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1 The Institute for Virology, Philipps University, Hans-Meerwein-Straße 3, 35043 Marburg, Germany
2 Crucell Holland BV, PO Box 2048, 2301 CA Leiden, The Netherlands
Correspondence
Jan ter Meulen
j.termeulen{at}crucell.com
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Eichler et al., 2003). Both cleavage fragments are transported to the cell surface, where probably homotrimeric complexes of GP-1 are linked non-covalently to homotrimeric complexes of membrane-anchored GP-2 (Eschli et al., 2006). LASV interacts via GP-1 with the 
subunit of the transmembrane dystroglycan complex, thereby initiating uptake of the virus into the cell (Cao et al., 1998; Kunz et al., 2002). Fusion of viral and cellular membranes is thought to be mediated by the low pH of the endosomal compartment, because entry of the related lymphocytic choriomeningitis virus (LCMV) is inhibited by lysosomotropic agents and mixing of LCMV virions with fluorescently labelled liposomes led to their dequenching at low pH (Di Simone et al., 1994; Di Simone & Buchmeier, 1995). GP-2 of LASV was recently shown to belong structurally to the class I viral fusion protein family, which includes fusion proteins of retroviruses, orthomyxoviruses, paramyxoviruses, filoviruses and coronaviruses (Eschli et al., 2006). They are characterized by a hydrophobic N terminus and two antiparallel helices separated by a glycosylated antigenic apex. After insertion of a short hydrophobic peptide located at the N terminus of the fusion protein into the membrane of the target cell, the helices form thermodynamically favoured six-helix bundles that bring the viral and cellular membranes into apposition, leading ultimately to their fusion (Earp et al., 2005). Whilst the principal features of LASV and LCMV GP-2 correspond to those of other class I fusion proteins, their highly conserved N-terminal regions (residues 260–266) with the canonical fusion tripeptide sequence Gly–X–Phe are only weakly hydrophobic and it was therefore proposed that their function may differ from that of classical fusion peptides (Gallaher et al., 2001). Experimentally, an internal hydrophobic sequence of LASV GP-2 comprising residues 276–298, which is also highly conserved among all arenaviruses, has been shown to fuse liposomal membranes under acidic conditions (Glushakova et al., 1992). For the New World arenavirus Junín virus (JUNV) and very recently also for LCMV, it was shown that expression of the homologous signal peptide of GP-C is required for cell–cell fusion in a recombinant assay, possibly by stabilising the six-helix bundle and thereby being involved directly in the overall fusion process (York & Nunberg, 2006; Saunders et al., 2007). In order to map the region in the N terminus of LASV GP-2 required for fusion and infectivity, we established two highly sensitive assays based on reporter-gene activation by cell–cell fusion or transduction of target cells with LASV GP-C pseudotyped murine leukemia virus (MLV) particles. All hydrophobic amino acids in the two postulated fusion peptide segments (FPSs) of LASV GP-C were substituted individually by alanine, and the mutant glycoproteins were investigated for intracellular cleavage, cell-surface expression, fusogenicity and infectivity. |
METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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NXgp, constitutively expressing MLVgagpol (kindly provided by G. P. Nolan, Department of Microbiology and Immunology, Stanford University School of Medicine, CA, USA), were grown in Dulbecco's modified Eagle medium (DMEM) with 1 mM pyruvate and supplemented with 10 % heat-inactivated fetal calf serum (FCS), 100 units penicillin ml–1, 0.1 mg streptomycin ml–1 and 2 mM L-glutamine. Chinese hamster ovary (CHO)-K1 cells (ATCC CRL-61) were grown in DMEM nutrient mixture F12 Ham with 1 mM pyruvate supplemented with 10 % heat-inactivated FCS, 100 units penicillin ml–1, 0.1 mg streptomycin ml–1 and 2 mM L-glutamine, whereas the SKI-1/S1P-deficient CHO subclone SRD-12B (kindly provided by J. L. Goldstein, Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, TX, USA) was maintained in the same medium as the CHO cells except for the addition of 5 µg cholesterol ml–1, 1 mM sodium mevalonate and 20 µM sodium oleate (Rawson et al., 1999).
Expression vectors, antibodies and construction of GP-C mutants.
Synthetic codon-optimized LASV GP-C (strain Josiah, GenBank accession no. AAA46286
[GenBank]
) was purchased from GeneArt and cloned into the eukaryotic expression vectors pAdApt (long cytomegalovirus promoter) and pCAGGS (chicken 
-actin promoter), which were described previously (Havenga et al., 2001; Niwa et al., 1991). Construction of pCAGGS vectors for the expression of LASV nucleoprotein (NP) and LASV matrix protein (Z) and generation of anti-peptide rabbit serum for detection of NP, Z and GP-2 were described previously (Lenz et al., 2001; Strecker et al., 2003). Eighteen single amino acid exchanges to alanine (Fig. 1) were introduced into the proposed FPS of GP-2 as well as at position G271 by recombinant PCR and confirmed by DNA sequencing (Higuchi et al., 1988). G260 was also changed to arginine and leucine. The 100 % conserved proline residue at position 275 was changed to the hydrophilic amino acid arginine, because substitution of prolines in internal fusion peptides has been shown to affect the fusogenicity of Ebola virus and avian sarcoma/leukosis virus significantly (Delos et al., 2000; Gómara et al., 2004). The plasmid pCDNA-SKI-1/S1P for the expression of SKI-1/S1P was kindly provided by N. G. Seidah, Laboratory of Biochemical Neuroendocrinology, Clinical Research Institute of Montreal, Quebec, Canada (Seidah et al., 1999). The reporter-gene expression plasmids for the transient cell–cell fusion assay, encoding the human immunodeficiency virus 1 (HIV-1) tat protein (pL3tat) or the HIV-1 long terminal repeat linked to the -galactosidase gene (HIV-1-LTR--gal, pJK2), were a kind gift of V. Bosch (DKFZ Heidelberg, Germany) and have been described previously (Schwartz et al., 1990; Kimpton & Emerman, 1992). As internal positive controls for the fusion assay, the GP-C of the New World arenavirus JUNV and the haemagglutinin (HA) of influenza virus were used, because they have been evaluated in similar assays (Huang et al., 1981; York & Nunberg, 2006; Saunders et al., 2007). Construction of the pCAGGS plasmid for expression of LCMV GP-C (strain WE) was described previously (Beyer et al., 2003). The expression plasmid and antibody for detection of influenza virus HA, subtype H7 (H7-HA), were kindly donated by R. Wagner, Paul-Ehrlich-Institut, Langen, Germany, and described before (Wagner et al., 2005). The GP-C of JUNV, strain MC2, cloned into the expression vector pCDNA-3.1+, was a kind gift of J. Nunberg (Montana Biotechnology Center, The University of Montana-Missoula, MT, USA) (York et al., 2004). The pHIT-derived packageable MLV genome vector pCnBg, encoding the -gal reporter gene, which was used for the production of retroviral MLV pseudotypes (rPT) expressing foreign viral surface proteins, and the polyclonal goat anti-MLV p30 antibody (Quality Biotech), for titration of the pseudotyped particles, were generous gifts from P. M. Cannon (University of Southern California, Los Angeles, CA, USA) and described previously (Soneoka et al., 1995; Bruett & Clements, 2001). The input quantity of equal amounts of cells in the individual assays was tested with a mouse monoclonal anti--actin antibody (Abcam).
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-gal reporter gene lysis buffer (Roche) according to the manufacturer's instructions. A 100 µlvolume of 1 mM chlorophenol red -D-galactopyranoside (CPRG; Roche) was added to the cleared lysate supernatants as a -gal substrate and the colorimetric -gal-induced conversion of CPRG was measured as A570 in an Dynatech ELISA plate MR7000 reader. CHO and SRD-12B cells were treated as described above for Vero cells, except for the modification of a triple transfection with pAdApt-LASV-GP-C, pJK2 and pCDNA-SKI-1/S1P. In all experiments, the amount of transfected plasmid DNA was equalized by co-transfection of the respective amounts of empty vector. All assays were performed at least three times in triplicate (i.e. at least nine times). Fusion activities of all FPS mutants were normalized with respect to the level of processed, cell-surface-expressed GP-2 as measured by quantitative Western blotting (WB), resulting in the normalized fusion activity (NFA). Mutants showing 
10 % of wild-type (wt) activity after normalization were regarded as positive for fusion.
WB analysis.
After electrophoretic transfer of proteins onto nitrocellulose membranes, the membranes were blocked at 4 °C overnight with 10 % milk powder in PBS. The blots were incubated for 1 h with the respective primary antibody diluted in PBS supplemented with 1 % milk powder and 0.1 % Tween 20, followed by incubation with a secondary antibody coupled with horseradish peroxidase (Dianova). Bound antibodies were visualized by using the SuperSignal chemoluminescence substrate as described by the supplier (Pierce). Quantitative WB analysis was done using the TINA 2.09g software (Raytest Isotopenmessgeräte).
Cell-surface biotinylation and immunoprecipitation.
Vero cells in six-well plates were transfected with pAdApt-LASV-GP-C (wt or FPS mutants) using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Control cells were transfected with the empty pAdApt vector. After a 24 h period following lipofection, the cells were incubated on ice for 30 min, washed twice with cold PBS containing 1 % NP-40 and centrifuged in a Heraeus Biofuge at 13 000 r.p.m. (16 060 g) for 30 min; the biotinylated proteins in the supernatant were precipitated with streptavidin–Sepharose beads (Amersham Biosciences) overnight at 4 °C. The beads were washed four times with lysis buffer and bound biotinylated proteins were eluted with reducing Laemmli SDS sample buffer, boiled and resolved by SDS-PAGE (12 % gel) followed by transfer to a nitrocellulose membrane. LASV GP-C (uncleaved) and GP-2 (processed subunit) were detected with polyclonal rabbit anti-GP-2 antibody (anti-GP477).
Infectivity assay with rPT harbouring LASV GP-C.
MLV was pseudotyped with recombinantly expressed LASV GP-C (wt or FPS mutants) by using a modified transient plasmid expression system described previously (Soneoka et al., 1995). Briefly, NXgp cells were co-transfected in 10 cm dishes with equal amounts of pCnBg and pAdApt-LASV-GP-C (wt or FPS mutants) or empty pAdApt vector. rPT titration was performed by quantitative WB analysis, comparing protein-band intensities of the MLV p30 capsid protein 2 days post-transfection in the supernatants of NXgp cells cleared of cellular debris. The presence of LASV GP-C (wt and mutants) on rPT was tested by WB analysis using the anti-GP477 antibody. Vero cells were infected with rPT-containing cell-culture supernatant and transduction efficacy was measured 2 days post-infection as described above for the cell–cell fusion assay. To enhance adsorption of rPT to target cells, polybrene (Sigma-Aldrich) was added to the rPT-containing supernatants during infection at a final concentration of 8 µg ml–1 (Sena-Esteves et al., 2004). Infectivity of all FPS mutants was normalized according to rPT titre and incorporation of processed GP-2 in the rPT, resulting in normalized infectivity (NINF). Mutants showing 10 % of wt activity after normalization were regarded as being positive for infectivity.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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-gal), overlaid with 293 cells transfected with pL3tat (HIV-1-tat), and exposed to different pHs. If the resulting conformational changes in the cell-surface-expressed viral glycoproteins result in fusion of the two different cell populations, the HIV-1 tat protein will transactivate the HIV-1 LTR. This leads to expression of -gal, which is detected by conversion of the chromogenic substrate CPRG. Fig. 2 shows that recombinant influenza virus H7-HA and recombinant JUNV GP-C as positive controls induced cell–cell fusion at a pH optimum of 5.0, as described previously (Huang et al., 1981; York & Nunberg, 2007). Vero cells expressing codon-optimized LASV GP-C fused with 293 cells only at pH
4.5, with maximal fusion occurring at pH 4.0. LCMV GP-C induced cell–cell fusion over the same range and with the same pH optimum as LASV GP-C.
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shows that fusion did not occur in the absence of cleavage, whereas co-transfection of LASV GP-C with SKI-1/S1P restored cleavage and resulted in fusion at pH 4.0.
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). We therefore investigated their influence on cell–cell fusion by co-transfection experiments. Fig. 4 shows that, compared with cell–cell fusion induced by GP-C alone, co-transfection with Z and NP did not change the pH requirements for fusion, and reduced fusion activity slightly.
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) were processed correctly (Fig. 5a). WB analysis revealed that all mutants, except the G260L mutant, were cleaved, but four were cleaved with a much lower efficiency than that of the wt molecule (T261A, M284A, L285A and F293A; Fig. 5a). We next determined whether mutated GP-2 was expressed on the surface of transiently transfected Vero cells by cell-surface biotinylation and immunoprecipitation. All 22 mutants were detected as uncleaved GP-C on the cell surface, whereas only 14 mutants were also transported as cleaved molecules to the cell surface (Fig. 5b). For all mutants with reduced GP-C cleavage and four mutants with wt-like cleavage no cell-surface expression of GP-2 was detected (Fig. 5b).
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shows that no GP-C alanine mutant was fusogenic above 30–40 % of wt GP-C activity. Fusogenicity was abolished for all mutants without cell-surface expression of subunit GP-2 (G260L, T261A, L266A, M284A, L285A, I286A, C292A and F293A). For mutants that showed GP-2 cell-surface expression, fusogenicity was either abolished completely (F262A, W264A, G277A, Y278A and L280A) or reduced to 10–40 % of wt activity (G260R, G260A, G271A, P275R, G276A, W283A, L290A, G294A and V298A). This includes three of the five alanine mutations introduced in the proposed N-FPS (at amino acid positions 260, 262 and 264) and eight of the 13 alanine mutations introduced in the proposed I-FPS (at amino acid positions 276, 277, 278, 280, 283, 290, 294 and 298). All fusion-active mutants were tested over a range from pH 7 to 3, but no change in pH optimum compared with wt GP-C was observed (data not shown). These results are compatible with an involvement of both the proposed N-FPS and I-FPS in fusogenicity of GP-2.
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-gal gene (rPT). The results were normalized according to the amount of processed GP-2 incorporated into the rPT. Uncleaved GP-C was always incorporated into rPT, but only 11 of the 22 mutant GP-2 molecules were detected as subunits on the particle surface (data not shown). Six of these (G260R, G260A, G271A, P275R, W283A and L290A) transduced Vero cells in the absence of polybrene, whereas all other mutants did not (Fig. 7). Addition of polybrene to the assay resulted in additional infectivity in three of the remaining mutants (G276A, G294A and V298A), which had also shown activity in the fusion assay. The data suggest that these mutations influence the secondary structure of GP-2 such that activity in the fusion assay is retained, but attachment or other pre-fusion steps are impaired. Of the GP-2 mutants that show both cell-surface expression and incorporation into rPT, six of 11 clearly correlate in the cell–cell fusion and infectivity assay (nine of 11 taking the polybrene results into account). The results therefore demonstrate that fusogenicity is a prerequisite for infectivity.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). In this study, we demonstrate that GP-2 acts as the pH-dependent fusion protein of both LASV and LCMV by testing the functionality of wt and mutated viral glycoproteins in a recombinant cell–cell fusion and a pseudovirus infectivity assay. Unexpectedly, cell–cell fusion mediated by LASV and LCMV GP-2 required a pH of <5.0 with an optimum at pH 4.0, which is much lower than that reported for other class I fusion proteins and not observed within the endosomal compartment. However, HA of influenza virus A and GP-C of JUNV mediated fusion in our assay at a pH optimum close to 5.0, which corresponds to previously reported data from similar assays (York & Nunberg, 2006; Huang et al., 1981). As fusion of the murine astrocytoma cell line DBT by recombinantly expressed GP-C of LCMV was recently reported to occur at pH 5.0 (Saunders et al., 2007), cell-line-specific differences may account for the observed disparities. As reported for the fusion glycoproteins of other enveloped viruses, cleavage of LASV GP-C and cell-surface expression of GP-2 were found to be required for fusion activity in our assay.
The highly conserved hydrophobic N terminus (aa 260–266) of LASV and LCMV GP-2, comprising the canonical fusion tripeptide Gly–X–Phe, was proposed as an N-FPS (Gallaher et al., 2001). However, the in vitro fusion of liposomes at pH 4.5–5.5 with a peptide corresponding to an internal hydrophobic sequence (aa 276–298) suggested the existence of an additional I-FPS (Glushakova et al., 1992). Because functional viral FPSs are sensitive to mutations, we performed an alanine scan of all hydrophobic amino acids of both predicted FPSs. Fusogenicity was reduced or abolished in all 14 mutants that were expressed as GP-2 on the cell surface. Four of these mutations were located in the N-FPS, eight in the I-FPS and two in between (G271 and P275), thus clearly demonstrating involvement of the whole N terminus of GP-2 in the fusion process. Conservative glycine to alanine changes tended to have the least pronounced effect, reducing fusogenicity compared with wt by 65–80 %, including the N-terminal G260A substitution. Interestingly, all New World arenaviruses have an alanine at this position (Fig. 1). The most pronounced effect on fusogenicity was observed for alanine substitutions of aromatic amino acids. Based on 100 % conservation among all Old and New World arenaviruses and on the loss of fusogenicity in the assay when substituted with alanine, we identified four critical hydrophobic amino acid positions in the N terminus of GP-2: W264, located in the proposed N-FPS, and G277, Y278 and L280, located in the proposed I-FPS. Interestingly, hydrophilic substitution of P275 reduced but did not abolish fusogenicity.
In the recombinant pseudotype assay, nine of 11 GP-C mutants with processed GP-2 detectable on the surface of the particles showed reduced or abolished rPT infectivity; two mutants were hyperinfectious (G260A and G271A). Three mutants with significant, albeit reduced, fusogenicity (G276A, G294A and V298A) showed infectivity only in the presence of polybrene, which non-specifically enhances adsorption of viral particles to cells (Davis et al., 2002). We therefore speculate that the alanine substitutions resulted in structural changes in GP-2 that abolished attachment (or internalization) and led to loss of epitopes.
Taken together, the data demonstrate that (i) GP-2 is the functional fusion protein of LASV, (ii) processing of GP-C is required for fusogenicity, which in turn is a prerequisite for infectivity, and (iii) amino acids from both highly conserved hydrophobic regions in the N terminus of GP-2 are critical for fusion and infectivity. This finding can probably be generalized to all arenaviruses, because several amino acid positions are 100 % conserved within the family. Similar findings of a hierarchical correlation of processing of LCMV GP-C, fusogenicity and infectivity were published recently (Saunders et al., 2007).
Exactly how the N terminus of GP-2 interacts with the cellular membrane remains to be investigated. The canonical fusion tripeptide sequence Gly–X–Phe is clearly important, as shown by the mutational analysis; however, the glycine tolerated an exchange to a polar amino acid, as opposed to the N-FPS of influenza HA2, for example. All New World arenaviruses have an alanine at this position and, interestingly, the alanine exchange, whilst reducing fusogenicity, increased the infectivity of Lassa GP-C pseudotypes. Based on its comparatively weak hydrophobicity and the fact that post-translational cleavage of GP-C occurs not at dibasic amino acids, but within the hydrophobic site (LL/GTFTWTL) (Lenz et al., 2001), its candidacy as a functional fusion peptide analogous to those of influenza and HIV-1 has been questioned (Gallaher et al., 2001). The primary role of the arenavirus N-FPS may therefore not be as a definitive anchor for GP-2 in the endosomal target membrane, but rather to enable contact with the internal hydrophobic sequence. This sequence, however, differs structurally from previously identified I-FPSs, which are segmented into two ordered regions with an intervening turn or loop, usually containing one or more proline residues (Delos et al., 2000). The Robson–Garnier algorithm predicts an order–turn–helix structure for the N terminus of GP-2, with the turn at P275, which is located between the putative N-FPS and I-FPS. The predicted turn region would be approximately 10 aa, which falls within the range of the predicted turn regions for other viral I-FPSs (Delos et al., 2000). However, with respect to fusogenicity and infectivity, P275 was less sensitive to hydrophilic substitution than the prolines located in the I-FPSs of other viruses, e.g. Ebola virus (Ito et al., 1999).
We therefore propose that the two hydrophobic regions within the N terminus of GP-2 resemble N-terminal and internal fusion peptides, respectively, but that both need to interact with the cellular membrane to initiate fusion.
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ACKNOWLEDGEMENTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Received 25 February 2007;
accepted 19 April 2007.
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N terminus determined experimentally (Lenz et al., 2001
N terminus and SKI-1/S1P cleavage site predicted (York et al., 2004
, LASV GP-C; 
, LCMV GP-C; 
, JUNV GP-C; 
, influenza virus HA) and a plasmid expressing HIV-1-LTR-
) served as positive and negative controls, respectively. One experiment, representative of three experiments performed independently and in triplicate, is shown. Bars represent SD.
