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Journal of Virology, November 2000, p. 10658-10669, Vol. 74, No. 22
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Life Cycle of an Endogenous Retrovirus,
ZAM, in Drosophila melanogaster
P.
Leblanc,1
S.
Desset,2
F.
Giorgi,3
A. R.
Taddei,3
A. M.
Fausto,3
M.
Mazzini,3
B.
Dastugue,2 and
C.
Vaury2,*
ENS/INSERM U 412, 69364 Lyon Cedex
07,1 and Unité INSERM U384, 63000 Clermont-Ferrand,2 France, and
Interdepartmental Center for Electron Microscopy, Tuscia
University, 00100 Viterbo, Italy3
Received 3 May 2000/Accepted 4 August 2000
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ABSTRACT |
ZAM is an env-containing member of the
gypsy family of retrotransposons that represents a
possible retrovirus of invertebrates. In this paper, we traced
ZAM mobilization to get information about a potential path
a retroelement may take to reach the germ line of its host. In situ
hybridization on whole-mount tissues and immunocytochemistry analyses with antibodies raised against
ZAM Gag and Env proteins have shown that all
components necessary to assemble ZAM viral particles,
i.e., ZAM full-length RNAs and Gag and Env polypeptides,
are coexpressed in a small set of follicle cells surrounding the
oocyte. By electron microscopy, we have shown that
ZAM viral particles are indeed detected in this somatic lineage of cells, which they leave and enter the closely apposed oocyte. Our data provide evidence that the vesicular traffic and yolk
granules in the process of vitellogenesis play an important role in ZAM transfer to the oocyte. Our data support the
possibility that vitellogenin transfer to the oocyte may help a
retroelement pass to the germ line with no need of its envelope product.
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INTRODUCTION |
ZAM is a 8.4-kb
retroelement that resides within the genome of Drosophila
melanogaster (11). On the basis of sequence
similarity and gene organization, ZAM is a member of a group
of retrotransposons that bears a striking resemblance to the
vertebrate retroviruses. These elements are flanked by long
terminal repeats (LTRs) that direct the transcription of full-length
RNAs representing potential templates for reverse transcription during
mobilization. The LTRs flank three open reading frames (ORFs) analogous
in position and coding potential to the retroviral gag,
pol, and env genes (Fig. 1). Among the diverse classes of
eukaryotic retrotransposons, the presence of a third
env-like ORF (ORF3) is unique to ZAM and a small
group of other members of this family, including gypsy, 297, 17.6, Idefix, and
nomad in D. melanogaster (3, 8, 14, 19,
26), tom in Drosophila ananassae
(25), Osvaldo in Drosophila buzzatii
(15), TED in the lepidopteran Trichoplusia
ni (5), and Yoyo in the medfly
Ceratitis capitata (28). An envelope protein
expressed in vivo has been identified for only three of these elements
(gypsy, tom, and TED) (16, 21,
24, 25), and only one of them, gypsy, has been shown
to date to have infectious properties (9, 22). Although
retroviral Env proteins are known to be involved in viral infectivity
through host cell receptor recognition and fusion of viral and cellular
membranes, the role of the Env glycoproteins encoded by these elements
is still unclear since no budding has ever been visualized for any of
them.
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FIG. 1.
Genetic organization of the ZAM retroelement.
The retrovirus-like gag, pol, and env
ORFs are flanked by 5' and 3' LTRs. The pol and
env riboprobes used for in situ hybridization experiments
are shown, as are ZAM polypeptides used for preparing
polyclonal antibodies directed against Env and Gag proteins (pEnv and
pGag, respectively). 1 and 2, oligonucleotides used for the PCR
amplification of the gag ORF. The BglII and
DraI restriction sites in the env ORF are those
used for subcloning the envelope-encoding region into the pRSETC
expression vector.
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ZAM was first identified as a spontaneous insertion at the
white locus, giving rise to the
wIR6RevI allele in a line of D. melanogaster subsequently called RevI (11).
This mutation occurred in the course of a massive amplification of
ZAM elements in this line due to their mobilization, which remains active in this stock of flies (3). The existence of RevI and its parental line, wIR6,
which displays a low copy number of stable ZAM elements,
offers a good genetic system where the control of ZAM
mobilization and its relationship with its host genome may be studied.
Indeed, we previously reported that ZAM transcription is
active in RevI and inactive in wIR6.
Two kinds of transcripts similar to mRNAs synthesized from a vertebrate
retrovirus involved in a replication cycle were identified in
RevI. One corresponds to a full-length genomic RNA, and the other corresponds to a subgenomic transcript of the ORF3 gene able to
encode a protein which displays all the features of retroviral envelope
proteins. Due to the presence of these transcripts in the course of
ZAM mobilization, an important issue is to know whether its
entire replication cycle is identical to that of infectious retroviruses and involves an extracellular step before ZAM
integration in the genome.
We initially reported that ZAM is mobilized through a
reverse transcription process occurring in the germ line of flies
(11). In this paper, we searched for tissues where
ZAM is transcribed, translated, and potentially assembled in
viral particles.
We report that ZAM RNAs are detected in a very specific
somatic lineage of cells located around the oocyte in the ovaries of
the RevI line. Using polyclonal antisera raised against
bacterial ORF1- and ORF3-encoded ZAM fusion proteins, we
show that both proteins are coexpressed with the full-length
ZAM RNAs in these follicle cells surrounding the oocyte.
Furthermore, we provide evidence that particles of ZAM
are formed in these follicle cells and pass to the oocyte via the
vitelline granule traffic with no apparent need for its Env protein.
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MATERIALS AND METHODS |
Fly stocks.
The wIR6 and
RevI strains (low copy number and high copy number of
ZAM, respectively) are from the collection of the Institut National de la Sante et de la Recherche Médicale U384.
In situ hybridization.
Embryos at different stages were
collected on grape juice agar plates, and fly stocks were maintained on
cornmeal-glucose-yeast media at 20°C.
Ovaries and testes were dissected in 1× phosphate-buffered saline
(PBS). Dissected ovaries, testes, and embryos were fixed
in
heptane-saturated 4% paraformaldehyde-0.1 M HEPES (pH 6.9)-2
mM
MgSO
4-1 mM EGTA for 20 min. Ovaries were rinsed with PBT
(PBS,
0.1% Tween 20) before proteinase K treatment was begun.
Hybridization
with
ZAM env or
pol
digoxigenin-labeled RNA probes was performed
at 55°C overnight and
was followed by washes in hybridization
solution (55.5% formamide,
0.25× SSC [1× SSC is 0.15 M NaCl plus
0.015 M sodium citrate], 0.5 mg of heparin, 0.1 mg of salmon sperm
DNA, and 0.1 mg of tRNA/ml, 0.1%
Tween 20), in a 1/1 mixture of
hybridization solution and PBT at 55°C
for 30 min each, and in
PBT at room temperature (two washes for 20 min
each). The hybridized
probe was detected using the Genius kit
(Boehringer).
DNA constructs, protein purification, and generation of
polyclonal antibodies.
The ZAM gag ORF was amplified
with the Expand long-template PCR system (Boehringer) on
RevI genomic DNA with oligonucleotide 1 (5'-GAGATCTCAAACAACTCGCTCCGTGTTA-3'; positions 1819 to 1839) and oligonucleotide 2 (5'-GGAATTCCTTCTATGTTGTGTAGCCC-3';
positions 2805 to 2823) (Fig. 1). Oligonucleotides 1 and 2 display at their 5' ends BglII and EcoRI
restriction sites, respectively. The gag PCR product was
inserted into the pGEX4T2 vector (Pharmacia Biotech) for glutathione
S-transferase (GST)-Gag fusion protein production in the
bacterial BL26 strain. The GST-Gag fusion protein was purified by
chromatography with glutathione immobilized on cross-linked 4% beaded
agarose (Sigma). In order to test the anti-Gag polyclonal antibody, the
ZAM gag PCR product was subcloned into the pRSETB vector for
His-Gag fusion protein production (see Results).
A 0.7-kb
BglII-
DraI DNA fragment (Fig.
1)
encoding the N-terminal part of
ZAM Env protein was
subcloned from the BH clone
(
11) into the pRSETC vector for
histidine fusion protein production
in the bacterial BL21 strain
according to the manufacturer protocol
(Invitrogen). The histidine-Env
fusion protein was purified according
to the manufacturer protocol by
chromatography on a nickel affinity
resin
(Invitrogen).
The purified GST-Gag and histidine-EnvN
t fusion proteins
were used for generation of polyclonal antibodies in rabbits and rats,
respectively
(Eurogentec).
Whole-mount immunocytochemistry.
Ovaries were dissected in
cold 1× PBS and fixed in 5% formaldehyde-1× PBS-50 mM EGTA-25%
(vol/vol) heptane for 20 min. They were treated in methanol and
copiously rinsed in 1× PBS. Immunodetections were performed with the
ABC-Vectastain kit (Vector Biosys) according to the manufacturer
protocol. Primary antibodies (pAbGag or pAbEnv) were
added at 1/1,000 dilution. Preadsorbed secondary antibodies (goat
anti-rabbit horseradish peroxidase (HRP) or goat anti-rat HRP)
were added at a dilution of 1/400. After coloration ovaries were
analyzed by optical microscopy (Zeiss Axiophot microscope).
For fluorescence stainings, we have used fluorescein
isothiocyanate-conjugated antirat or Texas red-conjugated antirabbit
antibodies at dilutions of 1/200 and 1/600, respectively (Molecular
Probes). Ovaries were embedded in Mowiol 4.88 (Calbiochem) prepared
as
described by the manufacturer at pH 8.5. Whole-mount ovaries
were
scanned on the Leica confocal microscope. Optical sections
were 2 µm
thick.
Ultrastructural studies.
For ultrastructural studies 2- to
3-day-old flies were dissected in PBS, and the ovaries were quickly
fixed for 2 h in ice-cold 5% glutaraldehyde-4% formaldehyde in
0.1 M cacodylate buffer at pH 7.2. Individual ovarian follicles were
separated from the ovaries while in the fixative. Following a prolonged
rinse in the same buffer, the ovarian follicles were postfixed for
2 h in 1% osmium tetroxide in 0.1 M cacodylate buffer at pH 7.2 and rinsed again in the same buffer. Ovarian follicles were then
dehydrated in a graded series of alcohols, passed through propylene
oxide, and eventually polymerized in epoxy resin for 3 days at 60°C.
For immunocytochemical detection of viral antigens, ovarian follicles
were fixed for 2 h in 1% glutaraldehyde-4% formaldehyde
in 0.1 M buffer at pH 7.2. After dehydration in alcohols, ovarian
follicles
were embedded in Unicryl resins and allowed to polymerize
under a UV
lamp at 4°C for 3 days. Sections were obtained with
an LKB
ultramicrotome and mounted over uncoated nickel grids.
To detect the
presence of viral antigens by gold immunocytochemistry,
a number of
ovarian follicles were dissected and fixed in formaldehyde
and then
incubated for 3 h in primary mouse (pAbGag) or rat
(pAbEnv)
antibodies diluted 1:500 in PBS. Ovarian follicles were
then thoroughly
rinsed in PBS and incubated for an additional hour at
room temperature
with either gold-tagged secondary goat anti-mouse
immunoglobulin
G (20 nM) or antirat (10 nM) antibodies (NCI) diluted
1:200 in
PBS. Grids were conventionally stained with uranyl acetate and
lead citrate and eventually observed in a Jeol EM transmission
electron
microscope.
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RESULTS |
ZAM is transcribed in the somatic follicle cells
surrounding the oocyte.
The pattern of spatial expression of
ZAM was determined by in situ hybridization with an
antisense-specific riboprobe of the ZAM env gene labeled
with digoxigenin. (Fig. 1 and 2; see
Materials and Methods). This probe potentially recognizes the two
ZAM transcripts identified through Northern blot analyses,
i.e., the full-length 8.6-kb transcript and the 1.7-kb subgenomic
transcript of the env gene (11). A
ZAM-specific expression pattern was observed with this
probe in the RevI strain, where ZAM mobilization
is active. These transcripts were detected in the gonads and in the central nervous systems (CNS) of late embryos (>10 h). No
hybridization was detected in early embryos (<8 h). The signal
observed in the CNS was detected in almost all the embryos, while the
prominent expression in gonads occurred in about 50% of the embryos,
suggesting that this signal could be restricted to one sex (Fig. 2B).
When a sense strand-specific probe for the env gene of
ZAM was used to probe RevI embryos, no signal was
observed in the gonads of the embryos (data not shown).
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FIG. 2.
In situ hybridization to whole-mount embryos and ovaries
to visualize the distribution of ZAM transcripts. (A)
Schematic representation of an adult ovariole. The ovariole is composed
here of the germarium (early stages of oogenesis) and later of two
follicles in stages 9 and 10. The germ line cell nuclei (nurse cells
and oocyte nuclei) are in grey, and the somatic cell nuclei (follicle
cell nuclei) are in black. (B to F) In situ hybridizations of
ZAM RNAs with the antisense env riboprobe (Fig.
1); (G) similar experiment with a sense env riboprobe. (B)
Late embryos (up to 10 h at 20°C) of the RevI strain.
Arrowheads, hybridization signals located in gonads. (C) Ovariole of
RevI female. Arrowheads, hybridization signals located in
the somatic follicular cells at the posterior part of each follicle and
in the germarium. (D) Higher magnification of RevI follicles
(stage 10). Strong hybridization signals are observed (arrowheads) in
the posterior follicular cells. (E) Late embryo of the
wIR6 strain. After a long time of revelation, a
leaky signal is observed in the gonads (arrowheads). (F) Ovariole of
the wIR6 strain. No signal is detected in the
follicular cells and the germarium. (G) Negative control of
RevI follicle hybridized with the sense env
riboprobe. In all panels, the anterior part of an embryo or ovariole is
at the left margin.
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In situ hybridization of late embryos of the
wIR6 strain, in which
ZAM elements
are stable, did not give any signal with the
antisense-specific
riboprobe of the
ZAM env gene although a very
faint
hybridization may be detected in gonads after a very long
exposure time
(Fig.
2E). This result corroborates those found
by Northern blotting
analysis (
11), indicating that
ZAM mobilization
is accompanied by elevated RNA levels in
RevI.
Since
ZAM mobilization is known to occur in the germ line of
flies, we then investigated
ZAM transcription in the genital
apparatus of adult flies. Testes and ovaries were dissected from
wIR6 and
RevI strains (see Materials
and Methods) and subjected to
in situ hybridization experiments with
the riboprobes described
in Fig.
1.
ZAM RNAs were visualized
in
RevI ovaries (Fig.
2C and
D). Whatever the probe used, no
transcript was detected in
RevI or
wIR6 testes or in
wIR6
ovaries (Fig.
2F).
In insects, ovaries are composed of developing egg chambers arranged in
tubular structures called ovarioles (Fig.
2A). The
Drosophila ovary consists of 15 to 18 ovarioles. Each
ovariole
contains a series of egg chambers at progressively
more-advanced
stages of oogenesis (
10,
23). At the tip of
each ovariole,
the stem cells of the germ line and the follicle cell
precursors
reside in the germarium. During oogenesis, the germ line
stem
cells and follicle cells go through a defined set of division
cycles and become organized into egg chambers, which progressively
leave the germarium and continue developing as they move posteriorly
within the ovariole. The mature egg chamber consists of the oocyte
and
15 nurse cells, which are both surrounded by a monolayer of
somatic
follicle cells (
13,
23).
During oogenesis,
ZAM transcription occurs very early in the
germarium of each ovariole and then is detected in the follicular
cells
of each egg chamber. However,
ZAM RNAs are not present in
all the follicle cells but are restricted to a patch of follicle
cells
located at the posterior side of the oocyte.
ZAM expression
persists until late stages of oogenesis (Fig.
2C and D). Similar
experiments performed on
RevI ovaries with the sense
riboprobe
did not reveal any hybridization signal (Fig.
2G).
Since the
env riboprobe used to detect these transcripts did
not allow discrimination between the presence of the full-length
transcript and the presence of the ORF3 subgenomic transcript,
a
specific
ZAM riboprobe of the
pol gene (Fig.
1)
was used for
additional in situ hybridizations. The same pattern of
expression
as the one described above with the ORF3 probe was observed,
indicating
that full-length
ZAM RNAs are present in the
follicle cells (data
not
shown).
We then addressed the question whether proteins encoded by
ZAM ORFs could be detected in the cells where
ZAM
transcripts have
been
visualized.
The ORF1-encoded ZAM polypeptide is present in
cells where ZAM transcription is occurring.
Retroviral Gag
proteins are synthesized from full-length RNAs as Gag and Gag-Pol
fusion polyproteins and are assembled into virus-like particles in
cells where these RNAs are detected. Gag structural polypeptides
constitute the core of the viral particle. In order to know
whether ZAM Gag proteins are synthesized in tissues where
full-length transcripts have been detected, we performed immunocytochemistry experiments.
A purified bacterial GST-Gag fusion protein encompassing the whole
length of
ZAM Gag was prepared and injected into rabbits
(see Materials and Methods). A polyclonal antibody denoted
pAbGag,
which potentially recognizes the pGag region (Fig.
1) of
Gag,
was obtained. We verified that the pAbGag antibody is
raised against
the Gag protein and not exclusively against the GST
peptide present
in the fusion protein. For that purpose, the
gag ORF was subcloned
into the Tag-histidine pRSETB vector
and a histidine-Gag fusion
protein was expressed. From Western blotting
experiments, we found
that pAbGag clearly reacts with the
histidine-Gag fusion protein
(data not
shown).
We then examined the pattern of spatial and developmental accumulation
of Gag products during different stages of
Drosophila development where full-length
ZAM RNAs had been previously
identified.
Using pAbGag for immunocytochemical experiments, we
detected
ZAM Gag proteins in all egg chambers of the
RevI strain with the same
distribution as
ZAM
RNAs (Fig.
3A and B). A strong
immunostaining
was revealed in a few follicle cells located at the
posterior
part of each egg chamber (Fig.
3A). At stage 10, Gag
immunostaining
strongly underlined an area located at the frontier of
the follicle
cells and the oocyte. This signal tended to extend around
the
oocyte from stage 10 (Fig.
3B).
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FIG. 3.
Expression of Gag and Env proteins during
Drosophila oogenesis. Shown is the immunolocalization of Gag
and Env proteins using polyclonal pAbGag and pAbEnv antibodies,
respectively, in ovaries of RevI and
wIR6 females. (A to C) Gag of ZAM
revealed with the pAbGag antibody in a RevI ovariole.
(A) Strong immunostaining is detected in each follicle in a few somatic
follicle cells which surround the oocyte and in all follicles. (B)
Higher magnification of the posterior part of an early stage 10 follicle. Staining is indicated by arrowheads. (C) Stage 10 follicle
from a wIR6 female. No Gag proteins are detected
by the pAbGag antibody. (D to F) ZAM Env revealed with the
pAbEnv antibody in a RevI ovariole. (D) High level of
staining can be observed in a few somatic follicle cells at the
posterior part of early stage 10 follicles (arrow). (E) High
magnification of the posterior region of the follicle. (F) Stage 10 follicle from a wIR6 female. No Env proteins are
detected by the pAbEnv antibody.
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No immunostaining was detected in
RevI embryos or larvae or
when controls were performed using pAbGag antibody on
wIR6 ovaries (Fig.
3C). Preimmune serum on
RevI ovaries did not produce
any immunostaining (data not
shown).
Translation of ZAM ORF3 is restricted to a defined
developmental window of oogenesis.
ZAM encodes a subgenomic
mRNA of 1.7 kb whose sequence predicts a protein with structural motifs
typical of retroviral Env proteins, i.e., a signal peptide, a potential
transmembrane domain, putative N-glycosylation sites, and
cysteine residues. In order to determine whether this predicted Env
protein is indeed synthesized in tissues where ZAM
RNAs have been detected, immunocytochemistry experiments on late
embryos, larvae, and dissected ovaries from RevI female
strains were performed.
To this end, a bacterial histidine-ORF3 peptide fusion protein, in
which the coding sequence for the ORF3 peptide extended
from nucleotide
6385 to 7105 of
ZAM ORF3, was synthesized (pEnv;
Fig.
1).
After purification by chromatography on a nickel affinity
resin, it was
injected into rats (see Materials and Methods).
A polyclonal
antibody denoted pAbEnv, which recognizes the recombinant
histidine-ORF3 peptide in Western blot analyses whereas the preimmune
serum does not, was obtained (data not
shown).
Using pAbEnv, we then determined the temporal and cell
type-specific expression of
ZAM Env protein in flies. No
immunoreactivity
was detected with pAbEnv in embryos or larvae
(data not shown).
Env proteins revealed with pAbEnv were only
detected in the ovaries
of the
RevI strain (Fig.
3D and E).
This translation of
ZAM ORF3
was restricted to the very
small patch of follicle cells surrounding
the oocyte where
ZAM transcripts were identified. However, the
signal was
detected in a more defined subset of these somatic
cells since it was
present at the posterior parts of stages 9
and 10 and not at
earlier stages of follicle development. At a
higher magnification
a strong immunostaining highlighted the area
along the follicle
cells and the oocyte in addition to Env presence
inside the follicle
cells
(E).
In order to verify the specificity of this immunostaining, controls
were performed using the pAbEnv antibody and the preimmune
serum on
wIR6 and
RevI ovaries, respectively.
No Env protein was detected in
ovaries of the
wIR6 strain (Fig.
3F), and no immunostaining was
observed with the
rat preimmune serum on
RevI ovaries (data
not
shown).
Gag and Env proteins encoded by ZAM are coexpressed in
the same somatic lineage.
To verify that Gag and Env proteins are
indeed coexpressed in the follicle cells of RevI egg
chambers, we used confocal microscopy on RevI ovaries
stained with anti-Gag antibody and anti-Env antibody (Fig.
4).
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FIG. 4.
ZAM gag and env genes are
coexpressed in the follicle cells of RevI follicles. Double
staining of RevI follicles with Gag antibody (red) and Env
antibody (green) in early stage 10 of oogenesis (upper panels) and in
later stage 10 (lower panels). Bars: upper panels, 10 µm; lower
panels, 30 µm. oo, oocyte; fc, follicle cells.
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As expected, double staining revealed that Gag and Env proteins are
coexpressed at stages 9 and 10 A of oogenesis in the follicle
cells
located at the posterior part of the ovarian follicle (Fig.
4, upper
panels). The detected fluorescence is specific to these
follicle cells
and almost absent within the other follicle cells
surrounding
the oocyte. At this stage of oogenesis, Gag and Env
proteins are
visualized within the cytoplasm of the
cells.
At a later stage, around stage 10 B, of oogenesis (Fig.
4, lower
panels). Gag and Env are detected as a thick line visualized
at the
boundary of the follicle cells and the oocyte. No Gag or
Env signal was
clearly detected further within the ooplasm of
the oocyte or in the
nucleus, which is located at the opposite
side of this germ cell. At
this stage of development, an Env signal
persists within the follicle
cells while the Gag product is no
longer visualized. This picture of
the Gag signal detected within
the follicle cells and then concentrated
at the follicle cell-oocyte
border at a later stage of development is
consistent with movement
of Gag-containing particles between the
two.
Viral particles are detected in the follicle cells of the
RevI line.
Since all components necessary to assemble
particles have been found in a small patch of cells clearly identified,
we then searched for potential ZAM particles by an electron
microscopy approach. A number of ovarian follicles from
Drosophila strain RevI were examined by electron
microscopy and compared with those from wIR6
flies. Ellipsoidal or ring-shaped particles about 45 nm in diameter with an electron-translucent center were regularly seen in the posterior follicle cells of ovarian follicles at stages 8 to 10. The
region of the follicle cell cytoplasm most highly enriched in viral
particles is the one close to the apical plasma membrane (Fig.
5A and B). Some particles have also been
occasionally observed inside the nucleus.
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FIG. 5.
Ultrastructural identification of ZAM viral
particles in ovarian follicles of the RevI strain from
D. melanogaster. (A) A posterior follicle cell (fc) facing
the oocyte (oo) from a stage 9 ovarian follicle is shown. Vm, vitelline
membrane. Bar, 0.5 µm. (B) Enlargement of panel A to show the apical
end of a follicle cell, where numerous roundish viral particles
(arrows) (average diameter, 45 nm) can be clearly seen. Bar, 0.3 µm.
(C) The apical end of a posterior follicle cell from a stage 10 ovarian
follicle of the wIR6 strain showing several
vitelline membrane precursors (pVm) and the vitelline membrane itself,
but not viral particles. Bar, 0.9 µm. (Inset) Enlargement of the
vitelline membrane precursors.
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No viral particle was detected in any of the ovarian tissues or
intercellular spaces examined in the
wIR6 line
(Fig.
5C). This result is in good agreement with data presented
above
concerning the
wIR6 line in which no
ZAM mobilization or any
ZAM RNA or Gag or Env
polypeptides have been observed and strongly supports the idea
that the
presence of the particles detected in
RevI is correlated
with
ZAM mobilization.
At stages 8 to 10 of oogenesis,
Drosophila oocytes
accumulate large amounts of yolk. Yolk precursor proteins are
synthesized
in the fat body and are transported via the hemolymph to
the oocyte
membrane, where they are subsequently taken up by endocytic
vesicles.
However, a significant amount of yolk protein is also
synthesized
in the follicle cells themselves (
17).
Interestingly, most of the viral particles detected within the follicle
cells have been seen in contact with the membrane
enclosing the
secretory granules containing the vitelline membrane
precursors or even
bound to the granule content itself (Fig.
6C).
In the
wIR6
strain, no particles are associated with the secretory granules
(Fig.
5C, inset).
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FIG. 6.
Cytochemical detection of viral particles on vitelline
membrane precursors and in yolk granules of the RevI strain.
(A) A vitelline membrane precursor (pVm) along the apical end of a
posterior follicle cell (fc) from a stage 9 ovarian follicle. Note the
presence of numerous viral particles (arrowheads) around the granule
periphery. Bar, 0.25 µm. Vm, vitelline membrane. (B) The apical end
of a posterior follicle cell from a stage 11 ovarian follicle. Note the
presence of numerous viral particles (arrowheads) along the margin of
the vitelline membrane. Bar, 0.2 µm. (C) A forming yolk granule (y)
from the cortical ooplasm of a stage 9 ovarian follicle of
RevI following a 1-h exposure to HRP. Note that viral
particles (arrowheads) are present along the superficial layer
underneath the limiting membrane. A peroxidase-labeled endocytic
vesicle is also visible along the membrane (arrow). oo, oocyte. Bar,
0.5 µm. (D) A forming yolk granule from a stage 9 RevI
ovarian follicle fixed for 4 h with zinc osmium iodide (OZI). Note
the presence of several viral particles (arrowheads) along the
superficial layer among several electron-dense spots of OZI
precipitates. Bar, 0.1 µm.
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Although the mobilization of
ZAM occurs within the germ line
of
RevI, no virus budding along the apical plasma membranes
of
the follicle cells was observed in this study. In this context,
it
is interesting to note that the vitelline membrane precursors
synthesized within the follicle cells are released from this somatic
lineage and pass to the extracellular region bordering the oocyte.
Thus, the viral particles may benefit from their association with
the
vitelline membrane precursors to sort out this somatic lineage.
Viral
particles detected in more developmentally advanced ovarian
follicles
are stockpiled along the apical follicle cell plasma
membrane, as if
extracellular release of residual viral particles
would have indeed
been impeded by completion of the vitelline
membrane (Fig.
6B).
Viral particules were also detected within the oocyte at stages 8 to 10 of oogenesis. At these stages, the
Drosophila oocyte
is
heavily involved in taking up vitellogenin from the hemolymph
by
receptor-mediated endocytosis (
18). The endocytic apparatus
at these developmental stages comprises a plethora of vesicles
including coated vesicles, transitional yolk bodies, and mature
yolk
granules (
6). Viral particles can be easily recognized
by
size and shape among these vesicles in the cortical ooplasm
of
RevI. Viral particles were located over the yolk granules,
where they appear uniformly dispersed along the superficial layer
(Fig.
7C) or enclosed within a vesicular
membrane (Fig.
6C and
D).
View larger version (126K):
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|
FIG. 7.
Immunocytochemical detection of Gag viral antigens. (A)
The follicle cell-oocyte border from a stage 9 RevI ovarian
follicle tested with anti-Gag antibody. fc, posterior follicle cell; N,
follicle cell nucleus; oo, oocyte; Vm, vitelline membrane. Bar, 4 µm.
(B) Enlargement of panel A to show numerous 20-nm gold grains of the
secondary antibody along the apical end of the follicle cell. Bar, 1 µm. (C) Portion of the cortical ooplasm from a stage 10 RevI ovarian follicle showing gold grains (arrowheads) due
to anti-Gag antibody along the oolemma. Bar, 0.5 µm. (D) A forming
yolk granule (y) from a stage 9 RevI ovarian follicle. Note
the presence of gold grains due to anti-Gag antibody (arrowheads) over
the superficial layer among viral particles (arrows). Bar, 0.4 µm.
|
|
Gold immunocytochemical experiments localize Gag and Env proteins
of ZAM at sites where particles accumulate.
To
ascertain that the ring-shaped particles observed in the posterior-most
follicle cells of the RevI ovaries are indeed due to
ZAM expression, a number of ovarian follicles at stages 9 and 10 were treated for the immunocytochemical detection of
ZAM proteins. When tested with anti-Gag antibodies, the most
heavily labeled sites of the follicular epithelium appeared to be those
cells that face the posterior pole of the oocyte (Fig. 7A). Within the follicle cell cytoplasm gold label accumulated along the apical end,
even though the basolateral borders were also labeled to some
extent (Fig. 7A and B). Along the apical border, gold particles were preferentially associated with the vitelline membrane precursors or, extracellularly, with the deposited vitelline membrane.
In the cortical ooplasm, gold label appeared dispersed among endocytic
vesicles (Fig.
7C). Yolk granules were also labeled,
but the gold
particles over these organelles occurred more frequently
inside
the so-called superficial layer than within the enclosed
main
body (Fig.
7D). In ovarian follicles at a more advanced developmental
stage of oogenesis than stage 10, the label tended to gradually
disappear both from the follicle cell cytoplasm and the oocyte
(data not
shown).
When tested with anti-Env antibodies, ovarian follicles appeared
labeled over both the follicle cell cytoplasm and the oocyte
(Fig.
8A), with gold particles occurring along
the apical follicle
cell membrane (Fig.
8B) and the cortical
ooplasm among the endocytic
vesicles (Fig.
8C). Label appeared to
persist along the oocyte
plasma membrane even in ovarian
follicles with a complete vitelline
membrane and no endocytic uptake
(Fig.
8D).
View larger version (189K):
[in this window]
[in a new window]
|
FIG. 8.
Immunocytochemical detection of Env viral antigens. (A)
The follicle cell (fc)-oocyte border from a stage 10 RevI
ovarian follicle exposed to anti-Env antibody. Gold grains are
dispersed over the vitelline membrane (Vm). y, yolk granule. Bar, 0.5 µm. (B) The apical end of a posterior follicle cell from a stage 9 RevI ovarian follicle showing several gold grains
(arrowheads) along the plasma membrane. Bar, 0.4 µm. (C) The
posterior-most cortical ooplasm from a stage 9 RevI ovarian
follicle tested with anti-Env antibody. Arrowhead, gold-labeled coated
vesicle. oo, oocyte. Bar, 0.4 µm. (D) Portion of a stage 11 RevI ovarian follicle showing the vitelline membrane and the
underneath oolemma. Vitellogenic uptake has ceased by this
developmental stage in D. melanogaster, and yet gold grains
due to the anti-Env antibody are still seen bound along the microvilli
of the oolemma. Bar, 0.6 µm.
|
|
As a general rule, the gold labeling due to anti-Env antibodies is low
and does not spatially coincide with viral particles,
indicating that
the 45- to 50-nm particles may correspond to
ZAM particles
devoid of an envelope. Although the
ZAM Env protein
is
associated with the plasma membrane, as expected for a functional
Env
protein, these results suggest that the Gag-Env interaction
may not
have an obligatory role for the cell-to-cell transmission
of
ZAM.
| |
DISCUSSION |
The present work reports data about the mobilization of retroviral
particles produced in a somatic lineage and passing to the germ line.
Analysis of the cell-to-cell transmission of the ZAM
retroelement of Drosophila permits us to propose a possible mechanism for such a mobilization.
The mobilization of ZAM correlates with the production
of all components necessary to assemble virus-like particles in the
follicle cells.
A previous study had reported that ZAM
displays all the structural features of a vertebrate retrovirus
(11). However, this first study failed to determine whether
the predicted products of ZAM were indeed synthesized in the
course of its mobilization. This was achieved in this study.
Expression of retroviruses necessitates transcription of a full-length
RNA and synthesis of retrovirally encoded proteins
Gag, Pol, and Env.
All these components encoded by
ZAM have been
detected in
the ovaries of a strain where
ZAM mobilization is
known to
be high, the
RevI strain, and are absent in the
wIR6 line, in which
ZAM mobilization
does not occur. Full-length
ZAM transcripts have been
detected in a group of cells of somatic
origin that are the follicle
cells surrounding the posterior part
of each
oocyte.
Polyclonal antibodies raised against the putative full-length Gag
protein recognized a
ZAM Gag product in adult ovaries in
a
distribution pattern similar to that of
ZAM RNAs. Gag was
detected
in each follicle starting from early stages of oogenesis. At
stages
9 and 10A, the antibody revealed Gag proteins at the border
between
the oocyte and the follicle cells, in addition to their
location
in the follicle cells. Later, in stage 10B, Gag proteins were
present around the oocyte while they were then absent from the
follicle
cells. These data are consistent with movement of Gag-containing
particles between the follicle cells and the oocyte. Antibodies
raised
against the
ZAM Env led us to visualize the presence
of
such a protein in a specific group of follicle cells at the
posterior
part of the oocyte. Although
ZAM RNA and Gag
proteins have been
visualized in these cells, the Env pattern of
expression is detected
in a more restrictive pattern of development
since Env proteins
are only present at stages 9 and 10 of oogenesis and
are absent
in earlier
stages.
Virus-like particles of ZAM may benefit from exocytic
and endocytic exchanges to pass from the follicle cells to the germ
line.
Previous experiments had indicated that novel ZAM
insertions frequently occur within the germ line of RevI
(11). Owing to the fact that all the components necessary
for ZAM's mobilization had been detected in a somatic
lineage and that movement of Gag-containing particles had been
suggested by our immunocytochemical approach, we then searched for a
potential pathway leading ZAM to the oocyte. Through an
ultrastructural study, ring-shaped or ellipsoidal viral particles of
about 45 nm in mean diameter were detected in RevI. These
particles are similar to defective human immunodeficiency virus
particles that exhibit an electron-dense ring corresponding to a Gag
protein not yet cleaved to yield the mature viral form (7).
Several lines of evidence strongly argue that these particles correspond to ZAM particles: (i) they are detected within
cells where immunostaining and confocal analysis with anti-Gag and
anti-Env antibodies have revealed the presence of ZAM
products, i.e., the follicle cells facing the posterior pole of the
ovarian follicle; (ii) they are absent in wIR6,
where no ZAM mobilization has been observed; (iii)
immunogold cytochemistry with antibody pAbGag confirms this
staining pattern by showing that the labeling of the follicular
epithelium is primarily due to the ZAM product lying close
to the vitelline membrane precursors.
This structural analysis brought three pieces of information that help
to trace ZAM mobilization. First, the particles occur
in close
association with the vitelline membrane precursors along
the apical
cytoplasm of the posterior-most follicle cells. These
data indicate
that the particles will be able to sort out the
follicle cells when
these vesicles are secreted. Second,
ZAM particles
are also
detected within the cortical ooplasm, indicating that
ZAM
particles have been able to pass from the follicle cells to
the oocyte.
In the oocyte, the particles display a very specific
distribution.
Indeed, almost all of them are embedded within the
superficial layer of
the yolk granules along the cortical ooplasm.
Third, as for
ZAM Gag, Env is observed within the follicle cells
specifically along the apical follicle cell membrane. However,
no
budding within the extracellular compartment between the follicle
cells
and the oocyte has ever been
detected.
These overall data support the following pathway by which
ZAM particles enter the
RevI germ line. The
initial step is to form
and accumulate
ZAM particles in the
follicle cells. At early stage
10 of oogenesis, these particles are
secreted along the apical
end of the follicle cells in close
association with the vitelline
membrane precursors. In more
developmentally advanced ovarian
follicles, extracellular release of
residual particles is then
impeded by completion of the vitelline
membrane leading to viral
particles stockpiled along the apical
follicle cell plasma membrane.
Once released into the follicle
cell-oocyte interface, they are
transferred to the oocyte and
eventually conveyed to the yolk
granules, where most of them are
detected.
Surprisingly, the scenario deduced from our data supports the idea that
ZAM may not need its envelope for an extracellular
transmission. It has already been reported that retroviruses may
not
require their Env proteins for budding to take place. As an
example, in
polarized cells, the human immunodeficiency virus
type 1 Gag protein
has been found to direct budding from cell
membranes with no necessity
for the Env glycoproteins (
2).
The retroviral Gag proteins
play a part in the incorporation of
Env into the viral particle, but
they also have the capacity for
packaging foreign glycoproteins
(
4,
27). In that context,
it is interesting to suggest that
Gag proteins of
ZAM could recognize
the vitellogenin
proteins as foreign glycosylated proteins and
benefit from their
release out of the posterior follicle cells
to sort out this somatic
lineage.
This potential way for a retroelement to pass from one cell to another
may explain the results obtained with retroviruses
expressed in a
somatic lineage close to the germ line or other
retroelements from
insects such as
gypsy. Indeed, in a genetic
context
permissive for
gypsy mobilization, particles containing
gypsy RNA have been described as preferentially clustered
along
the plasma membranes of the anterior follicle cells.
When these
cells were tested with anti-Env antibodies, gold
labeling appeared
almost exclusively associated with the plasma
membrane but no
viral particle budding or extracellular release from
the follicle
cells could be observed (
12). In addition,
recent data from
a genetic approach have clearly demonstrated that
invasion of
the female germ line by
gypsy retroviruses may
occur in an Env-independent
manner (
1). Although no direct
evidence was presented, the
authors proposed that nonenveloped
particles might enter the oocyte
by endocytosis as a cytochemical
tracer. One can predict that
the vitellogenic traffic is potentially
involved in
gypsy mobilization,
as suggested for
ZAM from our
observations.
What is then the function of the
env genes of both these
elements? Song et al. (
21) reported that at least some
gypsy elements
can be enveloped and display infectious
properties. Research into
the role of
ZAM Env and the
formation of enveloped particles will
certainly be the next step in
understanding the
ZAM life cycle.
Indeed, if
ZAM
particles are "homed" by their Gag proteins to
regions of the
plasma membrane where the Env glycoproteins of
ZAM reside,
they may have been undetected in our
experiments.
When
ZAM is in the oocyte, the next step in the
ZAM cycle is for it to reach the oocyte nucleus. Although
this part of the
ZAM cycle remains to be elucidated, the
data reported in this
paper bring the interesting observation that
ZAM Gag may enter
the nuclei of the follicle cells. Indeed,
gold cytochemistry performed
with pAbGag detected a nuclear
staining in the follicle cells.
These data could indicate that the
ZAM Gag structural protein
displays a specific motif
responsible for directing the protein
into the nuclei as already
reported for foamy viruses (
20).
Is such a motif responsible
for the entry of
ZAM into the nucleus
of the oocyte? Future
experiments will have to clarify the pathway
of
ZAM to the
oocyte
nucleus.
| |
ACKNOWLEDGMENTS |
We thank Tom Eickbush for reading a first version of the
manuscript and providing helpful suggestions. We are grateful to members of the group, and especially Caroline Conte, for critically reading the manuscript and for valuable comments. We thank Francis Harper for his involvement in a preliminary electron microscopy analysis, Nathalie Gauthier for assistance in obtaining antibodies, M. Grammont for her schematic representation of an adult ovariole, and P. Giraud for assistance with confocal microscopy.
This work was supported by grants from the INSERM (U384), by a project
grant from Programme Génome CNRS (Intégrité et
plasticité des génomes), and from ARC 1999 to C.V. and
partly by the Italian Ministry for Research and Technology in the
University (MURST), Program on Cell Interactions, to F.G. and M.M.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Unité
INSERM U384, BP 38, 28 Place Henri Dunant, 63000 Clermont-Ferrand,
France. Phone: (33) 4 73 60 80 24. Fax: (33) 4 73 27 61 32. E-mail:
Chantal.VAURY{at}inserm.u-clermont1.fr.
| |
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Journal of Virology, November 2000, p. 10658-10669, Vol. 74, No. 22
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Top
Abstract
Introduction
