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Journal of Virology, October 1999, p. 8884-8889, Vol. 73, No. 10
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Walleye Dermal Sarcoma Virus: OrfA N-Terminal End
Inhibits the Activity of a Reporter Gene Directed by Eukaryotic
Promoters and Has a Negative Effect on the Growth of Fish and
Mammalian Cells
Z.
Zhang and
D.
Martineau*
Département de Pathologie et
Microbiologie, Faculté de Médecine
Vétérinaire, Université de Montréal,
Saint-Hyacinthe PQ J2S 7C5, Canada
Received 13 January 1999/Accepted 22 June 1999
 |
ABSTRACT |
Walleye dermal sarcoma virus (WDSV) is a fish retrovirus causing a
skin tumor termed walleye dermal sarcoma, which develops and regresses
on a seasonal basis. The WDSV genome contains three short open reading
frames designated orfA, orfB, and
orfC in addition to the viral structural genes,
gag, pol, and env. orfA
and orfB transcripts are detected in tumors by reverse
transcription-PCR. Recently, OrfA, whose amino acid sequence is similar
to that of cyclins A and D, has been shown to complement a
cyclin-deficient yeast strain. We report that expression of the
accessory gene orfA inhibited nonspecifically the activity
of a reporter gene directed by various eukaryotic promoters. In
addition, stable transfection with the wild-type orfA
generated substantially fewer G418-resistant colonies in both fish and
mammalian cells than the parent vector. An orfA mutant
expressing only the first N-terminal 49 residues of the full-length
protein had the same negative effect on the activity of the reporter
gene and on the number of stably transfected colonies as the
full-length OrfA. Thus, OrfA inhibits cell growth and/or causes cell
death, and the first 49 N-terminal residues of this protein are
sufficient to cause these negative effects.
| |
TEXT |
The walleye dermal sarcoma virus
(WDSV) is a retrovirus etiologically associated with a skin tumor
termed walleye dermal sarcoma (WDS) that is endemic in walleye fish
throughout North America (2, 14). In contrast to other
tumors induced by animal retroviruses, WDS cyclically develops and
regresses (12, 13). Tumors develop in the fall, when viral
expression and virion production are minimal (3), and then
persist and increase in size through winter until spring, when viral
expression and virion production are maximal and when tumors
synchronously undergo coagulation necrosis and show a second type of
cell death morphologically consistent with apoptosis (13).
Viral transmission presumably occurs at that time, when fish congregate
for spawning (2, 18, 19).
Retroviruses are currently classified into simple and complex
retroviruses. The latter group includes lentiviruses and spumaviruses, whose larger genome contains accessory genes in addition to the structural genes gag, pol, and env.
Accessory genes are involved in pathogenicity, in oncogenesis, and in
the control of viral gene expression (5, 6, 10). The large
size of WDSV genome (12.708 kb [DNA]) and the presence of three
accessory genes place WDSV in the complex retrovirus group (8,
12).
orfA and orfB are two nonoverlapping open reading
frames located immediately downstream of the env gene. Their
high transcription levels in regressing tumors and their low
transcription levels in developing tumors support that they play a
major role in tumor development and regression (11, 16).
The similarity of orfA to cyclin D at the amino acid
sequence level is reflected functionally by its ability to complement a
cyclin-deficient yeast strain mutant. Since cyclins are involved in
both neoplasia and cell death, orfA might play a role in the benign biological behavior of WDS in nature (11). The
objective of the present study was to determine the role of
orfA in tumor development and/or regression.
W12 cells, derived from WDS tissue, were cultured at 25°C without
CO2 supplement. The firefly (Photinus pyralis)
luciferase gene was used as a reporter gene and was placed under the
control of the simian virus 40 (SV40) early promoter in pSV40 (Promega, Madison, Wis.). The pCMV reporter plasmid was constructed by placing the HindIII-BamHI 2.7-kb fragment containing
the luciferase gene from pGL2-basic (Promega) under the control of the
cytomegalovirus (CMV) immediate early promoter in pcDNA3 (Invitrogen,
Carlsbad, Calif.). pActin was derived from the plasmid pFV6a-CAT
(provided by P. B. Hackett [University of Minnesota]), which
contains the promoter and the first intron of the carp 
-actin gene.
pActin was constructed by replacing the CAT gene of pFV6a-CAT with the BamHI-NheI 2.7-kb fragment containing the
luciferase gene. pMMTV was derived from the pMSG vector (Pharmacia
Biotech Inc., Baie D'Urfé, Quebec, Canada) by placing the
NheI-SalI fragment containing the luciferase gene
downstream of the mouse mammary tumor virus (MMTV) full-length
promoter. To construct pLTR, two primers, LTR-F (forward,
5'-CTCGGTACCAAATGAGAAACTAA) and LTR-R (reverse,
5'-CGGAAGCTTTGTTAATTCAAATT), were used to PCR amplify the
WDSV long terminal repeat (LTR) (590 bp) from a WDSV clone
(12). The 5' ends of the LTR-F and LTR-R primers contained a
KpnI site and a HindIII site, respectively. The purified PCR amplicon was inserted in the polylinker sequence upstream of the firefly luciferase gene in the promoterless vector pGL2-basic (Promega). To construct porfA (Fig.
1), the entire orfA gene (894 nucleotides [nt]) was PCR amplified from a WDSV full-length clone
(12) by using the primers ORFA-F (forward, 5'-ATAAGACTACTACAGGGTACGTCC) and ORFA-R (reverse,
5'-AGTTATCCTATTGGATCGACGACG) and subcloned into pSVK3
(Pharmacia Biotech Inc.). To generate pEcoRV, EcoRV was used
along with SmaI to remove the orfA ATG initiation
codon. pNdeI was generated by NdeI digestion of
porfA, end repair with Klenow fragment, and intramolecular
ligation; the resulting 2-bp insertion created a +2 frameshift at 144 bp (NdeI site) and fused five out-of-frame codons.
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FIG. 1.
(A) WDSV genomic structure and restriction sites of
orfA (894 bp). The WDSV genome is 12.7 kb long (DNA) and
contains three accessory genes, orfA, orfB, and
orfC. (B) Genetic organization and mutants of the WDSV
accessory gene orfA. The enzymes used to generate deletions
are shown on the top of the full-length orfA
(porfA). Dark boxes indicate the original open reading
frame, whereas empty boxes illustrate disruption of the original open
reading frame. The mutant pNdeI was generated by insertion of 2 bp at
the NdeI site, resulting in the disruption of the open
reading frame and fusing 5 out-of-frame codons [wt orfA:
139-GCA (A) ACC (T) CAT (H) ATG (M) GTC (V) CTG (L) TTA (L) AAA
(K)-163; pNdeI: 139-GCA (A) ACC (T) CAT (H) ATA (I) TGG (W)
TCC (S) TGT (C) TAA (stop codon)-163]. The fidelity of the mutants was
confirmed by sequencing.
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W12 and NIH 3T3 cells were transfected with Lipofectamine (GIBCO BRL,
Gaithersburg, Md.), and luciferase activity was determined with a Lumat
LB 9501/16 (Berthold). pCMV, which contains a neo selection
marker gene, was used to cotransfect W12 and NIH 3T3 cells (from the
American Type Culture Collection) for stable transfection with
wild-type (wt) orfA and orfA mutants. Transfected
cells were selected with G418 for 12 days, fixed, and stained with
Giemsa, and G418-resistant colonies were counted. Total cellular RNA
was isolated from cells stably transfected with porfA and
pNdeI and treated with DNase. cDNA was generated and PCR amplified with the primers ORFA-F and ORFA-R. The reverse transcription-PCR products were analyzed by Southern blotting with an orfA-specific
probe generated by PCR from a WDSV full-length clone
(12). In order to detect apoptosis, we examined transiently
transfected cells to detect DNA fragmentation by agarose gel
electrophoresis. Cells floating in medium and adherent cells were
collected, pelleted, and resuspended in 20 µl of phosphate-buffered
saline containing 50 µg of RNase per µl. The suspension was
directly loaded into a 1.8% agarose gel well containing 10 µl of 4%
sodium dodecyl sulfate. The well was sealed with low-melting-point
agarose and incubated at room temperature for 45 min. Following
electrophoresis, genomic DNA was examined under UV light.
The BLAST program was used to search databases. Amino acid alignments
were carried out visually. The amino acid substitutions used to
determine sequence similarities were as follows: D = E, F = Y = W, M = L = V = I, R = K = H, and
S = A, S = T.
orfA expression nonspecifically inhibits luciferase
activity directed by eukaryotic promoters.
In fish cells,
orfA expression inhibited the luciferase activity directed
by WDSV-LTR and the carp -actin promoter 18-fold and 3.5-fold
respectively (Fig. 2). To further define
this negative effect, several reporter vectors containing eukaryotic
promoters (CMV immediate early promoter, SV40 early promoter and MMTV
full-length LTR) were constructed and cotransfected with
porfA. Wt orfA expression decreased the
luciferase activity directed by the CMV, SV40 and MMTV promoters 278-, 34-, and 317-fold, respectively (Fig. 2).
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FIG. 2.
Effect of orfA expression on luciferase
reporter gene activity driven by different promoters in a fish cell
line, W12. The effector plasmid, porfA, was cotransfected
with reporter genes consisting of the luciferase gene directed by the
CMV immediate-early promoter, the MMTV full-length LTR, SV40 early
promoters, the WDSV full-length LTR, and a carp -actin promoter. The
parent plasmid of porfA (pSVK3) was used as a negative
control.
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To further characterize OrfA functionally, a deletion mutant (pEcoRV)
and an insertion mutant (pNdeI) were constructed. pEcoRV
lost
orfA inhibitory activity in both NIH 3T3 and W12 cells,
while
in contrast, pNdeI showed the same inhibitory activity as the
full-length
orfA (Fig.
3).
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FIG. 3.
Mutational analysis of orfA gene. The
luciferase reporter plasmid pCMV and pLTR were respectively
cotransfected with full-length orfA and the orfA
mutants. The parent plasmid of porfA (pSVK3) was used as a
negative control. (A) W12 cells; (B) NIH 3T3 cells.
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orfA expression decreases the number of G418-resistant
cell colonies.
To determine if orfA expression affects
cell growth, we conducted stable-transfection experiments. In W12
cells, the number of G418-resistant colonies resulting from
cotransfection with orfA or pNdeI was reduced by 34%
compared to the number of colonies stably transfected with the parent
vector pSVK3. In NIH 3T3 cells, the number of G418-resistant colonies
was reduced 4 times by the expression of wt orfA and 5 times
by the pNdeI mutant (Fig. 4; Table
1).
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FIG. 4.
Effect of orfA expression on the number of
G418-resistant cells transfected with the neo resistance
gene. Full-length orfA (porfA) (panels 2 and 5)
and the mutant pNdeI (panels 3 and 6) were cotransfected with plasmid
pcDNA3, which expresses the selection marker gene, neo.
Transfected cells were selected for two weeks with G418. G418-resistant
colonies were fixed with methanol and stained with Giemsa.
Transfections in panels 1, 2, and 3 were conducted in NIH 3T3 cells and
transfections in panels 4, 5, and 6 were carried out in W12 fish cells.
In panels 1 and 4, cells were transfected with the parental plasmid
pSVK3.
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orfA is expressed in G418-resistant cells.
Total
cellular RNA was isolated from G418-resistant cells after 2-week drug
selection, and cDNAs were synthesized by using a poly(dT) primer and
PCR amplified. Hybridization of PCR products with a WDSV
orfA probe demonstrated that orfA was expressed
in G418-resistant cells generated by porfA and pNdeI
transfection (Fig. 5). We observed DNA
fragmentation into oligonucleosome-sized DNA fragments in some
regressing tumors (20), indicating that tumor regression may
occur through apoptosis. Next, we asked if the reduction in the number
of G418-resistant colonies resulting from orfA expression
was the result of apoptosis. NIH 3T3 cells were transfected with
porfA and the mutant pNdeI. Transfected cells were collected
at different times (0, 6, 12, 18, 24, 30, and 36 h)
posttransfection. Control cells treated with actinomycin showed typical
DNA fragmentation. No DNA fragmentation was detected from cells
transfected with either wt orfA or the mutant pNdeI (data
not shown).
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FIG. 5.
Southern blot analysis of PCR-amplified orfA
cDNA expressed in G418-resistant cells. orfA cDNA was
synthesized from total RNA isolated from G418-resistant W12 cells and
PCR amplified with the primer pair ORFA-F-ORFA-R. The PCR product was
electrophoresed, blotted onto a nylon membrane, and hybridized with a
WDSV orfA-specific probe. Lane 1, cells transfected with the
parent plasmid; lanes 2 and 3, cells transfected with porfA
and pNdeI, respectively. The molecular size of orfA is
indicated on the right.
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We demonstrated that transfecting cells from widely different classes
of vertebrates with the full-length
orfA inhibited the
activity of a reporter gene directed by various eukaryotic promoters
and that this inhibition did not require any other viral gene
products
(Fig.
2 to
4). In addition,
orfA expression resulted
in
fewer piscine and mammalian cells surviving G418 selection.
Removal of
the
orfA initiation codon (pEcoRV) abolished
orfA
inhibitory
activity, and a frameshift at codon 50 preserved the
inhibitory
activity shown by the full-length
orfA.
Considered together, these
observations indicate that
orfA
expression inhibits cell growth
and/or causes cell death and that
the first 49 N-terminal residues
of this protein are sufficient to
cause these negative effects.
These observations also suggest that
these effects are mediated
by OrfA protein, not by
orfA
mRNA. The expression of
orfA in cells
that survived G418
selection is best explained by an arrest in
the cell cycle, by low
levels of
orfA expression, or by the selection
of
orfA mutants.
A database search using the BLAST program showed that the pNde mutant
sequence was similar to that of the N-terminal helix,
the
1 helix,
and a large part of the
2 helix of the first cyclin
box of cyclin A. The first N-terminal amino acids of OrfA corresponded
exactly to the
second N-terminal amino acid sequence of the human
cyclin A N-terminal
helix (amino acids [aa] 179 to 190), and the
first 12 aa of OrfA were
very similar to those of the cyclin A
N-terminal helix (Fig.
6; Table
2). Other similarities were the
presence
of alanines 235, 259, and 264, characteristic of the
cyclin fold
(numbering from the human sequence), the presence
of the corresponding
alanine 363 and alanine 359 present within
the second repeat, the
presence of lysine 266 and glutamic acid
295, which contact cdk2 in the
cyclin A-cdk2 complex, and the
presence of a hydrophobic patch in the
putative
1 helix, corresponding
to the MRAIL sequence region of the
human cyclin A (
9) (Fig.
6). The degree of similarity
between OrfA and the first cyclin
box of human cyclin A (aa 208 to 303)
as a whole was the same
as that obtained with cyclin D (
11).
However, the similarity
between OrfA amino acid sequence and that of
cyclin D is low in
the N-terminal region, upstream of the cyclin D
helix A (
11).
In contrast, this region was very similar to
the N-terminal region
(including the N-terminal helix) of cyclin A
(Fig.
6; Table
2),
and the similarity of cyclin A
3 and
5 helices
with the corresponding
regions of OrfA is greater than the
similarity of cyclin D helices
C and E with the corresponding regions
of OrfA (Table
2). Thus,
the OrfA amino acid sequence is most
comparable to that of cyclin
A truncated at its N-terminal end.
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FIG. 6.
Amino acid sequence alignment of the orfA
accessory gene in WDSV, WEHV1, and WEHV2 with cyclin A of humans (Hum),
goldfish (Gfish), clam, Chlorohydra viridissima (Hydra), and
Drosophila melanogaster (Dros.). Identical and similar amino
acids are in boldface. The 10 helices that compose the cyclin box
are indicated by boxes. Conserved residues are indicated by asterisks,
notably alanines 234 and 263, lysine 265, and glutamic acid 284 (numbering is from the human sequence).
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TABLE 2.
Comparison of OrfA amino acid sequences with those of D-
and A-type cyclins within the third and fifth -helix of the first
repeat of the cyclin box
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|
Cyclin A has been one of the first cyclins identified as being involved
in cancer (
4). Paradoxically, cyclin A expression
and cdk2
activation have also been associated with apoptosis (
7,
15).
Similarly, OrfA might arrest the cell cycle or cause apoptosis,
or on
the contrary trigger cell proliferation (
11). Strikingly,
a
region of ODV-EC27 (EC27), a protein of
Autographa
californica nucleopolyhedrovirus, which is very similar to the
cyclin box
1 and
2 helices, corresponds exactly to
orfA mutant pNdeI. EC27
arrests the cell cycle and has
functional features of both cyclin
B (it binds cdc2) and cyclin D (it
binds cdk6) (
1). By binding
cdc2, EC27 might be responsible
for cell cycle arrest at G
2/M
by forming an active cyclin
B-like-cdc2 complex that resists degradation
(
1).
Alternatively, a putative OrfA-cdk complex may be inactive.
In the
cdk2-cyclin A complex, the N-terminal cyclin box fold (residues
208 to
306) and the N-terminus helix are both involved in contacting
cdk2.
Thus, the OrfA N terminus (pNde), which contains the N-terminal
helix
and a large part of the first cyclin box of cyclin A, might
bind cdk2
and inactivate it, thereby preventing mitosis. Since
cyclins determine
the substrate specificity of cyclin-cdk complexes,
OrfA could also
modify cdk2 specificity by recruiting different
substrates
(
17) (Fig.
6; Table
2).
Our inability to detect apoptosis in the current transfection studies
might be due to a number of factors.
orfA constitutive
expression might cause apoptosis only at specific steps of the
cell
cycle. Since cell cycles were not synchronized in the current
study,
transfected cells would become apoptotic only as they enter
a specific
step of the cell cycle, and thus only a small number
of transfected
cells would be apoptotic at any given time. It
is also possible that
the low transfection efficiency (5%), as
determined by transfection
with the pCMV-
-Gal plasmid, did not
allow the detection of
apoptosis. OrfA might trigger apoptosis
after a period longer than that
used in our assay. Alternatively,
OrfA may cause cell death by
nonapoptotic mechanisms. Finally,
it is possible that other proteins,
viral or cellular, are involved
in the apoptosis observed in regressing
tumors in addition to,
or instead of,
orfA.
| |
ACKNOWLEDGMENTS |
We thank P. B. Hackett (University of Minnesota) for providing
plasmids and P. Bowser and J. W. Casey for tumor samples.
This work was supported by a grant from the Natural Sciences and
Engineering Research Council of Canada (no. 138236). Z.Z. was partly
supported by a grant from Université de Montréal.
| |
FOOTNOTES |
*
Corresponding author. Mailing address:
Département de Pathologie et Microbiologie, Faculté de
Médecine Vétérinaire, Université de
Montréal, Saint-Hyacinthe PQ J2S 7C5, Canada. Phone: (450) 773-8521, ext. 8307. Fax: (450) 778-8113. E-mail:
martinea{at}ere.umontreal.ca.
| |
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Journal of Virology, October 1999, p. 8884-8889, Vol. 73, No. 10
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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