,
and has proved to be a convenient and sensitive
approach[9].
In agreement with these findings, our result provides
further evidence to show that the GFP-based indicator cell
line system is indeed sensitive and can be applied for the
detection of BFV infections, even at non-cytopathic
conditions. Our results show that in the co-culture
experi-ment, the percentage of GFP-positive BICL infected by
FBL-BFV was about 5fold more than that of HeLa-BFV and
293T-BFV. Western immunoblotting result also show different
expression levels of BTas, and the ratio among these 3 cells
shows the same trend as that of BICL detection. However,
in the transfection experiment, the pCMV-BFV-transfected
FBL cells were only about 1.5-fold higher than that of HeLa
and 293T in the BICL system. This discrepancy is likely due
to the low transfection efficiency of plasmid DNA as
compared with the high efficiency of viral genome delivery
during viral infection. As the β-gal report result shows, the
transfection efficiency of FBL cells is lower than those of
HeLa and 293T cells. Moreover, the calibrated ratio
correlates well with that of BFV infection experiment. As a result,
in both experiments, the infectivity detected in FBL cell was
significantly higher than that in human cells such as 293T
and HeLa cells, indicating that the propagation of BFV in
these human cells was limited. This restriction was
suspected to be because of the defects in viral entry, viral
replication, the aberrant expression of either Tas or
Bet[25], or epigenetic modification of BFV proviral
genomes[9].
Using a potent RT inhibitor AZT, Rethwilm and colleagues
restricted HFV and FeFV at a late stage of virus
infection[17,18]. Similarly, we employed the
GFP gene as an indicator to replace the beta-galactosidase gene in the AZT assay, which
allowed us to observe viral infection in a living cell system.
Our results are consistent with their studies and indicate
that RT was a necessary and essential step for BFV
production that took place before the virus release from an infected
cell.
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