1 Fundación Ciencia para la Vida, Zañartu 1482, Santiago, Chile
2 Instituto Milenio MIFAB, Zañartu 1482, Santiago, Chile
3 Centro de Genómica y Bioinformática, Pontificia Universidad Católica, Zañartu 1482, Santiago, Chile
4 Universidad Andrés Bello, Zañartu 1482, Santiago, Chile
Correspondence
Nicole D. Tischler
nicole.tischler{at}bionova.cl or
nicoletis{at}yahoo.com
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ABSTRACT |
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Published online ahead of print on 24 August 2005 as DOI 10.1099/vir.0.81083-0.
The GenBank accession number for the ANDV glycoprotein precursor sequence reported in this paper is 30313865 (nucleotide sequence, AY228238).
Supplementary figures and tables are available in JGV Online.
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INTRODUCTION |
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A common feature of the family Bunyaviridae is the presence of two glycoproteins, which are anchored in the viral envelope membrane by their C-terminal transmembrane regions. These envelope proteins are derived from a single open reading frame of the genomic single-stranded () RNA medium-size segment and their size and location in the open reading frame varies between and within genera in the family Bunyaviridae (Elliott, 1990). After translation, the glycoprotein precursor (GPC) is cleaved into two glycoproteins, termed Gn and Gc according to their position in the precursor. It has been suggested that these glycoproteins associate as heterodimers and accumulate in the Golgi apparatus, where viral budding takes place (Antic et al., 1992
; Chen & Compans, 1991
; Persson & Pettersson, 1991
).
Viral glycoproteins anchored to the envelope membrane are responsible for receptor recognition and entry into target cells through fusion between viral and cellular membranes. After binding to a receptor (Gavrilovskaya et al., 1998; Kim et al., 2002
), hantaviruses enter cells through clathrin-dependent receptor-mediated endocytosis (Jin et al., 2002
) and are thought to fuse with endosomal membranes when the pH is below 6·3 (Arikawa et al., 1985
; McCaughey et al., 1999
). Although the fusogenic activity of hantavirus glycoproteins has been demonstrated, its assignment to Gn or to Gc has not been resolved (Ogino et al., 2004
).
It has been proposed that the active centre of viral fusogenic proteins consists of fusion peptides (FPs), which drive the initial partitioning of the fusion protein into the target membrane and subsequently disrupt the bilayer architecture (reviewed by Epand, 2003; Nieva & Agirre, 2003
). Based on high-resolution X-ray diffraction data, two fusion machineries have recently been identified (Jardetzky & Lamb, 2004
). Class I encompasses fusion proteins of several unrelated viral families, among them the influenza virus haemagglutinin, the human immunodeficiency virus gp41, the paramyxovirus F and the Ebola virus GP2. Their common structural characteristics include a trimeric coiled-coil fold adjacent to the N-terminally located fusogenic unit, which is composed of amino acids in
-helical conformation (reviewed by Skehel & Wiley, 2000
). In contrast, class II fusion proteins are distinguished by three domains of antiparallel
-sheet structures (Rey et al., 1995
), containing an internal FP, which is formed by a loop flanked by two
-sheets (Allison et al., 2001
; Levy-Mintz & Kielian, 1991
). This second fusion class has been described for alphavirus E1 and flavivirus E proteins, belonging to the families Togaviridae and Flaviviridae, respectively. In spite of the differences between these fusion classes, FPs share several common physico-chemical and topological parameters (reviewed by Epand, 2003
; Hernandez et al., 1996
; Nieva & Agirre, 2003
; White et al., 1983
), such as high sequence conservation within the viral family, a length of 1525 residues, a high Gly residue content and location in the ectodomain of envelope proteins.
Here, we identify and characterize an FP candidate sequence within the Gc envelope glycoprotein of hantaviruses and of associated genera. In addition, a three-dimensional molecular-model structure derived for the Gc ectodomain supports the compatibility of the hantavirus Gc glycoprotein with a class II fusion-protein fold. These results suggest a role of Gc in fusion and associates hantaviruses with the class II viral fusion machinery.
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METHODS |
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Hidden Markov models (HMMs) of hantavirus Gc, alphavirus E1 and flavivirus E proteins were extracted directly from the PFAM database seed groups (Bateman et al., 2004). HMM graphical representation was performed by HMM Logos (http://logos.molgen.mpg.de/), which incorporates both emission and transition probabilities in a graphical manner (Schuster-Böckler et al., 2004
).
Fold-recognition methods.
The ANDV GPC sequence was divided into the glycoproteins Gn and Gc, according to the WAASA cleavage site (Löber et al., 2001). From the resultant Gc protein (487 aa), transmembrane and adjoining regions were excluded to reduce the false-positive ratio of fold-recognition programs and, hence, the 414 N-terminal residues were submitted to the following fold-recognition programs: 3D-PSSM version 2.6.0 (Kelley et al., 2000
), LOOPP version 3.00 (Meller & Elber, 2001
) and FUGUE version 2.0 (Shi et al., 2001
). Outputs were ranked according to each program's score values and the results were subsequently inspected for cross-matches.
Comparative molecular modelling.
Hydropathicity-profile comparisons were conducted through the protein hydrophilicity/hydrophobicity comparison server (http://bioinformatics.weizmann.ac.il/hydroph/). Hydropathicity indices were obtained according to the KyteDoolittle scale (Kyte & Doolittle, 1982) using a 21-residue window size and allowing gap insertion to encourage divergent comparisons.
MODELLER-6 (ali & Blundell, 1993
) was used to develop several comparative models of the ANDV Gc ectodomain (residues 1414), using as template the E protein crystallographic data (Rey et al., 1995
) of Tick-borne encephalitis virus (TBEV) (PDBid: 1SVB). The input local alignment was optimized manually to maximize the secondary-structure overlap (15·3 % identity; 24·4 % similarity). Alternatives for disulfide bridges of the model were defined heuristically by HMM comparisons of Gc and class II fusion proteins and the alignment proximity of Cys residues with the template. Twenty models were generated and ranked according to analysis of their stereochemistry by using PROCHECK (Laskowski et al., 1993
). Each model was additionally ranked by the VERIFY3D score (Eisenberg et al., 1997
) and potential energy values were computed by MODELLER. From the ensemble, the top 10-ranked structures were selected as starting structures for molecular-dynamic simulations.
Molecular-dynamic simulations.
Template and model structures were subjected to molecular-dynamic simulations by using the GROMOS-96 force field (Van Gunsteren et al., 1996) within the Gromacs 3.1 software (Van der Spoel et al., 2002
). Structures were embedded in a solvent box with the simple-point charge water model to obtain a periodic boundary condition. Long-range electrostatics were calculated with the particle-mesh Ewald method. Lennard-Jones and short-range Coulombic interactions were both cut off at 0·9 nm. Simulations were performed under normal pressure and temperature conditions, applying a constant pressure of 1 bar independently in all three directions with a coupling constant of 0·5 ps and compressibility of 4·5x105 bar1. Additionally, ions were added to compensate the net charge of the whole system. Temperature was controlled by independently coupling the protein, solvent and counterions in a bath at a temperature of 300 K with a coupling constant of 0·1 ps. Energy minimization to reduce close contacts was achieved through the steepest-descents minimization protocol, until the maximum force decayed to 100 kJ mol1 nm1. The system was then equilibrated to 300 K over 100 ps with 2 fs integration steps, using the LINCS algorithm to restrain all bond lengths (Hess et al., 1997
). After this, constraints over bonds were removed and a full 1 ns molecular-dynamic simulation at 300 K was performed with 1 fs integration steps. Trajectory frames were collected each 1 ps and root mean square deviation of backbone atoms and root mean square fluctuation per residue were calculated.
Liposome preparation.
Liposomes were prepared following the method of Hope et al. (1985). Briefly, dried lipid films were hydrated with 5 mM HEPES, 150 mM NaCl, 0·1 mM EDTA (pH 7·4) and subjected to five cycles of freezing and thawing. Subsequently, vesicles were sized by extrusion through a polycarbonate filter with a pore size of 0·1 µm. Liposomes consisted of a 1 : 1 : 1 : 1·5 molar ratio of phosphatidylcholine (from egg yolk), phosphatidylethanolamine (prepared from egg phosphatidylcholine by transphosphatidylation), sphingomyelin (from bovine brain) and cholesterol. All lipids were purchased from Avanti Polar Lipids. The concentration of liposome suspensions was determined by phosphate analysis (Böttcher et al., 1961
).
Fluorescence anisotropy of peptides.
Peptides that represent the identified conserved fusion cd loop-region residues 103130 of the ANDV Gc sequence were synthesized in two sizes. Sequences are as follows: Gc-cd1 (residues 104125), FFEKDYQYETGWGCNPGDCPGV; Gc-cd2 (residues 103133), CFFEKDYQYETGWGCNPGDC PGVGTGCTACG. A negative-control peptide derived from the ANDV Gn protein (residues 8094), VEWRKKSDTTDTTNA, was also used. All fluorescence measurements were performed with a Perkin-Elmer LS50 spectrofluorimeter equipped with polarizers in excitation and emission beams. Temperature was maintained at 20 °C. Small aliquots of liposomes (2·5 mM) were added to a 10 µM peptide solution. The suspension was incubated for 10 min before recording anisotropy through excitation at 295 nm (5 nm bandpass) and emission in L-format at 355 nm (10 nm bandpass). Anisotropy values were averaged from 1015 measurements. Light scattering produced by liposomes was measured by incubation of the corresponding liposome concentrations with tryptophan and subtracted. The concentration of liposomes at half saturation was calculated by the dissociation constant KD of the best-fitted hyperbola: r=(rmaxxL)/(KD+L), where r is the anisotropy of peptides at a given liposome concentration (L) and rmax is the anisotropy of bound peptides at liposome saturation.
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RESULTS |
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As FPs are assumed to be conserved sequences within viral families, the five blocks conserved in the family Bunyaviridae were analysed for possible FP characteristics (Fig. 1a). The first (I) and fourth (IV) blocks (ANDV GPC residues 575594 and 606613, respectively) were discarded as FP candidates, because of their location in the predicted endodomain of Gn. The sequences of blocks II, III and V (ANDV GPC residues 763780, 863877 and 766780, respectively) fulfil the requisites of conservation, ectodomain localization, minimum length and lack of putative glycosylation sites and, hence, represent suitable FP candidates.
For further studies, the sequence corresponding to blocks II and V (ANDV GPC residues 763780) was selected based on the convergence criteria between the Gibbs and MOTIF block-search algorithms (Fig. 1a, red-underlined region in Fig. 1b
). This region has 26·7 % identity and 86·7 % similarity within the analysed sequences of members of the family Bunyaviridae, showing conserved Cys, Gly and Trp residues (Fig. 1b
). As seen in Fig. 1(a)
, the predicted predominant secondary structure of the FP candidate region is randomly coiled, in accordance with the high content of secondary-structure breakers such as Gly and Pro residues (eight of 15 residues within ANDV). The location of this FP candidate within the first 130 residues of Gc (ANDV Gc residues 115129), together with the predominant
-sheet secondary structure predicted for Gc, suggests that this sequence has characteristics similar to those described for class II FPs.
HMM comparisons of FPs
To provide further evidence that the identified sequence shares properties with class II FPs, and to obtain position-specific probabilities of the presence of key amino acids within the selected conserved region, HMMs were compared qualitatively. Fig. 1(c) (top) shows the HMM corresponding to the hantavirus Gc region that comprises the putative FP (upper red line). The strongest position-specific probabilities belong to Trp, Cys, Gly and Pro residues. In comparison, class II FPs of flavivirus E proteins (middle) and alphavirus E1 proteins (bottom) show residues that are essential for FP functionality (see Discussion). Similarities among these three HMMs extend far beyond the putative FP region, which includes highly conserved Cys residues of alphaviruses, flaviviruses (indicated by asterisks) and hantaviruses (positions 12, 24 and 28, using the aromatic Trp 115 as reference).
Fold recognition of the ANDV Gc protein
To determine whether the ANDV Gc protein, which includes the putative FP, may adopt class II fusion-protein structural features, the fold of the first 414 residues of the ANDV Gc protein sequence was studied by using three fold-recognition programs (3D-PSSM, LOOPP and FUGUE). Results obtained with 3D-PSSM showed, as a first hit, the class II fusion protein E1 of Sindbis virus (PDBid: 1LD4) with an E value of 0·03 (95 % confidence). In the case of LOOPP, the dengue 2 virus fusion protein E was obtained as a hit (PDBid: 1OK8), with a threading-energy value of 164·3 (95 % confidence). Similar results were found with FUGUE: the Semliki Forest virus fusion protein E1 (PDBid: 1RER) had a hit with a Z-score value of 2·6 (see Supplementary Tables S1S3, available in JGV Online). Hence, class II fusion proteins are the only cross-matched results. Furthermore, the majority of the threading output protein hits belong to the mainly class, according to the CATH database classification.
Development and evaluation of an ANDV Gc molecular model
To further analyse whether the ANDV Gc protein may support a class II fusion-protein fold, a three-dimensional comparative model was derived. In order to develop such a model, the crystallographic structure of a well-known class II fusion protein was used. To select such a structure, hydropathicity profiles among ANDV Gc and several class II fusion proteins with available crystallographic data were compared. These proteins included dengue virus E, TBEV E, Semliki Forest virus E1 and Sindbis virus E1. Despite the low sequence conservation among hantavirus Gc proteins and class II fusion proteins (approx. 20 %), an unexpectedly high hydropathic profile correspondence was found. The best matching profile for ANDV Gc was obtained with the E protein of TBEV (PDBid: 1SVB) (Fig. 2). As expected, the Gc FP candidate and the well-characterized FP of the TBEV E protein present an amphipathic nature (Fig. 2
), in accordance with their requirement for partition from aqueous milieux into membranes (Nieva & Agirre, 2003
). Taking into account the fact that E and E1 fusion proteins share the same topology (Lescar et al., 2001
), further criteria to choose the TBEV E protein as a crystallographic template were based on: (i) high-quality resolution for the crystallographic structure of the TBEV E protein at 1·90 Å; (ii) similar hydropathicity profiles along the complete sequences (Fig. 2
); and (iii) consistent overlap between the predicted and observed secondary-structure elements among ANDV Gc and TBEV E proteins (Supplementary Fig. S1, available in JGV Online). Fig. 3
(a) (top) shows the best-scored model of the ANDV Gc ectodomain (residues 1414) in comparison with the TBEV E template crystallographic structure (bottom). As seen, the modelled structure retains a high content of
-sheet secondary structure along its three domains (Fig. 3a
), in accordance with the two-dimensional prediction (Fig. 1a
). Moreover, the FP candidate contained in the cd loop is located in an equivalent position to the FP of TBEV E, exposing the conserved tryptophan side chain. Putative disulfide bonds involve Cys residues 87122 and 103129, in a manner that resembles the 1SBV structure in the FP region (Fig. 3b
and compare with HMMs, Fig. 1c
). A potential third disulfide bond was assigned to the third domain between Cys residues 321351, based on the proximity in the alignment of these residues with known disulfide pairs in the reference crystal structure (see Supplementary Fig. S1, available in JGV Online).
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Binding of the FP candidate to artificial membranes
To study whether the putative FP identified in the Gc protein of hantaviruses has the potential to interact with membranes, experiments with synthetic peptides in the presence of artificial membranes were performed. Given the fact that small molecules present a higher rotational movement free in solution than bound to macromolecules, the intrinsic fluorescence anisotropy of peptides was measured in their free state and in the presence of lipid vesicles. It has been shown that the fusion of flaviviruses with artificial membranes is facilitated by the presence of sphingolipids and cholesterol (Corver et al., 2000) and that these lipids are absolutely necessary for alphavirus fusion (Waarts et al., 2002
). For this reason, vesicles of two different compositions, containing only phosphatidylcholine or containing phosphatidylcholine, phosphatidylethanolamine, sphingomyelin and cholesterol, were prepared.
As seen in Fig. 3(b), the conserved FP region of class II fusion proteins (Fig. 1c
) encompasses a loop, termed the cd fusion loop (Rey et al., 1995
). To cover the cd fusion loop (ANDV Gc residues 103130), two peptides of different length were synthesized (see Methods). For fluorescence experiments, the single tryptophan residue present in each peptide was employed as fluorophore. Vesicle-dependent anisotropy changes as high as 0·12 for the short Gc-cd1 peptide and 0·1 for the longer Gc-cd2 peptide were observed after incubation with vesicles made of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin and cholesterol (Fig. 4
). These findings show that peptides free in solution decreased their rotational movement upon addition of liposomes, reflecting their binding to these molecules. In comparison, no significant anisotropy changes were detected when a control peptide derived from the ANDV Gn sequence was incubated with the liposomes (Fig. 4
).
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DISCUSSION |
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The presence of an internal FP in the hantavirus Gc protein is consistent with biochemical data from other members of the family Bunyaviridae, in which the Gc protein has been associated with the viral fusion activity. Antibodies against Gc but not against Gn of California encephalitis virus block syncytium formation without preventing viral attachment to the cell surface (Hacker & Hardy, 1997). In addition, an avirulent Gc variant of La Crosse virus bears a defective fusion function (González-Scarano et al., 1985
). Furthermore, the native Gc protein presents conformational changes at the fusion pH (Pekosz & González-Scarano, 1996
), as shown for activation of other fusion proteins.
FPs have traditionally been described as hydrophobic sequences, but class II FPs also comprise charged and polar residues (see Fig. 1c). They are supposed to be anchored to the target membrane by aromatic residues and are estimated to penetrate the membrane bilayer by 6 Å (Modis et al., 2004
). The exposed carbonyls and charged residues on the outside rim of the fusion loop are thought to impede further penetration and may interact tightly with the phospholipid heads (Gibbons et al., 2004
; Modis et al., 2004
). This concept coincides with the observation that fusion of class II viruses with liposomes is a non-leaky process (Smit et al., 2002
). Therefore, a hemifusion intermediate has been proposed, in which the outer leaflets of the interacting membranes have merged, while the inner leaflets are still apart (Smit et al., 2002
).
To provide additional arguments for a class II FP in hantaviruses, HMMs of well-known flaviviruses and alphaviruses were compared with the HMM of hantaviruses (Fig. 1c). It is remarkable that HMMs within the known class II FPs of alphaviruses and flaviviruses are not statistically comparable (data not shown). This might not be surprising when the low sequence conservation of approximately 20 % is considered. Therefore, their classification into class II fusion proteins is based on their unexpected structural similarity, as revealed by crystallographic analysis of alphavirus E1 and flavivirus E proteins (Gibbons et al., 2004
; Lescar et al., 2001
; Modis et al., 2004
; Rey et al., 1995
). Clearly, then, three-dimensional structures of proteins may be more conserved than suggested by sequence comparison.
Hence, sequences of class II FPs seem to be quite variable among different viral families, except for particular residues involved in their functionality. These residues seem to be more conserved in terms of physico-chemical properties, rather than in terms of sequence. In this sense, the identified FP candidate of hantaviruses shows a high conservation of the required aromatic and Gly residues necessary for interaction with membranes, as well as Cys residues that are presumably involved in structure stabilization (Fig. 1b and 1c). The location of conserved Cys residues in the hantavirus HMM in positions homologous to those of Cys residues of class II FPs far beyond the conserved FP candidate region may imply similar roles (Fig. 1c
). Cys residues included in class II FPs are known to stabilize the fusion loop, which is located at the tip of domain II (Rey et al., 1995
; Lescar et al., 2001
), by three to four disulfide bridges. In summary, the similarity in sequence composition between the hantavirus FP candidate and class II FPs reinforces the hypothesis that the hantavirus Gc protein may contain a class II-like FP.
Evidence for the functionality of the identified FP candidate region is provided by its potential to interact with artificial membranes. The intrinsic fluorescence anisotropy assays clearly demonstrated the interaction of synthetic hantavirus FP candidates with lipid vesicles (Fig. 4). Their higher affinity to liposomes containing sphingomyelin and cholesterol coincides with membrane compositions known to facilitate fusion (Corver et al., 2000
) or to be required for the fusion of class II viruses with liposomes (Waarts et al., 2002
).
To study whether the hantavirus Gc protein may adopt a class II fusion-protein fold, a three-dimensional model was derived. Despite the low sequence similarity between hantavirus Gc and class II fusion proteins (approx. 20 %), encouraging results on prediction of class II fusion proteins by three different fold-recognition programs (Supplementary Tables S1S3, available in JGV Online) persuaded model development. Although no unique set of outputs was generated by these programs, these results strongly support the notion of a -fold structure of Gc and suggest the compatibility of the ANDV Gc sequence with a class II fusion-protein fold in terms of energy values and secondary-structure arrangement. In addition, a strong correspondence of hydropathicity profiles and secondary structures between hantavirus Gc and class II fusion proteins was observed, achieving the best consensus with the TBEV E protein (Fig. 2
and Supplementary Fig. S1, available in JGV Online).
The ANDV Gc model satisfies the acceptance requirements of applied stereochemical parameters and structural stability by means of molecular-dynamic simulations with backbone-coordinate deviations below 3 Å in a 1 ns trajectory analysis (Supplementary Fig. S2, available in JGV Online). As, in the Gc model, some Cys residues appear unpaired despite their close location in the three-dimensional space, further model improvements need to be focused on disulfide-pair formations (Supplementary Fig. S3, available in JGV Online). However, trajectory-analysis data indicate that three proposed disulfide bridges seems to be the minimal number that guarantees the structural stability of the proposed model. In summary, these results suggest that the hantavirus Gc protein is compatible with a class II fusion-protein fold and confirm its possible association with this class of viral fusion machinery.
The association of bunyavirus Gc proteins with class II fusion proteins coincides, moreover, in their overall arrangement. Class II fusion proteins are synthesized as polyproteins together with an N-terminal companion glycoprotein that acts as a chaperone to prevent their aggregation (Marquardt & Helenius, 1992) and probably suppresses their activation in the Golgi network (Guirakhoo et al., 1991
, 1992
), as is the case for p62 in alphaviruses (Andersson et al., 1997
) and prM in flaviviruses (Konishi & Mason, 1993
; Lorenz et al., 2002
). Such a role could also be ascribed to bunyavirus Gn proteins, as Gc does not enter the Golgi apparatus when expressed in the absence of Gn (Persson & Pettersson, 1991
; Shi & Elliott, 2002
).
Class II fusion proteins are not merely responsible for fusion processes, as they also determine the viral envelope protein shell of icosahedral symmetry in either homodimeric or heterodimeric associations (Strauss & Strauss, 2001). Hence, the proposed similarity of Gc proteins of members of the family Bunyaviridae to class II fusion proteins would also influence the viral morphology. In line with this notion, regularly spaced surface projections have been described for some bunyavirions (Martin et al., 1985
; McCormick et al., 1982
; White et al., 1982
) and even an icosahedral surface organization has been proposed for these viruses (von Bonsdorff & Pettersson, 1975
; Ellis et al., 1981
; Lee & Cho, 1981
). Furthermore, based on the observation that the proteolytic removal of the glycoproteins produces highly deformable virion shapes, it has been hypothesized that the structural stability of bunyavirions might be conferred by the spike glycoproteins themselves (von Bonsdorff & Pettersson, 1975
). In summary, and due to the absence of a matrix protein that mediates the association and stabilization between viral envelope and nucleocapsid, a highly organized structure of the surface glycoproteins of bunyaviruses may be plausible and of clear advantage for virion stability.
In conclusion, the characteristics described here for the Gc proteins of hantaviruses and other members of the family Bunyaviridae suggest their role in cell fusion and associate them with class II fusion proteins. Furthermore, these findings raise the question of whether Gn and Gc envelope glycoproteins may be involved in distinctive roles, such as receptor binding, nucleocapsid interaction and fusion, as described for alphavirus E1 and E2 proteins. To confirm the participation of the proposed FP in the hantavirus fusion activity, additional studies are required, including site-directed mutagenesis. Finally, the proposed three-dimensional Gc model may be of value in the development of cell-entry inhibitors that could be useful in therapy.
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ACKNOWLEDGEMENTS |
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Received 5 April 2005;
accepted 6 August 2005.
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