Subcellular localization analysis of bovine foamy virus Borf1 protein

Abstract

The Borf1 protein is encoded by an immediate-early gene of the bovine foamy virus (BFV) and plays a key role in the viral
life cycle. Borf1 is a DNA binding protein which can transactivate both the long terminal repeat (LTR) and the internal promoter
(IP) of BFV by specifically binding to the transactivation responsive element (TRE). To analyze the subcellular localization
of Borf1 during the BFV life cycle, this gene was cloned into a prokaryotic expression vector and expressed in a soluble form.
After the purification and immunization, we raised the mouse anti-Borf1 serum with a high titer based on ELISA results. Western
blot analysis showed that the antiserum could specifically recognize the Borf1 protein that was expressed in 293T cells. With
this specific serum, we revealed the nuclear and cytoplasmic localization of Borf1 in HeLa cells that was transfected with
Borf1. Moreover, the immuno-fluorescence assay also showed that the localization of Borf1 during the infection and transfection
of BFV was identical.

Full text

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VIROLOGICA SINICA, February 2008, 23 (1):37-42
DOI 10.1007/s12250-008-2893-3

CLC number: S852.65 Document code: A Article ID: 1674-0769 (2008) 01-0037-06
Subcellular Localization Analysis of Bovine Foamy Virus Borf1 Protein*
Juan TAN, Kai WU, Rui CHANG, Qi-min CHEN, Yun-qi GENG and Wen-tao QIAO**
(The Key Laboratory of Molecular Microbiology and Techaology, Ministry of Education and Tianjin Key
Laboratory of Microbial Functional Genomics, College of Life Sciences, Nankai University, Tianjin 300071, China)

Abstract: The Borf1 protein is encoded by an immediate-early gene of the bovine foamy virus (BFV) and plays a
key role in the viral life cycle. Borf1 is a DNA binding protein which can transactivate both the long terminal
repeat (LTR) and the internal promoter (IP) of BFV by specifically binding to the transactivation responsive
element (TRE). To analyze the subcellular localization of Borf1 during the BFV life cycle, this gene was cloned
into a prokaryotic expression vector and expressed in a soluble form. After the purification and immunization, we
raised the mouse anti-Borf1 serum with a high titer based on ELISA results. Western blot analysis showed that the
antiserum could specifically recognize the Borf1 protein that was expressed in 293T cells. With this specific
serum, we revealed the nuclear and cytoplasmic localization of Borf1 in HeLa cells that was transfected with
Borf1. Moreover, the immuno-fluorescence assay also showed that the localization of Borf1 during the infection
and transfection of BFV was identical.
Key words: Bovine foamy virus (BFV); Borf1; Antiserum

Foamy viruses (FVs; also known as spumaviruses
or spumaretroviruses) were once classified as one of
the genera of the Retroviridae. However, they possess
different replication and gene regulation strategies
from other retroviruses and consequently these charac-
teristics led to the creation of a distinct viral subfamily
known as the Spumaretrovinae by the International
Committee on Virus Taxonomy in 2002. In fact, their
replication may bridge the gap between retroviruses
and hepadnaviruses (1). However, they share no
commonalities in their pathology, because FVs have
not been found to cause any apparent diseases (5, 7).
So research concerned with their distinct gene regu-
lation strategies and interaction with host cells has
generated much interest. Furthermore, their distinct
characteristics may contribute to the construction of
gene vector based on FVs and benefit work on disease
therapy.
Tas, the transactivator of FVs, plays a key role in
viral replication and gene regulation by binding with
LTR and IP (4). Recently, it has been reported that the
interaction of Tas and some cellular proteins, such as
the promyelocytic leukemia protein, could affect both
viral gene expression and infectivity (6). The 249aa
Borf1 (or BTas, the Tas equivalent of PFV) is encoded
by the first open reading frame (ORF). Previously, we

Received: 2007-07-23; Accepted: 2007-12 -20
* Foundation items: National Natural Science Foundation of
China (30570072, 30770097) and Natural Science Foun-
dation of Tianjin (05YFJM- JC01000).
** Corresponding author. Tel: +86-22-23504547,
Fax: +86-22- 23501783,
E-mail: wentaoqiao@nankai.edu.cn

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38 Virol. Sin. (2008) 23: 37-42

had demonstrated that Borf1 was the transactivator of
BFV (2) and could regulate viral gene expression by
binding to LTR and IP (3, 8). To further investigate its
structural localization and function, we raised a high-
titer anti-Borf1 serum against the recombinant soluble
Borf1 protein. Western blot analysis and immno-
fluoresence assay showed that this antiserum could
recognize the eukaryotic Borf1 with high specificity.
Also, the nuclear and cytoplasmic localization of
Borf1 was confirmed in HeLa cells that were
transfected with Borf1. Furthermore, with this specific
antibody, we showed the nuclear and cytoplasmic
localization of Borf1 in Fetal Bovine Lung (FBL)
cells that were infected by BFV. These results further
confirmed our findings about the transactivator role of
Brof1 in BFV infection, and provided an effective way
to monitor the activities of Borf1 with this specific
antibody, as well as a way to further investigate the
molecular mechanism of this key process in
infection.

METHODS AND MATERIALS
Cells and virus
Fetal Bovine Lung (FBL), HeLa and 293T cells
(conserved by our lab) were grown in DMEM (Gibco)
supplemented with 10% (v/v) fetal bovine serum
(HyClone) and antibiotics.
Plasmid Construction
The complete coding region of Borf1 was amplified
by PCR from Hirt DNA of BFV. The following two
primers, which incorporate the EcoR I or Sal I site,
were designed for PCR: Forward, 5’-cag gaa ttc gag
atg gct agc gg-3’; Reverse, 5’-cag gtc gac cgc aac aca
tat c-3’. The resulting PCR product was cloned into
pGEX-6p-1 to construct prokaryotic expression plas-
mid pGEX-Borf1, and also cloned into pCMV-Tag2B
to construce eukaryotic expression plasmid Pcmv-
Tag-Borf1. The resultant plasmids were checked by
restriction enzyme analysis and sequencing.
Expression and purification of Borf1
The prokaryotic expression plasmid pGEX-Borf1
was transformed into E. coli BL21 (DE3) (Invitrogen),
and the positive colonies were selected by antibiotics.
Briefly, the desired clone was grown overnight at 37
℃, inoculated into fresh media and grown for an
additional 3 h (OD600 = ~ 0.7) and then induced by
IPTG at a final concentration of 1 mmol/L in culture
medium for 4 h. The expressed fusion proteins were
subsequently purified by Glutathione Sepharose 4B
(Amersham) with PreScission Protease according to
the manufacturer’s instructions. In addition, an anion
exchange column and molecular sieve chromato-
graphy were used to purify this protein.
Generation of Antibody
The purified Borf1 protein was used as the antigen
to raise antiserum in mice. Five BALB/C mice were
firstly immunized with 50 μg purified protein that was
emulsified in an equal volume of Freund’s complete
adjuvant. The inoculations were performed by subcu-
taneous injection in the belly. Freund’s incomplete
adjuvant and 30 μg purified protein were used in
subsequent injections. Three booster injections were
given every other week after the first injection. Three
days after the last immunization, the mice were
sacrificed to prepare the antiserum.
ELISA
First, purified Borf1 was incorporated in a 96-well
plate (0.04 μg per well). Then, each well was
incubated with different dilutions of the anti-Borf1
serum. After washing with PBS, a 1:3 000 dilution of

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Virol. Sin. (2008) 23: 37-42 39

goat anti-mouse peroxidase-labelled secondary antibody
was added. Finally, the wells were treated with TMB
and subsequently analyzed by a Precision Microplate
Reader.
Western blot analysis
293T cells were transfected with the pCMVTag-
Borf1. After 48 h, the cells lysates were electrophore-
tically separated on the SDS-PAGE and electrically
transferred to the PVDF Membranes (Amersham).
Nonspecific protein banding was blocked by treating
the membranes at room temperature for 45 min with
the phosphate buffered saline (PBS) containing 5%
skimmed milk powder. The membranes were washed
five times with the PBS containing 0.05% Tween 20
(PBST) and incubated at room temperature for 1 h
with a 1:3 000 dilution of the anti-Borf1 serum or the
mouse anti-Flag antibody in PBS. After washing five
times with PBST, the membranes were incubated with
a 1:3 000 dilution of the goat anti-mouse peroxidase-
labelled secondary antibody at room temperature for
45 min. The membrane was then washed five times
with PBST, treated with Western Blot Luminal Rea-
gent (Santa Cruz), and exposed to an X-Omat BT Film
(Kodak).
Immuno-fluorescence assay
HeLa cells were grown on coverslips and trans-
fected with the pCMVTag-Borf1 or pCMVTag2B
(Stratagene). After 48 h, cells were fixed in 4% para-
formaldehyde and permeabilized with 0.2% Triton
X-100. Coverslips were blocked with 3% BSA to
reduce the non-specific background. The cells were
reacted at 37℃ for 30 min with the anti-Borf1 serum
diluted 1:400 in PBS, washed in excess PBST and
then reacted with a 1:100 dilution of the Cy2-
conjucted AffiniPure Goat Anti-Mouse IgG (Jackson
ImmunoResearch). Cell nuclei were stained with 4, 6-
diamidino-2-phenylindole (DAPI), and the fluorescent
images were taken on a confocal microscopy.
RESULTS
Gene expression in E.coli
The prokaryotic expression plasmid pGEX-Borf1
was constructed and checked by the three restriction
enzymes Pst I, EcoR I/Sal I and Pvu I (Fig. 1). The
plasmid was then transformed into E. coli BL21
(DE3). Using pGEX-6p-1 transformed E. coli BL21
(DE3) as the control, SDS-PAGE analysis showed that
there was a specific band associated with the pGEX-
Borf1 transformed E. coli BL21 (DE3), which was
consistent with the predicted molecular weight of 54
kDa (Fig. 2).
Purification of Borf1 protein
To obtain purified Borf1, we used three steps in the
purification process. First, the GST-Borf1 was purified
from the bacterial lysates by the affinity chromato-
graphy using Glutathione Sepharose 4B. The cleavage
of the Borf1 protein from GST was achieved by using
PreScission Protease. Later, we purified the Borf1 by
an anion exchange column Resource Q and the

Fig. 1. Identification of construct pGEX-6p-Borf1. 1, DNA
marker; 2, Pst I: 4.0kb, 1.7kb; 3, EcoR I/Sal I: 4.9kb, 0.8kb; 4,
Pvu I: 2.9kb, 1.6kb, 1.2kb.

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40 Virol. Sin. (2008) 23: 37-42


Fig. 2. SDS-PAGE analysis of pGEX-6P-Borf1 expression in E.coli BL21 (DE3). 1, Protein Marker; 2, BL21 (DE3); 3, BL21 (DE3)
+ IPTG; 4, BL21 (DE3) transformed with pGEX-6P-1; 5, BL21 (DE3) transformed with pGEX-6P-1 + IPTG; 6, BL21 (DE3)
transformed with pGEX-6P-Borf1; 7, BL21 (DE3) transformed with pGEX-6P-Borf1 + IPTG..

molecular sieve chromatography (120 mL superdex
G200). Then the superdex elutes was analyzed with
SDS-PAGE. The final purification of the Borf1 exce-
eded 98% (Fig. 3).
Preparation of anti-Borf1 serum
Purified Borf1 protein was immunized in 5 BALB/
C mice. After four immunizations, the titer of anti-
serum was tested with ELISA. The five samples had a
titer of 1:4×104, 1:16×104, 1:4×104, 1:8×104 and
1:16×104, respectively. The results showed that the
antiserum could be used in western blot analysis. Then
the antiserum were harvested and stored in -20℃.
Specificity analysis of anti-Borf1 serum
To test the specificity of the antiserum, pCMVTag-
Borf1 was transfected into 293T cells. After 48 h, the
Flag-Borf1 protein was expressed in 293T cells. Then
hese cells were lysated and immunoblotted with anti-
Borf1 serum and anti-Flag antibody, respectively. The


Fig. 3. SDS-PAGE analysis of Superdex elutes. 1-4, Elutes
sample, elution volume is 12.5 mL, 13 mL, 13.5 mL, 14 mL; 5,
The condensed sample; 6, Marker.
result showed that there is a unique band of ~ 30 kDa
in both lanes (Fig. 4). This demonstrated that the anti-
orf1 serum can recognize the Borf1 protein expressed
in eukaryotic cells and has a similar specificity with
the commercial anti-Flag antibody.
Intracellular localization of Borf1
Using the antiserum, we next tested the intracellular
localization of Borf1. First, we tested the localization
of Borf1 in transfected cells. HeLa cells were
transfected with the pCMVTag-Borf1 or pCMVTag-
2B (negative control). Then, the indirect immuno-
fluorescence staining was used to confirm the locali-
zation of Borf1. The result in Fig. 5A shows that Borf1
was located in both the cytoplasm and the nu- cleus, but


Fig. 4. Western blot assay. Western blot analysis of the
Flag-Borf1 with anti-Flag antibody (lane 1) and the anti-Borf1
serum (lane 2).

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Virol. Sin. (2008) 23: 37-42 41

predominantly in the nucleus. In the negative control,
there was no green fluorescence (Fig. 5B).
Next, we tested the localization of Borf1 during
BFV infection. Because BFV can infect the FBL cells
and lead to the rapid syncytium formation, we chose
these cells as a suitable target. Forty-eight hours after
infection by BFV, the infected FBL cells were used to
perform the immunofluorescence assay with anti-
Borf1 serum. The result showed that Borf1 located in
both the nucleus and cytoplasm (Fig. 5C), but no
green fluorescence was shown in the negative control
(Fig. 5D). All these results show that the anti-Borf1
serum can correctly recognize the natural Borf1 and
can be used for immunofluoresence assay. In addition,
the localization of Borf1 was identical in both trans-
fected cells and infected cells.

Fig. 5. Intracellular localization of Borf1. A: The HeLa cells were transfected with the pCMVTag-Borf1. B: The HeLa cells were
transfected with the pCMV-Tag2B. C: The FBL cells were infected with BFV. D: The FBL cells were not infected with BFV.