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J Virol, January 1998, p. 853-856, Vol. 72, No. 1
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
A Moloney Murine Leukemia Virus-Based Retroviral
Vector Pseudotyped by the Insect Retroviral gypsy
Envelope Can Infect Drosophila Cells
Laure
Teysset,1
Jane C.
Burns,2
Hiroko
Shike,2
Barbara L.
Sullivan,2
Alain
Bucheton,1 and
Christophe
Terzian1,*
Centre de Génétique
Moléculaire, CNRS, Gif-sur-Yvette,
France,1 and
Department of
Pediatrics, University of California, San Diego, School of
Medicine, La Jolla, California2
Received 27 March 1997/Accepted 14 October 1997
 |
ABSTRACT |
The gypsy element of Drosophila
melanogaster is the first retrovirus identified so far in
invertebrates. Previous data suggest that gypsy
ENV-like ORF3 mediates viral infectivity. We have produced in the
293GP/LNhsp70lucL.3 human cell line a Moloney murine leukemia virus-based retroviral vector pseudotyped by the gypsy
ENV-like protein. We have shown by immunostaining that the
gypsy envelope protein is produced in 293GP/LNhsp70lucL.3
cells and that vector particles collected from these cells can
infect Drosophila cells. Our results provide direct
evidence that the infectious property of gypsy is due to
its ORF3 gene product.
| |
TEXT |
The gypsy (or
mdg-4) element is a 7,469-bp retroelement (11)
that resides within the genome of Drosophila melanogaster. gypsy shares several structural and functional
properties with the vertebrate retroviruses (reviewed in reference
14): (i) gypsy is flanked by long
terminal repeats (LTRs) with a U3-R-U5 retroviral structure that
directs transcription, (ii) gypsy has three open reading
frames (ORFs) analogous in size and position to the retroviral
gag, pol, and env genes (Fig.
1), and (iii) the organization of the
pol gene is similar to that of the vertebrate retroviral
pol genes (Fig. 1).
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FIG. 1.
(A) Organization of the gypsy element. Two
LTRs flank a central region which contains the three ORFs corresponding
to gag, pol, and env. The order of the
various domains of the pol gene is indicated (PR, protease;
RT, reverse transcriptase; RH, RNase H; IN, integrase). The predicted
54-kDa protein encoded by ORF3 is represented with potential
N-glycosylation sites (Y), the signal peptide (SP), the dibasic
cleavage site (arrow), and the transmembrane domain (TM). (B) Genetic
organization of pHCMV-gyp-ENV. Numbers refer to gypsy
nucleotides. CMV, human cytomegalovirus immediate-early promoter; SPL
and PA, splicing and polyadenylation signals, respectively, derived
from the rabbit beta-globin gene.
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|
The presence of a third ORF is characteristic of the invertebrate
gypsy family retroelements, including among others
17.6 (16), 297 (7), and
tom in Drosophila (18) and TED in
Trichoplusia (10). The predicted 54-kDa protein
encoded by gypsy ORF3 shares several general features with
vertebrate retroviral ENV glycoproteins (13, 17) (Fig. 1).
It contains a potential signal peptidase cleavage site at its N
terminus and a potential protease cleavage site Arg-Ser-Arg-Arg similar
to the vertebrate retroviral ENV cleavage motif (Arg/Lys)-X-Lys-Arg.
There are three consensus N-linked glycosylation sites and a C-terminal
transmembrane domain.
gypsy is controlled by the flamenco gene of
D. melanogaster (15). The gypsy ORF3
RNA and protein are detected only in females homozygous for
flamenco permissive alleles (13, 17). It has been
shown that extracts of homozygous flamenco permissive pupae and females can infect flamenco permissive larvae (9,
17) and that these female extracts contain infectious
gypsy associated with the product of gypsy ORF3
(17). These data support the concept that gypsy
is an autonomous retrovirus, the first identified so far in
invertebrates, and that gypsy ENV-like ORF3 mediates viral
infectivity. To analyze the infectious properties of gypsy ENV-like ORF3, we packaged a mammalian retroviral vector based on
Moloney murine leukemia virus (MoMLV) in the gypsy ENV-like protein.
MoMLV-based retroviral vectors can be pseudotyped with
gypsy envelope.
Previous studies have shown that the
MoMLV recombinant genome can be pseudotyped with the vesicular
stomatitis virus envelope glycoprotein (VSV-G) (2). To test
whether the MoMLV genome could be pseudotyped with the gypsy
envelope protein (gyp-ENV), the human cell line 293GP/LNhsp70lucL.3
(LNhsp70lucL) (8) (Fig. 2) was
transiently transfected with the pHCMV-gyp-ENV plasmid expressing the
gypsy ORF3 under the control of the strong immediate-early promoter of human cytomegalovirus. To construct pHCMV-gyp-ENV, a
BamHI-BglII fragment containing a part of the
leader region (nucleotides 535 to 568; accession no. M12927
[11]), ORF3, and a part of the 3' LTR (nucleotides
5551 to 7412) of gypsy were inserted into the unique
BamHI site in pHCMV-Bam (19).
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FIG. 2.
Genetic organization of LNhsp70lucL provirus. LTR, MoMLV
LTR; neo, neomycin phosphotransferase gene; hsp70, D. melanogaster heat shock 70 promoter; luc, firefly
luciferase gene. Arrows indicate direction of
transcription.
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The expression of
gypsy envelope protein in LNhsp70lucL
cells was assessed by immunostaining with the 7B3 monoclonal antibody
(Ab) raised against the
gypsy ORF3 product (
17).
Seventy-two
hours after transfection with pHCMV-gyp-ENV, cells were
washed
in phosphate-buffered saline (PBS), fixed in cold acetone,
washed
in PBS, and then incubated sequentially with the 7B3 monoclonal
Ab (diluted 1/2) for 1 h and fluorescein isothiocyanate
(FITC)-conjugated
anti-mouse immunoglobulin G (Vector Laboratories)
(diluted 1/100)
for 40 min. The expression of
gypsy envelope
protein was identified
in transfected LNhsp70lucL cells by confocal
microscopy (Fig.
3).
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FIG. 3.
Localization of the gypsy envelope protein by
confocal microscopy. (A) Negative control: nontransfected LNhsp70lucL
cells stained by anti-gyp-ENV and anti-mouse FITC antibodies; (B)
LNhsp70lucL cells transiently transfected with pHCMV-gyp-ENV and
stained with both anti-gyp-ENV and anti-mouse FITC antibodies. Bar, 10 µm.
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The infection efficiency of the retroviral vector packaged with gyp-ENV
was determined and compared to that observed with
the same
retroviral vector packaged with the VSV-G. These VSV-G-pseudotyped
vectors have previously been shown to infect and stably integrate
into
insect cells (
12). Briefly, 10
6
LNhsp70lucL cells were plated onto 100-mm-diameter plates overnight.
Cells were transfected with 20 µg of the appropriate plasmid by
standard calcium phosphate coprecipitation. At 72 h
posttransfection,
cell culture supernatants were collected, filtered
(pore size,
0.45 µm), and diluted 1/2 in
Schneider
medium (GIBCO) supplemented
with 10% fetal bovine serum and
2-µg/ml Polybrene, a polycation
that increases infection efficiency.
Pseudotyped vectors were
produced as previously described
(
19). Supernatants harvested
from LNhsp70lucL cells with no
envelope protein contain noninfectious
viral particles and were used as
a negative control. A Western
blot was performed in order to compare
the relative number of
particles produced by gyp-ENV-expressing
producer cells and producer
cells with no envelope protein. Virus
supernatants were concentrated
by ultracentrifugation (SW41 rotor,
30,000 rpm for 1 h at 4°C),
and equivalent volumes of
resuspended viral pellets were loaded
on a 10% polyacrylamide gel.
Immunostaining for the MoMLV viral
core protein with anti-p30 antibody
was performed as previously
described (
3) and revealed that
approximately equal numbers
of viral particles were present in each
viral pellet (Fig.
4).
Thus, the
noninfectious viral supernatant and the supernatant
containing the
pseudotyped vector contain approximately equal
numbers of viral
particles.
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FIG. 4.
Immunoblot of pellets of viral particles produced from
gyp-ENV-expressing cells and LNhsp70lucL cells with no envelope
protein. The blot was stained with anti-p30 antiserum to detect the CA
capsid protein.
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The
Schneider 2 D. melanogaster somatic cells were used as
recipient cells for the infection experiments. These cells do not
express the
gypsy envelope as determined by immunostaining
with
the 7B3 Ab (data not shown). Subconfluent
Schneider 2 cells were
incubated with these supernatants. Virus-containing
supernatant
was replaced by fresh
Schneider medium 24 h
after infection. Centrifugation,
at 1,000 ×
g for
1 h, of cells immediately after the addition
of the supernatant
was tested as a means to increase infection
efficiency (Table
1). The influence of gravitational force
during
centrifugation on the net direction of Brownian movement of
virus
particles can increase the probability of contact between the
virus and the cell surface (
1).
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TABLE 1.
Luciferase activity in Schneider 2 cells
infected by retroviral vector LNhsp70lucL pseudotyped by
gyp-ENV or VSV-G envelope
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To test for evidence of infection, cells were washed 4 days
postinfection in PBS, lysed, and briefly centrifuged, and supernatants
were assayed for luciferase activity according to the manufacturer's
(Promega) instructions. Infection with the gyp-ENV-pseudotyped
MoMLV
was clearly demonstrated following centrifugation (Table
1). The
luciferase activity measured after infection with the
VSV-G-pseudotyped
MoMLV was at least 100-fold higher than the
activity measured after
infection with the gyp-ENV-pseudotyped
MoMLV.
To examine the host range of the gyp-ENV-pseudotyped particles and to
confirm the persistence of proviral DNA, MOS-55 cells
from the mosquito
Anopheles gambiae were infected with supernatant
containing
noninfectious viral particles or gyp-ENV-pseudotyped
particles.
Infections were performed in triplicate, cells were
harvested at
72 h postinfection, and cell lysates and DNA were
prepared as
previously described (
7). No detectable luciferase
activity
was observed in MOS-55 cells infected with or without
centrifugation
(mean light units per microgram of protein: no
centrifugation, 1.3 ± 0.1; with centrifugation, 1.4 ± 0.3; negative
control, 1.0).
Amplification with nested primers specific for
the proviral LTR
(
7) yielded the expected 176-bp amplicon from
the mosquito
cell lines infected with the gyp-ENV-pseudotyped
vector. No PCR
product was seen following amplification of DNA
from mock-infected
cells, despite a positive signal following
amplification with muscle
actin primers (
7). These results
suggest that the
gyp-ENV-pseudotyped particles are weakly infectious
in MOS-55 cells.
Hence, our results demonstrate that the gyp-ENV-pseudotyped vector
was able to infect
Schneider 2 cells, although the infection
efficiency was lower than that for the VSV-G-pseudotyped
vectors.
gypsy envelope protein can be stably expressed in
LNhsp70lucL packaging cell lines.
Because expression of
gypsy envelope did not appear to be toxic in LNhsp70lucL
cells (no syncytia were observed), we generated a stable cell line that
constitutively expresses the gypsy envelope. Cells were
cotransfected with a plasmid conferring resistance to puromycin and
pHCMV-gyp-ENV, at a ratio of 1:5. Transfected cells were selected
in puromycin (1 µg/ml) and G418 (400 µg/ml). Colonies were screened for their ability to produce infectious pseudotypes by incubating culture supernatants with Schneider 2 cells and assaying for luciferase activity 4 days postinfection. The clone producing the highest virus titer was chosen, and expression of gyp-ENV was verified by staining with anti-gyp-ENV.
To increase the level of luciferase expression, infected
Schneider 2 cells were heat shocked (30 min at 37°C,
1 h of recovery
at 23°C, 30 min at 37°C, and 45 min at 23°C)
prior to analysis
of luciferase activity. The effects of different
concentrations
of Polybrene on infection efficiency were tested, and
maximal
luciferase activities were obtained with 8 µg/ml.
The luciferase activity observed following infection with culture
supernatants derived from the packaging cell lines constitutively
expressing the
gypsy envelope was markedly increased
compared
to infection with supernatants produced after transient
transfection
(Table
1). As expected, heat shock activated the hsp70
promoter,
thus increasing luciferase activity.
In this study, we show for the first time that the vertebrate
retroviral recombinant genome MoMLV can be pseudotyped by the
invertebrate retroviral
gypsy envelope protein. Our results
provide
direct evidence that the infectious properties of
gypsy (
9,
17) are due to its ORF3 gene product.
The fact that no cell fusion was observed in the LNhsp70lucL
cell line constitutively expressing the gyp-ENV protein may be
due to the absence of an appropriate receptor on the mammalian
cell
surface or to a low abundance of the
gypsy ENV protein
expressed
in these cells (
6).
Our data suggest that the
gypsy envelope is correctly
processed in human cells. Transport of the retroviral envelope
protein
to the cell surface and its proteolytic cleavage are
necessary
steps for incorporation into virus-like particles and
budding
of infectious particles. Among the endoproteases, it has been
shown that the furin protease has a role in cleavage of the human
immunodeficiency virus envelope proteins (
5). Dfur1 and
Dfur2
(
4) are two
D. melanogaster genes
exhibiting homology with
the human furin. This suggests that furin-like
function is conserved
among taxa, which could explain the
faithful processing of the
gypsy envelope protein
in mammalian cells.
Retroviral pseudotypes produced from our stable packaging cell line
expressing the
gypsy envelope protein provide a convenient
method to analyze the tropism of the
gypsy envelope in
different
insect species. The
gypsy envelope-pseudotyped
vector as a transformation
vector in invertebrates other than
D. melanogaster is under investigation
in our lab.
| |
ACKNOWLEDGMENTS |
We are grateful to Alain Pélisson for helpful discussions and
critical reading of the manuscript. We thank Fabienne Chalvet for
providing the gypsy fragment containing the ORF3 gene. We thank Bernard Mignotte's group for help with cell culture techniques, Spencer Brown for help with confocal microscopy, and Jean-Pierre Rousset for help with the luciferase activity determination. We thank
François-Loïc Cosset for helpful discussion and the p30 anticapsid Ab. The 7B3 Ab was kindly provided by Victor Corces, Baltimore, Md.
L.T. is a recipient of a fellowship from the Ministère de
l'Enseignement Supérieur et de la Recherche. This work
was supported by grants from the Centre National de la Recherche
Scientifique, the Association pour la Recherche sur le Cancer (contract
1132), and the Actions Coordonnées Concertées des Sciences
du Vivant.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Centre de
Génétique Moléculaire, CNRS, 91198 Gif-sur-Yvette
Cedex, France. Phone: 33-1 69 82 31 46. Fax: 33-1 69 82 43 86. E-mail:
terzian{at}cgm.cnrs-gif.fr.
| |
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J Virol, January 1998, p. 853-856, Vol. 72, No. 1
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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