Previous Article | Next Article 
J Virol, June 1998, p. 4906-4910, Vol. 72, No. 6
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
An Evolutionarily Conserved Splice Generates a Secreted
Env-Bet Fusion Protein during Human Foamy Virus Infection
Marie-Louise
Giron,
Hugues
de Thé, and
Ali
Saïb*
CNRS UPR 9051, University of Paris VII,
Hôpital Saint-Louis, 75475 Paris Cedex 10, France
Received 3 September 1997/Accepted 17 February 1998
 |
ABSTRACT |
Foamy viruses (spumaretroviruses) represent a retroviral genus
which exhibits unusual features relating it to pararetroviruses. Previously, we reported the existence of a protein species harboring Env, Bel, and Bet epitopes in human foamy virus (HFV)-infected cells
(M. L. Giron, F. Rozain, M. C. Debons-Guillemin, M. Canivet, J. Périès, and R. Emanoil-Ravier, J. Virol.
67:3596-3600, 1993). Here, we identify this protein as a 160-kDa
Env-Bet fusion glycoprotein (gp160) translated from an mRNA species
harboring a highly conserved splice site which deletes the membrane
anchor domain of Env and fuses the env open reading frame
with that of bel1/bet. While gp160 and Bet proteins were
both secreted into the supernatant, only Bet was taken up by recipient
cells. Since Bet plays a key role in the switch from lytic to chronic
infection, secretion of Bet and gp160, together with cellular uptake of
Bet, could be highly relevant for both immune response and development
of HFV infection in vivo.
| |
INTRODUCTION |
Foamy viruses (FV), or spumaviruses,
are complex retroviruses known to induce persistent infection in their
hosts without causing apparent disease (22, 25). Some
properties set the FV apart from all other retroviruses, such as the
formation of a specific pol mRNA and the presence of large
amounts of double-stranded viral DNA in the extracellular virion
(27); in these two features, FV resemble pararetroviruses.
Another property peculiar to the FV is their budding into the
endoplasmic reticulum (ER) rather than from the plasma membrane. This
is attributed to the presence of a dilysine ER-sorting motif in the
cytoplasmic domain of the Env protein (6, 7). Regulatory
genes are located between the 3' end of the env gene and the
3' long terminal repeat and are under the control of an internal
promoter (11). In human foamy virus (HFV), these regulatory
genes are bel1, bel2, bel3 and
bet. The nuclear transactivator Bel1 is essential for viral replication (15), while Bet, a fusion protein between Bel1
and Bel2, plays an important role in the establishment and control of
viral persistence, in vitro as well as in vivo (19, 20).
In a previous work, we have characterized viral polypeptides produced
during HFV infection (5). A specific env
monoclonal antibody (B4) immunoprecipitated four viral glycoproteins
from HFV-infected cells: gp160, gp130, gp70-80, and gp48
(5). We demonstrated that gp70-80 and gp48 correspond to the
surface (SU) and transmembrane (TM) mature Env glycoproteins,
respectively, and that the gp130 polypeptide represents the Env
precursor. However, the gp160 protein was immunoprecipitated by the
Env-specific monoclonal antibody as well as by a specific anti-Bet
antiserum, raising the issue of the existence of a putative Env-Bet
polypeptide.
The present study further characterizes this gp160 protein and
demonstrates that it represents a fusion protein between the env and bel regions derived from a spliced viral
mRNA. The evolutionarily conserved splice deletes the transmembrane
anchor of the TM Env protein as well as the ER retention motif and
fuses the env open reading frame (ORF) with those of
bel or bet. An Env-Bet fusion protein represents
the major form detected during HFV infection, although reverse
transcription-PCR (RT-PCR) experiments demonstrate the existence of an
env-bel1 transcript. While both gp160 and Bet proteins are
secreted into the supernatant, Bet appears to be the only protein taken
up by naive recipient cells. Secretion of viral proteins harboring
regulatory or structural domains or both could have major implications
for the biology of these viruses in an in vivo context both for immune
response and virus-cell interactions.
| |
MATERIALS AND METHODS |
Cells and virus.
Mycoplasma-free HFV stocks were grown on
U373-MG cells, a human neural cell line maintained in Dulbecco's
modified Eagle's medium supplemented with nonessential amino acids,
sodium pyruvate, and 10% fetal calf serum. COS-6, a simian cell line,
and BHK21, a hamster cell line, were maintained in the same medium.
Virus stocks were subjected to titer determination by the end-point dilution method on U373-MG cells as described previously
(20).
Transfection experiments.
COS-6 cells were transfected with
Lipofectin reagent (Gibco, BRL) as specified by the manufacturer. At
48 h posttransfection, the cells were lysed in lysis buffer and
proteins were studied by immunoprecipitation.
Protein analysis.
For immunoprecipitation assays, acutely
HFV-infected cells or transfected cells (107 cells) were
labeled with [35S]methionine-cysteine (50 µCi/ml; 1,245 Ci/mmol specific activity; Dupont NEN) for different times in minimal
essential medium lacking methionine-cysteine and supplemented with 5%
fetal calf serum. The cells were lysed in 50 mM Tris-HCl (pH 7.4)-100
mM NaCl-5 mM MgCl2-1% Triton X-100-0.5% deoxycholate,
0.05% sodium dodecyl sulfate [SDS]-3 mM phenylmethylsulfonyl
fluoride for 30 min at 4°C. After centrifugation, the supernatant was
collected and immunoprecipitated with a rabbit anti-whole-virus
antiserum as described previously (5). For
immunoprecipitation assays with supernatants of transfected cells, 75 µCi of [35S]methionine-cysteine per ml was used in
minimal essential medium lacking methionine-cysteine without fetal calf
serum, as previously described (23).
The antibodies (Ab) used were serum from HFV-infected rabbits
(20), a mouse monoclonal Ab (D11) against the Bet protein, a
mouse monoclonal Ab (B4) against the SU domain of the Env protein, and
rabbit polyclonal anti-Bel1 and anti-Bel2 antisera (kindly provided by
R. M. Flügel), all at a 1/100 dilution.
Peptide mapping and acid treatment.
U373-MG-infected cells
were immunoprecipitated with the rabbit anti-HFV polyclonal antiserum.
After immunoprecipitation and polyacrylamide gel electrophoresis
(PAGE), the slab gel was rinsed with water and dried without Amplify.
After autoradiography, the bands corresponding to Bet, gp160, and gp130
were cut out of the gel and placed on a second SDS-polyacrylamide slab
gel. The proteins were digested with 5 µg of V8 protease
(Worthington, Freehold, N.J.) in a stacking gel essentially as
described previously (3). For the acid treatment, the same
procedure was used to collect the gp160 and gp130 bands, which were
resuspended in 100 µl of glycine-acetate buffer (pH 4) and treated
for 1 h at 37°C in the presence of 0.2 mM phenylmethylsulfonyl
fluoride and 10 U of aprotinin. Then 100 µl of 2× electrophoresis
buffer was added and the samples were analyzed by SDS-PAGE.
Generation of eukaryotic expressing vectors.
The
Bet-expressing plasmid was obtained by blunting the
AatII-SalI (nucleotides [nt] 9341 to 12034 from
the infectious clone, pHSRV13) fragment with the Klenow enzyme and
subsequent subcloning into the unique SmaI site of the
pSG5M-expressing vector.
To study the formation of the Env-Bel fusion proteins, plasmid p2EB was
constructed by cutting pHSRV13 with
BanI and
SalI.
The insert was blunted with the Klenow enzyme and
subcloned in
the pSG5M-expressing vector at the
SmaI site of
the polylinker.
This plasmid contains the second ATG (nt 6526 on the
HFV map)
found at the 5' end of the
env gene. Plasmid p1EB,
which expresses
the Env protein from the first ATG (nt 6495), was
created by inserting
the
KpnI-
SpeI insert
fragment obtained by PCR (direct, ATT TT
G GTA CCA
TCT TGG
CAA C; reverse, TTG TGG AAT
ACT AGT CAT ATT TAC; the
KpnI
and the
SpeI sites are underlined) into p2EB
at the
KpnI-
SpeI
sites, replacing the 5' end of
this latter plasmid. Deletion mutants
with mutations in the pEB
plasmids were established by cutting
with
BamHI (nt 9675 from HFV map) and subsequent religation, leading
to plasmids
p1Env
Bel and p2Env
Bel, in which the
bel region is
missing.
Point mutants were obtained with the QuickChange site-directed
mutagenesis kit (Stratagene). The conservative mutation site
on the
donor splice site at the 3' end of
env was obtained with
the
following primers: direct, AAG GAA TTG G
CA ACT TTT TAT;
reverse:
ATA AAA AGT T
GC CAA TTC CTT (the point mutation is
underlined).
The
NheI-
XmnI insert containing the
mutation was sequenced and
subcloned in the same sites of the parental
plasmid. The resulting
plasmids, p1EctB and p2EctB, were used in
transfection experiments.
Plasmids p1EB
RGD and p2EB
RGD were
constructed by the same procedure
with the following primers: direct,
CCT TAT GGA GAT
GGG GGT GAT
GCA; reverse, TGC ATC ACC
CC
C ATC TCC ATA AGG, to insert the codon
mutation agg
ggg
(Arg
Gly) into the RGD motif in the
bet gene
on p1EB and
p2EB, respectively. The
BglII-
BglII insert was
sequenced
and recloned into the parental vector.
RT-PCR and PCR experiments.
For RT-PCR analysis, total
cellular RNAs from infected or transfected cell pellets were extracted
with an RNA extraction kit (Bioprobe Systems). RT-PCR experiments were
performed with the Access RT-PCR system (Promega). Briefly, 500 ng of
total RNA was used as the template for the synthesis of the
first-strand cDNA for 45 min at 48°C in the presence of avian
myeloblastosis virus reverse transcriptase. After denaturation at
94°C, the synthesis of the second strand and enzymatic amplifications
were carried out with Tfl DNA for 40 cycles of 94°C for
45 s, 54°C for 45 s, and 72°C for 1 min. The primers used
were as follows: direct, GAT TAC CAC ATT TGG TTG GAAT (nt 9091 to 9112 on the HFV map [primer A]); reverse, GTT TTG GAC CTT CTG AGC A (nt
10001 to 10019 [primer B]).
Southern blots, prepared by standard procedures (
24), were
hybridized overnight with an [
-
32P]dCTP-labeled probe
at 42°C in 5× SSC (1× SSC is 0.15 M NaCl
plus 0.015 M sodium
citrate)-0.1% SDS-5× Denhardt's solution-50%
formamide-100 µg
of denatured salmon sperm DNA per ml. Washing
was performed in 0.1×
SSC-0.1% SDS buffer at 60°C for 30 min twice.
Plasmid pHSRV13 was
used as a probe for PCR hybridization.
DNA sequencing.
DNA inserts or PCR products subcloned in the
pGEM-Easy vector (Promega) were sequenced with the ThermoSequenase kit
(United States Biochemicals) as specified by the manufacturer.
| |
RESULTS |
A gp160 protein is immunoprecipitated with anti-Bel1, anti-Bel2,
anti-Bet, and anti-Env antisera in HFV-infected cells.
Protein
extracts from U373-MG cells infected with 1 PFU/cell for 4 days were
immunoprecipitated with a set of different Ab raised against HFV
proteins. An antiserum obtained from HFV-infected rabbits and
recognizing all the HFV proteins gave rise to the classical pattern
previously described (5), corresponding to structural as
well as regulatory viral gene products. The B4 monoclonal Ab raised
against SU Env protein precipitated four distinct glycoproteins: the
gp130 Env precursor, SU as a smear between 70 and 80 kDa, the 48-kDa
TM, and a glycoprotein at 160 kDa (5). Interestingly, the
use of rabbit polyclonal anti-Bel1 and anti-Bel2 Abs as well as a mouse
monoclonal anti-Bet (D11) Ab precipitated the same gp160 polypeptide,
strongly suggesting that this protein species harbors both Env and
Bel/Bet epitopes (Fig. 1).
View larger version (60K):
[in this window]
[in a new window]
|
FIG. 1.
Identification of Env and Bel gene products by
immunoprecipitation of protein extracts from HFV-infected cells. M,
molecular mass markers. Rabbit anti-HFV polyclonal antiserum on protein
extracts from noninfected cells (negative control) or from infected
cells (positive control) were used as controls. Rabbit polyclonal
anti-Bel1 and anti-Bel2 Abs and mouse monoclonal anti-Bet (D11) and
anti-SU-Env (B4) Abs were used to detect the gp160 band.
|
|
To confirm that the gp160 protein presented both Env and Bel/Bet
epitopes, peptide-mapping experiments were performed. After
immunoprecipitation and autoradiography, the bands corresponding
to
gp130, gp160, and Bet proteins were cut out from the gel and
placed on
a second SDS-polyacrylamide slab gel. The proteins were
then digested
with V8 protease as previously described (
3).
Figure
2A shows that several polypeptides are
shared by gp130
and gp160 and others are shared by gp160 and Bet. These
results
are consistent with the presence of both Env and Bel/Bet
sequences
in the 160-kDa glycoprotein.
View larger version (52K):
[in this window]
[in a new window]
|
FIG. 2.
The gp160 band does not represent a protein complex. (A)
Peptide mapping of gp130, gp160, and Bet. Several bands are shared by
gp130 and gp160 and others are shared by gp160 and Bet (arrows). (B)
Low-pH treatment of gp160 and gp130. M, molecular mass markers. The
apparent molecular masses of the two proteins are not modified after
1 h of incubation at pH 4.
|
|
One hypothesis was to assume that the 160-kDa band could constitute a
complex between the gp130 Env precursor (or a major
part of it) and the
Bel or Bet proteins. This putative complex
might remain stable during
SDS-PAGE migration, as previously described
for homodimers of the Env
precursor of human immunodeficiency
virus type 2 (
16). In
this case, although the complex was resistant
to 1% SDS and reducing
agents, the authors succeeded in obtaining
protein dissociation after
acid treatment. Thus, after immunoprecipitation
with the rabbit
polyclonal anti-HFV antiserum, SDS-PAGE migration,
and autoradiography,
the gp130 and gp160 bands were extracted
from the gel and incubated for
1 h at pH 4. After neutralization,
the proteins were subjected to
SDS-PAGE on a second gel (see Materials
and Methods). As shown in Fig.
2B, acid treatment also failed
to dissociate the gp160. Moreover, in
protein extracts prepared
in the presence of 1% SDS or under highly
reducing conditions,
no dissociation of the gp160 protein was detected
(data not shown).
Altogether, these results strongly suggest that the 160-kDa band is not
a complex formed between Env and Bel/Bet proteins
but a glycoprotein
harboring Bel, Bet, and Env sequences.
An evolutionarily conserved splicing event generates the Env-Bel
fusion protein.
The presence of a high-molecular-weight fusion
Env-Bel/Bet glycoprotein could be accounted for by the existence of a
multiply spliced mRNA (derived from the long terminal repeat) which
would either suppress the stop codon in the env ORF or,
indirectly, change the ORF of the env gene upstream of the
stop codon, leading to the production of an Env-Bel or Env-Bet fusion
product. To assess this possibility, RT-PCR experiments were performed
with specific primers which flank the end of the env gene
and the bel region (Fig. 3A).
After 4 days of infection of U373-MG cells with wild-type HFV (0.5 PFU/cell), total RNAs were extracted and RT-PCR was performed as
described in Materials and Methods. With the primers used, we were able
to amplify four different RNA species at approximately 900, 780, 600, and 480 bp (Fig. 3A). PCR products were cloned, and three independent
clones of each amplification were sequenced. The 900-bp species
represents the full-length RNA. The 600-bp species corresponds to the
previously described 301-bp splice generating the Bet mRNA also
harbored by HFV (12, 21). Interestingly, the 780-bp
species corresponds to a 119-bp splice (nt 9307 to 9425) flanked by
typical donor and acceptor sites, as previously described
(12). This 119-bp deletion results in an ORF which deletes
the transmembrane domain of the Env protein and changes the
env ORF in its 3' end by suppressing the stop codon by
fusing in frame the env ORF to the bel/bet one.
Note that three amino acids (Asp-Cys-Ile) are inserted between Env and
Bel (Fig. 3B). These splice sites are highly conserved among the
spumaretroviruses sequenced to date (8, 9, 13, 14), even in
the remotely related feline foamy virus (26) (Fig. 3C), suggesting an important role of this specific splicing event in the
biology of this retroviral family. The 780-bp RNA could encode a
putative Env-Bel1 product, while the 480-bp species, which harbors the
119-bp as well as the 301-bp splice, generates an Env-Bet fusion
protein. Note that this latter RNA species and the full-length RNA are
the most abundant species detected by RT-PCR.
View larger version (21K):
[in this window]
[in a new window]
|
FIG. 3.
(A) RT-PCR experiments on total RNA from HFV-infected
cells. Four distinct classes of RNAs, which correspond to the four RNA
species of 900, 780, 600, and 480 bp, are amplified. Plasmid pHSRV13
was used as a probe for PCR hybridization. (B) Schematic representation
of the different mRNA species which can be detected between primer A
and primer B. Note that three new amino acids are created by the
splicing event (Asp-Cys-Ile in boldface type). The dilysine ER
retention motif is also depicted in the env ORF. (C)
Sequence comparisons of known and putative splice sites among different
sequenced FV. The donor and acceptor splice sites flank the
transmembrane anchor sequence of the Env TM protein and fuse the
env ORF with the transactivator one. SFVcpz, simian FV from
chimpanzee; FeFV, feline FV.
|
|
To obtain more insights into the formation of the gp160 protein, p1EB
and p2EB, harboring the entire
env and
bel ORFs,
as
well as the 3' LTR, under the transcriptional control of a simian
virus 40 early promoter were constructed (see Materials and Methods).
p1EB contains the first ATG (nt 6495 on the HFV map) of the
env ORF, while p2EB harbors only the second initiator ATG
codon (nt
6526). Since similar results were obtained with the two
constructs,
we report only those obtained with p2EB. After transfection
of
p2EB into COS-6 cells, we were able to immunoprecipitate not only
the 130-kDa Env precursor and the Bet protein but also the 160-kDa
glycoprotein (Fig.
4). Moreover, upon
transfection of the p2Env
Bel
plasmid (a mutant of p2EB with the
entire
bel and
bet gene sequences
deleted), the
130-kDa Env protein was clearly detected by immunoprecipitation,
but
neither Bet nor gp160 was detected. Furthermore, cotransfection
of p2Env
Bel and a Bet-expressing vector (pSGBet) did not lead
to the reappearance of the gp160 band, confirming our first biochemical
results that gp160 is not a complex between gp130 and Bet (Fig.
4).
View larger version (64K):
[in this window]
[in a new window]
|
FIG. 4.
Identification of the gp160 protein in pEB-transfected
cells. Immunoprecipitation was performed with the rabbit anti-HFV
antiserum. M, molecular mass markers. Cells were transfected with p2EB,
p2EnvBel, p2EnvBel plus pSGBet, and p2EctB. Mutation of the
splice acceptor site abolishes the formation of gp160. Note the absence
of the gp160 species but the detection of Bet in p2EctB-transfected
cells.
|
|
To directly prove that the 160-kDa protein was generated from a 119-bp
splice event at the 3' end of the
env gene, a conservative
point mutation was generated on the 5' donor splice site, changing
a GT
into CT (nt 9307 and 9308 on the HFV map), leading to the
p2EctB
plasmid. As expected, after transfection of COS-6 cells
with this
construct, the Env precursor and the Bet protein were
both
immunoprecipitated by the rabbit polyclonal anti-HFV antiserum
but the
gp160 glycoprotein was no longer immunoprecipitated (Fig.
4). Moreover,
RT-PCR experiments performed on total RNA extracted
from
p2EctB-transfected cells did not detect the 780- and 480-bp
bands (data
not shown). These results directly demonstrate that
gp160 is translated
from an mRNA harboring the 119-bp splice in
the
env gene.
Note that in our immunoprecipitation assays, we
failed to detect
cleavage products of the gp130 or gp160 protein
from the
pEB-transfected COS-6 cells (Fig.
4).
The fusion protein is secreted into the supernatant.
Since the
gp160 protein possesses a signal peptide from the Env protein but lacks
its transmembrane anchor domain and the dilysine ER retention motif, we
wondered whether it could be secreted in the extracellular compartment
by the cellular secretory pathways. Therefore, we transfected COS-6
cells with p2EB and labeled the transfected cells with
[35S]Met-Cys-containing medium 48 h
posttransfection. After overnight labeling, we immunoprecipitated the
cytoplasmic extracts of transfected cells or filtered culture
supernatant (0.45-µm-pore-size filter [Corning]) with the rabbit
anti-HFV antiserum and performed SDS-PAGE. Although gp160, gp130, and
Bet were immunoprecipitated in transfected cells, only gp160 and Bet
were detected in the supernatant (Fig. 5,
lane 3). This result has been confirmed by the use of the D11 monoclonal Ab directed against Bet (data not shown). The absence of the
gp130 protein in the acellular culture medium and the use of p2EB
instead of the lytic wild-type virus strongly suggest that the
detection of Bet and gp160 in the cell culture supernatant is due not
to cell lysis but, rather, to their secretion from the transfected
cells. However, in HFV-infected BHK21 cells, gp160 and Bet were also
found in the culture medium (together with cleavage products from
gp160) prior to cell lysis (data not shown).
View larger version (63K):
[in this window]
[in a new window]
|
FIG. 5.
Secretion and uptake of Env-Bet and Bet proteins.
Cytoplasmic extracts from cells transfected with p2EB (lane 1),
p2EBRGD (lane 2) and supernatants from p2EB (lane 3)- and p2EBRGD
(lane 4)-transfected cells are shown. The upper (200-kDa) band probably
represents a cellular protein. After 18 h of labeling, filtered
culture medium from transfected cells was incubated with naive
recipient COS-6 cells for 4 h and subsequent immunoprecipitations
were performed with the rabbit anti-HFV antiserum on cytoplasmic
extracts. Lane 5, extracts from p2EB-transfected cells; lane 6, extracts from p2EBRGD-transfected cells.
|
|
Of the total HFV proteins immunoprecipitated in infected cells, 5 to
15% were found in the supernatant exclusively as Bet
and gp160. We
wondered whether these proteins could be internalized
by naive
recipient cells. To test this hypothesis, COS-6 cells
were transfected
with the p2EB vector. At 48 h posttransfection,
the cells were
labeled for 2, 6, 16, or 18 h and acellular supernatants
were
incubated with 10
6 recipient COS-6 cells at 37°C for 2, 4 or 6 h. After intensive
washes of the cell layer with
phosphate-buffered saline (to avoid
detection of viral proteins
adsorbed onto the plasma membrane),
the cells were collected and HFV
proteins were immunoprecipitated
with the rabbit polyclonal anti-HFV
antiserum. Approximately 10
to 20% of secreted labeled viral proteins
were taken up in 4 h.
As shown in Fig.
5, Bet is clearly taken up
whereas gp160 is undetectable.
However, such failure to detect gp160 in
recipient cells could
be a function of the sensitivity of our
immunoprecipitation assay.
Furthermore, some Bet was found in nuclear
extracts from recipient
cells, demonstrating that Bet is internalized
and transported
and not simply adsorbed on the plasma membrane (data
not shown).
The specificity, the in vivo relevance, and the putative
cellular
partners of the uptake mechanism will be the focus of future
studies.
Analysis of the Bet sequence reveals the presence of an Arg-Gly-Asp
(RGD) motif (amino acids 294 to 296), a sequence implicated
in complex
recognition mechanisms between cells, implying the
presence of proteins
from the integrin family (fibronectin, laminin,
and fibrinogen) at the
cell surface (reviewed in references
4 and
18). In some viruses, the presence of a functional
RGD motif
is required for infection or expression of their cytopathic
effects
(
1,
2,
17). We wondered whether this sequence could
be
involved in the uptake of Bet. A plasmid (p2EB
RGD) harboring
the
GGD motif instead of RGD in Bet was constructed and transfected
into
COS-6 cells as above. The mutated Bet, produced in the supernatant,
was
taken up by naive cells in the same way as the wild-type one
(Fig.
5,
lane 6). Detection of Bet from p2EB
RGD-transfected cells,
both
within the cell and in the supernatant, was weaker than from
p2EB-transfected cells, possibly reflecting a higher lability
of this
mutant (Fig.
5). Thus, while Bet and gp160 are secreted,
only Bet is
taken up by an RGD-independent mechanism.
| |
DISCUSSION |
In this report, a new HFV fusion protein species, translated from
an mRNA harboring a 119-bp splice at the end of the env ORF,
is described. This splice deletes the transmembrane anchor of the Env
protein, its dilysine ER retention motif and fuses the env
ORF to the bel/bet ORF. The 160-kDa Env-Bet glycoprotein and
Bet can both be secreted, but only Bet is taken up by naive recipient
cells via an RGD-independent mechanism.
This article describes a new LTR-derived mRNA, harboring a 119-bp
intron in the env gene, which, depending upon the presence or absence of the 301-bp intron in the bet gene, can lead to
the production of env-bel1 or env-bet mRNA. This
119-bp splice could be harbored by either the genomic, the
pol, or the env mRNA. While this very small
deletion precludes its detection on a Northern blot, the presence of
this splice in p2EB-transfected cells demonstrates that it can indeed
be found in env transcripts. The 119-bp intron is located
between exon 6 (which harbors the internal promoter) and exon 7 (encoding the bel genes) and is used in the synthesis of
bel1 and bet mRNAs from the internal promoter
(10-12). The fusion protein detected is an Env-Bet protein,
but RT-PCR experiments demonstrate the existence of an
env-bel1 transcript (Fig. 3). Interestingly, the different
spliced RNAs in Fig. 3A can also be directly amplified by PCR without
an RT step on extrachromosomal DNA extracted from HFV-infected cells,
as well as from molecular clones generated from chronically infected
cells (reference 21 and data not shown). This
suggests either that these viral mRNAs are nonspecifically
retrotranscribed in infected cells or that these splice events are part
of full-length viral DNAs, as is the case of the bet splice
in HFV (21).
The fusion protein is inserted into the ER membrane via the peptide
signal at the 5' end of Env but subsequently behaves differently from
the wild-type Env and is secreted. We found this protein in
supernatants of pEB-transfected cells and HFV-infected BHK21 cells,
consistent with the deletion of both the TM anchor and the dilysine ER
retention motif. Bet is also secreted and taken up by noninfected cells
(Fig. 5). Considering the role of Bet in viral resistance and our
demonstration that Bet-expressing stable cell lines are resistant to
HFV-induced lysis (23a), uptaken Bet protein could impair
viral replication in recipient cells and thus behave as a virokine.
| |
ACKNOWLEDGMENTS |
We thank Axel Rethwilm and Dirk Lindemann for communicating
results before publication. We warmly thank A. M. Poorters and N. Honoré for technical assistance, D. Sitterlin for help in some
experiments, and the Laboratoire Photographique de l'Institut d'Hématologie for the photographic work. We thank Libin Ma
(Leiden) for providing pSG5M.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: CNRS UPR 9051, University of Paris VII, Hôpital Saint-Louis, 75475 Paris Cedex
10, France. Phone: 33.1.53.72.40.79. Fax: 33.1.53.72.40.90. E-mail: alisaib{at}infobiogen.fr.
| |
REFERENCES |
| 1.
|
Bai, M.,
B. Harfe, and P. Freimuth.
1993.
Mutations that alter an Arg-Gly-Asp (RGD) sequence in the adenovirus type 2 penton base protein abolish its cell-rounding activity and delay virus reproduction in flat cells.
J. Virol.
67:5198-5205[Abstract/Free Full Text].
|
| 2.
|
Chang, K. H.,
C. Day,
J. Walker,
T. Hyypia, and G. Stanway.
1992.
The nucleotide sequences of wild-type coxsackievirus A9 strains imply that an RGD motif in VP1 is functionally significant.
J. Gen. Virol.
73:621-626[Abstract/Free Full Text].
|
| 3.
|
Cleveland, D. W.,
S. G. Fischer,
M. W. Kirschner, and U. K. Laemmli.
1997.
Peptide mapping by limited proteolysis in sodium dodecyl sulfate and analysis by gel electrophoresis.
J. Biol. Chem.
252:1102-1106[Abstract/Free Full Text].
|
| 4.
|
D'Souza, S. E.,
M. H. Ginsberg, and E. F. Plow.
1991.
Arginyl-glycyl-aspartic acid (RGD): a cell adhesion motif.
Trends Biochem. Sci.
16:246-250[Medline].
|
| 5.
|
Giron, M. L.,
F. Rozain,
M. C. Debons-Guillemin,
M. Canivet,
J. Périès, and R. Emanoil-Ravier.
1993.
Human foamy virus polypeptides: identification of env and bel gene products.
J. Virol.
67:3596-3600[Abstract/Free Full Text].
|
| 6.
|
Goepfert, P. A.,
K. L. Shaw,
G. Ritter, Jr., and M. J. Mulligan.
1997.
A sorting motif localizes the foamy virus glycoprotein to the endoplasmic reticulum.
J. Virol.
71:778-784[Abstract].
|
| 7.
|
Goepfert, P. A.,
G. Wang, and M. J. Mulligan.
1995.
Identification of an ER retrieval signal in a retroviral glycoprotein.
Cell
82:543-544[Medline].
|
| 8.
|
Herchenröder, O.,
R. Renne,
D. Loncar,
E. K. Cobb,
K. K. Murthy,
J. Schneider,
A. Mergia, and P. A. Luciw.
1994.
Isolation, cloning, and sequencing of simian foamy viruses from chimpanzees (SFVcpz): high homology to human foamy virus (HFV).
Virology
201:187-199[Medline].
|
| 9.
|
Kupiec, J. J.,
A. Kay,
M. Hayat,
R. Ravier,
J. Périès, and F. Galibert.
1991.
Sequence analysis of the simian foamy virus type 1.
Gene
101:185-194[Medline].
|
| 10.
|
Löchelt, M., and R. M. Flügel.
1995.
The molecular biology of human and primate spumaretrovirus, p. 239-292.
In
J. A. Levy (ed.), The Retroviridae. Plenum Press, New York, N.Y.
|
| 11.
|
Löchelt, M.,
W. Muranyi, and R. M. Flügel.
1993.
Human foamy virus genome possesses an internal, bel1-dependent and functional promoter.
Proc. Natl. Acad. Sci. USA
90:7317-7321[Abstract/Free Full Text].
|
| 12.
|
Muranyi, W., and R. M. Flügel.
1991.
Analysis of splicing pattern of human spumaretrovirus by polymerase chain reaction reveals complex RNA structures.
J. Virol.
65:727-735[Abstract/Free Full Text].
|
| 13.
|
Renshaw, R. W., and J. W. Casey.
1994.
Transcriptional mapping of the 3' end of the bovine syncytial virus genome.
J. Virol.
68:1021-1028[Abstract/Free Full Text].
|
| 14.
|
Renshaw, R. W.,
M. A. Gonda, and J. W. Casey.
1991.
Structure and transcriptional status of bovine syncytial virus in cytopathic infections.
Gene
105:179-184[Medline].
|
| 15.
|
Rethwilm, A.
1995.
Regulation of foamy virus gene expression.
Curr. Top. Microbiol. Immunol.
193:1-24[Medline].
|
| 16.
|
Rey, M. A.,
B. Krust,
A. G. Laurent,
L. Montagnier, and A. G. Hovanessian.
1989.
Characterization of human immunodeficiency virus type 2 envelope glycoprotein precursor during processing.
J. Virol.
63:647-658[Abstract/Free Full Text].
|
| 17.
|
Roivainen, M.,
T. Hyypia,
L. Piirainen,
N. Kalkkinen,
G. Stanway, and T. Hovi.
1991.
RGD-dependent entry of coxsackievirus A9 into host cells and its bypass after cleavage of VP1 protein by intestinal proteases.
J. Virol.
65:4735-4740[Abstract/Free Full Text].
|
| 18.
|
Ruoslahti, E., and M. D. Pierschbacher.
1986.
Arg-Gly-Asp: a versatile cell recognition signal.
Cell
44:517-518[Medline].
|
| 19.
|
Saïb, A.,
M. Koken,
P. van der Spek,
G. Périès, and H. de Thé.
1995.
Involvement of a spliced and defective human foamy virus in the establishment of chronic infection.
J. Virol.
69:5261-5268[Abstract].
|
| 20.
|
Saïb, A.,
M. Neves,
M. L. Giron,
M. C. Guillemin,
J. Valla,
J. Peries, and M. Canivet.
1997.
Long-term persistent infection of domestic rabbits by the human foamy virus.
Virology
228:263-268[Medline].
|
| 21.
|
Saïb, A.,
J. Périès, and H. de Thé.
1993.
A defective human foamy provirus generated by pregenome splicing.
EMBO J.
12:4439-4444[Medline].
|
| 22.
|
Saïb, A.,
J. Périès, and H. de Thé.
1995.
Recent insights into the biology of the human foamy virus.
Trends Microbiol.
3:173-178[Medline].
|
| 23.
|
Saïb, A.,
F. Saal,
M. L. Giron,
J. Valla,
F. Hojman,
J. Périès, and M. Canivet.
1993.
Comparative studies on IL-6 production status in HTLV-I chronically infected cell lines derived from different HTLV-I associated pathologies.
J. Biol. Regul. Homeostatic Agents
7:79-84.
|
| 23a.
| Saïb, A. Unpublished data.
|
| 24.
|
Sambrook, J.,
E. F. Fritsch, and T. Maniatis.
1989.
In
Molecular cloning: a laboratory manual, 2nd ed.
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
|
| 25.
|
Weiss, R. A.
1988.
A virus in search of a disease.
Nature
333:497-498[Medline].
|
| 26.
|
Winkler, I.,
J. Bodem,
L. Haas,
M. Zemba,
H. Delius,
R. Flower,
R. M. Flügel, and M. Löchelt.
1997.
Characterization of the genome of feline foamy virus and its proteins shows distinct features different from those of primate spumaviruses.
J. Virol.
71:6727-6741[Abstract].
|
| 27.
|
Yu, S. F.,
D. N. Baldwin,
S. R. Gwynn,
S. Yendapalli, and M. L. Linial.
1996.
Human foamy virus replication: a pathway distinct from that of retroviruses and hepadnaviruses.
Science
271:1579-1582[Abstract].
|
J Virol, June 1998, p. 4906-4910, Vol. 72, No. 6
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Delebecque, F., Suspene, R., Calattini, S., Casartelli, N., Saib, A., Froment, A., Wain-Hobson, S., Gessain, A., Vartanian, J.-P., Schwartz, O.
(2006). Restriction of Foamy Viruses by APOBEC Cytidine Deaminases. J. Virol.
80: 605-614
[Abstract]
[Full Text]
-
Schiffer, C., Lecellier, C.-H., Mannioui, A., Felix, N., Nelson, E., Lehmann-Che, J., Giron, M.-L., Gluckman, J. C., Saib, A., Canque, B.
(2004). Persistent Infection with Primate Foamy Virus Type 1 Increases Human Immunodeficiency Virus Type 1 Cell Binding via a Bet-Independent Mechanism. J. Virol.
78: 11405-11410
[Abstract]
[Full Text]
-
Shaw, K. L., Lindemann, D., Mulligan, M. J., Goepfert, P. A.
(2003). Foamy Virus Envelope Glycoprotein Is Sufficient for Particle Budding and Release. J. Virol.
77: 2338-2348
[Abstract]
[Full Text]
-
Meiering, C. D., Linial, M. L.
(2002). Reactivation of a complex retrovirus is controlled by a molecular switch and is inhibited by a viral protein. Proc. Natl. Acad. Sci. USA
99: 15130-15135
[Abstract]
[Full Text]
-
Lecellier, C.-H., Neves, M., Giron, M.-L., Tobaly-Tapiero, J., Saib, A.
(2002). Further Characterization of Equine Foamy Virus Reveals Unusual Features among the Foamy Viruses. J. Virol.
76: 7220-7227
[Abstract]
[Full Text]
-
Lecellier, C.-H., Vermeulen, W., Bachelerie, F., Giron, M.-L., Saib, A.
(2002). Intra- and Intercellular Trafficking of the Foamy Virus Auxiliary Bet Protein. J. Virol.
76: 3388-3394
[Abstract]
[Full Text]
-
Meiering, C. D., Rubio, C., May, C., Linial, M. L.
(2001). Cell-Type-Specific Regulation of the Two Foamy Virus Promoters. J. Virol.
75: 6547-6557
[Abstract]
[Full Text]
-
Pietschmann, T., Zentgraf, H., Rethwilm, A., Lindemann, D.
(2000). An Evolutionarily Conserved Positively Charged Amino Acid in the Putative Membrane-Spanning Domain of the Foamy Virus Envelope Protein Controls Fusion Activity. J. Virol.
74: 4474-4482
[Abstract]
[Full Text]
-
Tobaly-Tapiero, J., Bittoun, P., Neves, M., Guillemin, M.-C., Lecellier, C.-H., Puvion-Dutilleul, F., Gicquel, B., Zientara, S., Giron, M.-L., de Thé, H., Saïb, A.
(2000). Isolation and Characterization of an Equine Foamy Virus. J. Virol.
74: 4064-4073
[Abstract]
[Full Text]
-
Bansal, A., Shaw, K. L., Edwards, B. H., Goepfert, P. A., Mulligan, M. J.
(2000). Characterization of the R572T Point Mutant of a Putative Cleavage Site in Human Foamy Virus Env. J. Virol.
74: 2949-2954
[Abstract]
[Full Text]
-
Callahan, M. E., Switzer, W. M., Matthews, A. L., Roberts, B. D., Heneine, W., Folks, T. M., Sandstrom, P. A.
(1999). Persistent Zoonotic Infection of a Human with Simian Foamy Virus in the Absence of an Intact orf-2 Accessory Gene. J. Virol.
73: 9619-9624
[Abstract]
[Full Text]
-
Pietschmann, T., Heinkelein, M., Heldmann, M., Zentgraf, H., Rethwilm, A., Lindemann, D.
(1999). Foamy Virus Capsids Require the Cognate Envelope Protein for Particle Export. J. Virol.
73: 2613-2621
[Abstract]
[Full Text]
-
Linial, M. L.
(1999). Foamy Viruses Are Unconventional Retroviruses. J. Virol.
73: 1747-1755
[Full Text]
Top
Abstract
Introduction
