1 Institut de Génétique Humaine CNRS, 141 rue de la cardonille, 34396 Montpellier cedex 5, France
2 Laboratoire de Pathologie Comparée, 30380 Saint Christol les Ales, France
3 Ecole Pratique des Hautes Etudes UMR 7625, Université Paris 6, cc237, 7 quai Saint Bernard, 75252 Paris cedex 05, France
Correspondence
Christophe Terzian
cterzian{at}snv.jussieu.fr
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ABSTRACT |
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INTRODUCTION |
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It is usually assumed that ERVs originate from exogenous retroviruses that once integrated into the germ-line chromosomes (Löwer et al., 1996). This endogenization event is at the heart of important differences between exogenous retroviruses and ERVs. ERV replication is a non-infectious process, as these elements are transmitted vertically as part of the host genome. Their behaviour is therefore very similar to that of transposable elements, known as LTR retrotransposons. Exogenous retroviruses replicate only in the cells of infected individuals. They are not present in the germ line and therefore propagate horizontally as a result of their infectious properties. Virus entry is mediated by the envelope glycoprotein (Env), which is encoded by the retroviral genome. Hence, the fact that most ERVs are defective for the env gene can be explained by the non-essential role of Env in their propagation.
However, it has been shown that several ERVs have an intact envelope and retain the ability to encode Env proteins. This is the case for the human ERVs HERV-K (Tönjes et al., 1996) and HERV-Fc (Bénit et al., 2003
), porcine ERVs (Czauderna et al., 2000
), ovine ERV (Carlson et al., 2003
) and insect ERVs (Terzian et al., 2001
). Unexpectedly, infectious properties have been demonstrated experimentally for porcine ERV (Niebert & Tönjes, 2003
) and for the gypsy insect ERV (IERV) that is present in Drosophila melanogaster. gypsy infectivity has been demonstrated by experiments in which larvae lacking active copies of gypsy were fed with either crude extracts (Kim et al., 1994
) or purified particles (Song et al., 1994
) that contained actively transposing gypsy ERVs: new copies of gypsy were observed in the genomes of the progeny of the treated flies. In addition, it has been shown that particles of the Moloney murine leukaemia virus pseudotyped with gypsy Env are able to infect cultured Drosophila cells, albeit with a low efficiency (Teysset et al., 1998
). One can ask why some ERV genomes have kept the ability to encode a functional envelope. One step towards an understanding of the evolutionary history of infectious ERVs is to estimate the fusogenic properties of their Env proteins and to map their functional domains.
gypsy Env is produced from a subgenomic spliced RNA (Pélisson et al., 1994). Several putative domains that are typical of vertebrate retroviral glycoproteins have been described in this protein, namely a signal peptide, glycosylation sites and an RSRR endopeptidase cleavage site, although the latter is not followed by the hydrophobic region that is characteristic of a fusogenic region (see Fig. 1a
). Moreover, two reports have shown that there are significant similarities between IERV env gene products and a baculovirus fusion protein (Malik et al., 2000
; Rohrmann & Karplus, 2001
). These findings led us to reconsider the structural analogy with the surface/transmembrane subunit glycoproteins of vertebrate retroviruses that was suspected previously. Hence, a comparative analysis between gypsy Env and the fusion protein of baculoviruses allowed us to map putative functional domains (Rohrmann & Karplus, 2001
; Terzian et al., 2001
). Alignment of the predicted envelope fusion proteins of a number of baculoviruses, errantiviruses and paramyxoviruses has led to the identification of a region of high similarity (Misseri et al., 2003
). This region contains an RIAR furin-like consensus motif, contiguous with a hydrophobic sequence. This hydrophobic sequence contains a set of conserved amino acids followed by a predicted coiled-coil region, suggesting that it could be the fusion peptide.
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METHODS |
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The mAb 8E7 (Song et al., 1994) was provided by Victor G. Corces (Johns Hopkins University, Baltimore, MD, USA). Generation and purification of the polyclonal antibody E78P were done by synt : em (www.syntem.com). The following peptides corresponding to different parts of gypsy Env were synthesized: (1) RQIAINSETQKQINKLTDT, (2) siqnlmddvesegsprlwf and (3) FNMTEDGHKLEGGVVNN. Two rabbits were immunized with the three peptides according to the manufacturer's protocol. Fractions containing the immunopurified antibodies were detected by measuring A280 and mixed. Immunoreactivity was verified by ELISA and shown to be restricted to the two most C-terminal peptides (2 and 3).
Construction of recombinant baculoviruses.
For construction of the wild-type gypsy Env, a 1879 bp fragment encoding gypsy Env derived from the pDm111 plasmid (Bayev et al., 1984) by PCR amplification using synthetic primers WT BglII (5'-GAAGATCTGAAAAGCATGTTCACCCTCATGA-3') and WT Acc65I (5'-AAGGTACCATTATCACTAGCCTTGTGGCGATA-3') was produced. The resulting fragment contained BglII and Acc65I restriction sites (underlined) and was cloned into the pGmAc 217 vector (Gaymard et al., 1996
) under the control of the polyhedrin gene promoter. This construct was named pGmEnv2. The integrity of the Env fragment was confirmed by sequencing.
For construction of gypsy Env mutants, two putative furin-like cleavage sites (RxxR) that have been described previously (Misseri et al., 2003) were mutated. The RSRR putative cleavage site was mutated from pGmEnv2 by using a QuikChange site-directed mutagenesis kit according to the manufacturer's instructions (Stratagene). The mutation (shown in bold) was chosen in order to change Arg to Leu (RSRR
RSRL). Primers used were 5'-GAGCGGTCTCGGCTCGAAACTTGCGTG-3' and 5'-CACGCAAGTTTCGAGCCGAGACCGCTC-3' and the plasmid obtained was named pGmRSRL. The RIAR putative cleavage site was mutated to GIAG by PCR amplification from pGmEnv2 using the primers Mut EcoRV (5'-GCTGATATCATGGAGATAAT-3') and Mut XbaI (5'-ATCTAGACTTCCAGCTATTCCATGGAGGAC-3'), which contained EcoRV and XbaI restriction sites (underlined), respectively. The template pGmEnv2 and PCR product were then digested by EcoRV and XbaI and ligated together. The resulting plasmid was named pGmGIAG. The double mutant pGmRSRL/GIAG was obtained by digesting the pGmGIAG and pGmRSRL plasmids with EcoRV and XbaI and inserting the fragment containing the GIAG mutation into the pGmRSRL plasmid. The mutation sequences were confirmed by sequencing.
Recombinant gypsy Env baculoviruses were generated in Sf9 cells by cotransfection of Sf9 cells with recombinant baculovirus transfer vectors and Autographa californica multipolyhedrosis virus (AcMNPV) DNA, using the lipofection method (Felgner & Ringold, 1989). Recombinant baculoviruses were plaque-purified by standard techniques (O'Reilly et al., 1992
) and designated gypENV, gypRSRL, gypGIAG and gypRSRL/GIAG.
Env expression and maturation.
One hour prior to infection, cells were seeded in 25 cm2 flasks in 4 ml serum-free medium to allow attachment to the flask. Cells were then infected with a viral suspension at an m.o.i. of 10 and left for 1 h for virus adsorption. Next, 4 ml serum-rich medium was added and cells were incubated at 27 °C. Cells were collected 3 days later and washed with 500 µl PBS. A 10 µl aliquot was deposited in each well of an eight-well slide and treated for immunofluorescence.
Immunofluorescence.
Cells deposited in eight-well slides were fixed for 2 min in methanol at 20 °C and washed three times for 10 min each with PBT (0·5 % BSA, Triton X-100 in PBS). Cells were incubated with 5 % horse serum in PBT for 1 h and incubated overnight with the primary antibody E78P. Samples were then washed in PBT four times for 10 min each and incubated for 30 min with an fluorescein isothiocyanate (Jackson ImmunoResearch Laboratory)-conjugated anti-rabbit secondary antibody. The slide was mounted by using Mowiol (Calbiochem) for observation with a Leica SP2 DMIRB confocal microscope.
Western blotting.
Infected and uninfected cells were washed and resuspended in 200 µl RIPA buffer [150 mM NaCl, 1 % NP-40, 0·5 % deoxycholic acid sodium salt, 0·1 % SDS, 50 mM Tris/HCl (pH 8), 1 mM dithiothreitol] and left for 30 min on ice. Samples were sonicated twice for 5 s each. A 100 µl aliquot of each sample was deglycosylated with N-glycosidase F (PNGase F; New England Biolabs) and 5 µl of each sample was mixed with 5 µl 2x Laemmli. A 5 µl aliquot of each protein extract was run on a 12 % acrylamide gel and electroblotted onto nitrocellulose (Optitran BA-S 83; Schleicher & Schuell BioScience). Env was detected by using the mAb 8E7, provided by V. G. Corces (1 : 100 dilution for 1 h at room temperature). Antigen detection following incubation of blots with a horseradish peroxidase-conjugated second antibody (anti-mouse Ig from sheep, 1 : 3000 dilution for 20 min at room temperature; Amersham Biosciences) was carried out using ECL (Amersham Biosciences).
Ovaries were extracted from flies as described previously (Song et al., 1994). Proteins from ovaries were extracted following the same protocol as for cells.
Cell labelling and fusion assay.
For labelling of cells, infected and uninfected Sf9 cells were collected and resuspended in a serum-free medium. Calcein (485/525) acetoxymethyl (AM) ester and Vybrant DiI (549/565) were obtained from Molecular Probes. To monitor dye redistribution as a result of fusion, gypENV-expressing Sf9 cells were loaded with calcein-AM (green fluorescence) following the manufacturer's protocol. The AM ester penetrates into cells, where it is hydrolysed to form the impermeant dye. Uninfected cells grown in the same way were harvested and labelled with Vibrant DiI, a fluorescent lipophilic tracer (orange fluorescence). Once applied to cells, the dyes diffuse laterally within the plasma membrane, resulting in staining of the entire cell. Transfer of this probe between intact membranes is usually negligible.
Calcein-AM (0·5 µl of a 0·5 µg µl1 solution in DMSO) and Vybrant DiI (5 µl of a 1 mM solution) were added to 1 ml infected and uninfected Sf9 cells, respectively. Cells were incubated for 1520 min at 27 °C in the dark. Cells were then washed twice with serum-free medium and resuspended in serum-rich medium.
For the quantitative fusion assay, infected and uninfected cells were mixed in a 24-well culture dish at a final concentration of 5x105 cells and incubated for at least 2·5 h at 28 °C. Fusion was observed at pH 6. Different ratios of infected and uninfected cells were tested. Cells were then washed and resuspended in 500 µl PBS. Fusions were observed by using a confocal microscope and quantified by using a Becton Dickinson FACScan analytic flow cytometer.
Unlabelled cells were handled similarly and used as controls for intrinsic fluorescence. Compensation was made for optimal separation of fluorescence signals using labelled infected and uninfected Sf9 cells. Cell debris was excluded from the analysis. Each analysis was carried out on 10 000 cells.
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RESULTS |
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Sf9 cells were infected with each recombinant baculovirus. Three days after infection, cells were fixed and analysed for expression of the different Env proteins by using the E78P antibody raised against the C terminus of the protein. Observations using a confocal microscope (Fig. 2a) showed large amounts of wild-type Env expressed at the surface of cells infected with gypENV. Immunofluorescent detection also showed the presence of the mutated Env proteins at the surface of cells infected with gypRSRL, gypGIAG or gypRSRL/GIAG (Fig. 2b
d, respectively). No staining was detected in the negative control recombinant baculovirus Fab/AcMNPV, which expressed the Fab protein under the control of the polyhedrin promoter (Fig. 2e
) (Bès et al., 2001
). The signal and localization observed with the mutant proteins were identical to those observed with wild-type Env. The mutations did not seem to affect the general conformation of the protein significantly, as the antibody was able to detect each mutant protein at the cell surface.
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Taken together, these results showed that the in vivo cleavage site of gypsy seems to correspond to RIAR.
gypsy Env has fusogenic properties
We studied the fusogenic properties of gypsy Env in Sf9 cells. Cells infected with recombinant baculovirus expressing either the Env protein of gypsy or the Fab control protein were labelled with a cytoplasmic green fluorescent dye (Calcein-AM) as described in Methods. Uninfected cells were labelled with a membrane red fluorescent dye (Vybrant DiI). After 2·5 h co-culture of infected and uninfected cells, single-stained cells and double-fluorescent fused cells were observed by confocal microscopy. Several fused cells were observed, indicated by their increased size and dual fluorescence (data not shown).
The baculovirus vector has its own fusion protein, GP64, which has already been shown to be fusogenic (Monsma et al., 1996). Hence, cells infected with gypEnv had two glycoproteins at their membranes: GP64 and gypsy Env. Quantification of fusion was thus necessary to determine the effects of the presence of gypsy Env.
FACS analysis was carried out after 2·5 h co-culture of infected and uninfected cells. Sf9 cells labelled with Vybrant DiI and cells labelled with calcein-AM were analysed as controls for non-specific dye transfer or cellular autofluorescence. To determine the optimal fusion conditions, co-cultures with varying ratios of uninfected and infected Sf9 cells with gypENV or Fab/AcMNPV were prepared. The highest percentage of fusion was obtained with a ratio of 2 : 3 of infected versus uninfected cells (data not shown).
The ratio of percentage of fused cells after infection with gypENV to percentage of fused cells after infection with Fab/AcMNPV was determined. Seven independent repeats of the experiment were done and the mean±SEM was calculated. The results indicated that cells expressing both envelope glycoproteins, GP64 and gypsy Env, were twice as fusogenic as those expressing only GP64 or Fab/AcMNPV at their surface. Non-parametric statistical analyses of these results using both Kendall's and Spearman's tests gave highly significant scores (Kendall's test: z=2·553, P=0·01; Spearman's test: z=2·187, P=0·03). Therefore, the gypsy Env was able to mediate cell-to-cell fusion.
The fusion capabilities of the putative cleavage site mutants were also quantified in the same way. Six independent experiments were done for each mutant. The results showed that none of the mutants lost its fusion capabilities (Fig. 4; KruskallWallis test,
2= 3·1, df=3, P>0·30).
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DISCUSSION |
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The results also give new insight into the structure and presence of functional domains in gypsy Env. It was assumed previously that the cleavage site of gypsy Env was RSRR, by analogy with vertebrate retroviral surface and transmembrane subunit glycoproteins (Pélisson et al., 1994). In contrast to this assumption, our results indicated that cleavage occurs at the RIAR site in lepidopteran cells and suggest that this site is also used in Drosophila ovaries. However, gypEnv, the wild-type Env protein, and gypGIAG, gypRSRL and gypGIAG/RSRL, which contained mutations affecting the putative cleavage sites of this protein, had similar abilities to cause cell-to-cell fusion, suggesting that cleavage of the protein at these sites is not required for its fusogenic properties.
The fact that cleavage at the RIAR site is not necessary for the fusogenic properties of gypsy Env is unusual for an envelope protein. Indeed, other data have shown that these proteins are synthesized as inactive precursors that must be cleaved by host proteases for activity. It has been reported that the baculoviral fusion proteins LD130 and SE8 are truncated downstream of the RRHR and RSKR furin-like cleavage sequences, respectively, and that cleavage is necessary for fusogenic activity of LD130 (IJkel et al., 2000; Pearson et al., 2002
). However, it was shown recently that foamy viral cores that have incorporated cleavage-deficient Env proteins can be transferred from cell to cell (Heinkelein et al., 2003
). Three hypotheses could explain why the mutations that we introduced into the putative cleavage sites of gypsy Env did not affect the fusogenic properties of this protein: (i) cell-to-cell fusion does not require cleavage of gypsy Env, whereas this cleavage is required for virus-to-cell fusion, which was not taken into account in our experimental procedures; (ii) the overproduction of uncleaved gypsy wild-type and mutant Env proteins in the baculovirus expression system permitted cell-to-cell fusion, which would not be the case in physiological conditions; (iii) cleavage does not occur at the RIAR site, but an alternative site is recognized and cleaved by an endoprotease that is active only under specific conditions. In fact, detailed sequence analysis of gypsy Env allowed us to detect a subtilase-like cleavage sequence RKLL just upstream of RIAR (Fig. 1b
). This sequence is well-conserved among the Env proteins of gypsy from various Drosophila species. It was shown recently that the glycoprotein precursors of three different viruses were processed proteolytically by the subtilase SKI-1 (Beyer et al., 2003
; Lenz et al., 2001
; Vincent et al., 2003
), and a subtilase-like sequence is present in the D. melanogaster genome database (GenBank accession no. AAM20922). It would thus be interesting to test whether this site is cleaved in gypsy Env in vivo.
Why an ERV has maintained the capacity to encode a fusogenic glycoprotein remains a puzzling question. It has been shown previously that gypsy Env does not seem to be required for gypsy proviral multiplication in the germ line (Chalvet et al., 1999). One can suggest that gypsy of D. melanogaster is an infectious retrovirus that has only recently been endogenized. Hence, the evolutionary history and function of gypsy Env remain to be determined.
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ACKNOWLEDGEMENTS |
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Received 17 December 2003;
accepted 27 July 2004.