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Department of Microbiology and Immunology, Center for Molecular and Tumor Virology and Feist-Weiller Cancer Center, Louisiana State University Health Sciences Center, Shreveport, USA
Correspondence
Lindsey M. Hutt-Fletcher
lhuttf{at}lsuhsc.edu
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Present address: Department of Microbiology and Molecular Genetics, New England Primate Research Center, Harvard Medical School, 1 Pine Hill Drive, Southborough, MA 01772, USA 
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Primary infections acquired in childhood are usually asymptomatic, but with increasing age they are more likely to result in a self-limiting infectious mononucleosis. Of greatest concern, however, is that long-term carriage can be associated with the development of malignancies. These are predominantly of lymphoid and epithelial origin reflecting the fact that although the primary reservoir of virus in the infected host is in the B-lymphocyte population, infection of epithelial cells also appears to continue throughout life, and trafficking of virus between B cells and epithelial cells may be a common feature of persistence (Jiang et al., 2006; Pegtel et al., 2004; Sitki-Green et al., 2003).
Initiation of infection of B cells and epithelial cells, perhaps not surprisingly, involves different viral and cellular proteins. Fusion of virus with both cell types, as for all herpesviruses (Spear & Longnecker, 2003), requires the activity of the conserved glycoproteins gB and gHgL. These proteins are not only necessary but sufficient for EBV fusion with an epithelial cell (McShane & Longnecker, 2004). Fusion with a B cell, however, additionally requires glycoprotein gp42, a protein that is restricted to the lymphocryptoviruses. Epithelial cell fusion is triggered by a direct interaction between gHgL and an as-yet-unidentified co-receptor, gHgLR (Borza et al., 2004; Wang et al., 1998). B-cell fusion is triggered by an interaction between gp42 and HLA class II, which functions as a B cell co-receptor (Li et al., 1997b). The trigger is presumably transmitted to gHgL with which gp42 forms a complex. Activation of fusion by the two different co-receptors is mutually exclusive, requiring carriage of two different complexes in the virion. Only gHgL complexes that include gp42 can trigger B-cell fusion (Wang & Hutt-Fletcher, 1998) and only complexes that lack gp42 can trigger epithelial cell fusion because the presence of gp42 blocks the interaction of gHgL with gHgLR (Wang et al., 1998). In an HLA class II-positive B cell, but not in an HLA class II-negative epithelial cell, some three part complexes are lost as they interact with and accompany HLA class II to the protease-rich peptide-loading compartment. As a result epithelial cell virus is more B-cell tropic and B-cell virus is more tropic for epithelial cells, perhaps contributing to the movement of virus between the two cell types (Borza & Hutt-Fletcher, 2002).
The differences in the use of the gHgL complex for cell entry can be seen at the level of the gH sequence itself. Residues at both the amino terminus and the carboxyl terminus of the protein are important to the fusion of B cells and epithelial cells, but those that are involved in B-cell and epithelial cell fusion differ (Omerovic et al., 2005; Wu et al., 2005; Wu & Hutt-Fletcher, 2007). The limited mutational analysis of EBV gH that has been done to date has focused on regions of the protein that have been chosen either because of computer driven predictions (Omerovic et al., 2005) or because of mapping of sequences that are recognized by a monoclonal antibody (mAb) to gH that blocks epithelial cell but not B-cell fusion (Wu et al., 2005). Several non-human primate lymphocryptoviruses have been identified and the gH homologues of these viruses may offer additional insight into regions that are important to function. The rhesus lymphocryptovirus (Rh-LCV) isolated from the rhesus macaque (Cho et al., 2001) and the Callitrichine herpesvirus 3 (CalHV3) isolated from the common marmoset (Rivailler et al., 2002a) have been sequenced in their entirety (Rivailler et al., 2002a, 2002b). Rh-LCV is most similar to EBV. Each predicted open reading frame has a counterpart in EBV and the average homology between the two viruses is 76.6 %; the homology of the lytic cycle genes is even higher. EBV gH and Rh-LCV gH have 85.4 % identity and 92.6 % similarity. The average homology of EBV and CalHV3 genes is less at only 47.3 % and EBV gH and CalHV3 gH have 46.2 % identity and 64.6 % similarity. The sequence homology of Rh-LCV and CalHV3 proteins is also not high. The Rh-LCV gH has 45.8 % identity and 64.6 % similarity to CalHV3 gH. The gL homologues parallel this trend. EBV gL and Rh-LCV gL have 81.8 % identity and 89.8 % similarity, EBV gL and CalHV3 gL have 51.4 % identity and 63.2 % similarity and Rh-LCV gL and CalHV3 gL have 51.0 % identity and 62.2 % similarity. We explore here whether conserved sequences of the gH and gL homologues of EBV, Rh-LCV and CalHV3 are sufficient to enable them to substitute functionally for each other.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) were grown in RPMI medium. All culture media were obtained from Gibco-BRL Life Technologies and supplemented with 10 % heat inactivated fetal bovine serum.
Antibodies.
Antibodies used were monoclonal antibodies (mAbs) E1D1 to gHgL, CL59 and CL40 to gH (Molesworth et al., 2000), CL55 to gB and F-2-1 to gp42 (Li et al., 1995), and an anti-peptide antibody to EBV gL (Yaswen et al., 1993). All antibodies were affinity purified on protein A–agarose.
Plasmids.
Plasmids pCAGGS-EBV gH, pCAGGS-EBV gL, pCAGGS-EBV gB and pCAGGS-EBV gp42 (Wu et al., 2005) were made by cloning PCR-amplified EBV sequences into the pCAGGS/MCS vector (a gift of Martin Muggeridge, Louisiana State University Health Sciences Center, Shreveport, USA) for expression under the control of the 
-actin promoter in cooperation with the human cytomegalovirus immediate-early enhancer (Niwa et al., 1991). Plasmid pCAGGS-Rh-LCV gH was made by cloning sequences amplified by PCR from the cosmid DK12 (Rivailler et al., 2002b). Plasmid pCAGGS-Rh-LCV gL was made by cloning sequences amplified by PCR from the cosmid LV28. Plasmids pCAGGS-CalHV3 gH and pCAGGS-CalHV3 gB were made by cloning sequences amplified from cosmid A10 (Rivailler et al., 2002a), pCAGGS-CalHV3 gL was made by cloning sequences amplified by PCR from cosmid D6 and plasmid pCAGGS-CalHV3 gp42 was made by cloning sequences amplified by PCR from cosmid D4. All cosmids were a gift of Fred Wang (Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA). All plasmids were confirmed by sequencing. Cosmid A10 was discovered to include a single base pair change in the gH open reading frame at 34 451 bp of the genome that converted a tyrosine residue into a premature stop codon. This was repaired by cloning an XhoI–SacI fragment into the very small pSP72 vector (Promega) for repair of the mutation using the Quikchange II protocol (Strategene). The repair was confirmed by sequencing and the repaired sequences were recloned into pCAGGS plasmids. All plasmids (pTM1-EBV gH, pTM1-EBV gL and pTM1-EBV gp42) were made as described previously (Li et al., 1995) by cloning PCR-amplified sequences into the pTM1 vector (Moss et al., 1990) that contains a T7 promoter, the encephalomyocarditis virus cap-independent translation signal, a multiple cloning site and a T7 transcriptional terminator. Plasmid pTM1-CalHV3 gH was made by cloning PCR-amplified sequences into the same vector. The premature stop codon was not repaired in this construct.
Immunofluorescence.
For internal staining, slides bearing air-dried acetone-fixed cells were incubated at 37 °C for 30 min in a humidified atmosphere with mAbs, washed three times with PBS for 5 min each, reincubated for 30 min with the appropriate dilution of fluorescein isothiocyanate-conjugated sheep anti-mouse immunoglobulin (ICN/Cappel), washed three times and mounted in mounting medium (Kirkegaard Perry Laboratories).
Levels of cell surface expression.
The levels of expression at the cell surface of EBV gH, Rh-LCV gH and CalHV3 gH were measured by modification of previously described methods (Wu et al., 2005). Briefly, CHO-K1 cells were transfected with the appropriate pCAGGS-based plasmid together with pCAGGS-EBV gB and either pCAGGS-EBV gL or pCAGGS-Rh-LCV gL. DNA (3.2 µg) was mixed with 4 µl Target transfectin F2 and 12 µl Target peptide enhancer (Targeting Systems) in high glucose DMEM medium. Thirty-six hours later, transfected cells were removed from the plastic by scraping and recovered for 1 h at 37 °C. The cells were washed with ice-cold PBS containing 2 % fetal bovine serum and serially reacted with mAb E1D1 or mAb CL40 and phycoerythrin conjugated anti-mouse immunoglobulin (Jackson ImmunoResearch Laboratories) with washing between each step and analysed by flow cytometry.
AGS cell–cell fusion assay.
Fusion of AGS was measured as described previously (Wu et al., 2005). Cells were seeded in two-well chamber slides and were transfected at 70–80 % confluency for 4 h with 0.25 µg pCAGGS-based plasmids expressing EBV gH, Rh-LCV gH or CalHV3 gH, 0.25 µg plasmids expressing EBV gL, Rh-LCV gL or Cal HV3 gL and 0.6 µg of a plasmid expressing EBV gB or CalHV3 gB. Plasmids were mixed with 1 µl Target transfectin F2 and 2 µl Target peptide enhancer in high glucose DMEM medium. Twenty-four hours post-transfection, cells were fixed with ice-cold acetone. Cells transfected with EBV gB were stained with mAb CL55. Cells expressing CalHV3 gB, CalHV3 gH and CalHV3 gL were visualized as a result of expression of green fluorescent protein (GFP) from plasmid pmaxGFP (Amaxa). Fluorescent cells containing four or more nuclei were considered to have undergone fusion and were recorded as one fusion event. The extent of fusion was calculated as the number of fusion events as a percentage of the total number of fluorescent cells. The combination of EBV gH, gL and gB, which supported fusion of 49±12 % of the total number of fluorescent transfected cells was set at 100 % and fusion mediated by other combinations was expressed relative to this.
Epithelial cell–B-cell fusion assay.
B-cell fusion was measured as described previously (Wu et al., 2005). CHO-K1 cells were seeded in six-well plates and were transfected at 70–80 % confluency for 4 h with 0.8 µg pCAGGS-based plasmids expressing gH together with 0.8 µg pCAGGS-based plasmids expressing gL, 1.4 µg plasmids expressing gB, 1.2 µg plasmids expressing gp42 and 0.8 µg pST7-Luc containing the T7 promoter upstream of the luciferase gene (Ferrer et al., 1999) (a gift of Martin Muggeridge). Each well of transfected cells was then overlaid with two million Daudi 29 cells that expressed T7 RNA polymerase. Twenty-four hours later, cells were washed twice with PBS and lysed with 500 µl Passive Lysis buffer (Promega). Luciferase substrate (100 µl) was added to 20 µl supernatant of lysate. Luminosity readings were obtained by using a TD-20/20 luminometer (Promega).
Transfection infection protocol.
Expression from the pTM1-derived vectors was done as described previously (Li et al., 1995). Briefly, CV1 cells were grown to 90 % confluency in 100 mm diameter Petri dishes and infected with vvT7 at an m.o.i. of 5. Thirty minutes later the inoculum was removed, and the cells were washed twice in medium without serum and transfected with pTM1 plasmids. DNA (10 µg) was mixed with 30 µl Lipofectamine (Gibco-BRL) made to a total vol. of 2.5 ml with serum-free medium.
Radiolabelling and immunoprecipitation.
CV-1 cells that had been infected with vvT7 and transfected with pTM1 plasmids were labelled biosynthetically with 100 µCi Pro-Mix (70 % [35S]methionine, 30 % [35S]cysteine) and 300 µCi [35S]cysteine (>1000 Ci mmol–1; Amersham Biosciences) per dish (approx. 107 cells) as described previously (Li et al., 1995). Labelled cells were solubilized in radioimmunoprecipitation buffer (50 mM Tris/HCl, pH 7.2, 0.15 M NaCl, 1 % Triton X-100, 1 % sodium deoxycholate, 0.1 % SDS, 0.1 mM phenylmethyl-sulfonylfluoride and 100 U Aprotinin ml–1) and immunoprecipitated with antibody and protein A–Sepharose CL4B (Sigma). Immunoprecipitated proteins were washed, dissociated by boiling for 2 min in sample buffer with 2-mercaptoethanol and analysed by SDS-PAGE in 10 % polyacrylamide cross-linked with 0.28 % N,N’-diallyltartardiamide followed by fluorography.
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RESULTS |
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); it does not recognize gL alone (Li et al., 1995). We have previously reported that Rh-LCV gH cannot be recognized by CL59, but can be seen by CL40 and, in the presence of EBV gL, by E1D1 (Wu et al., 2005). We further explored the ability of the mAbs to recognize different combinations of EBV, Rh-LCV and CalHV3 proteins by indirect immunofluorescence of permeablized cells. EBV gH retained the ability to be recognized by E1D1, CL40 and CL59 if expressed with Rh-LCV gL (Table 1). It also retained the ability to be recognized by CL40 and CL59 if expressed with CalHV-3 gL, but lost the ability to be seen by E1D1. Rh-LCV gH retained the ability to be recognized by E1D1 and CL40 if expressed with Rh-LCV gL. It also retained the ability to be recognized by CL40, but lost the ability to be seen by E1D1 if expressed with CalHV3 gL. CalHV3 gH was not seen under any circumstances by either CL40 or CL59. It was recognized by E1D1 if expressed with either EBV gL or Rh-LCV gL but not if expressed with CalHV3 gL. Thus, expression of the E1D1 epitope was conditional on the expression of either EBV or Rh-LCV gL.
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). To determine the effects of different combinations of gH and gL homologues on trafficking of gH to the cell surface, the appropriate vectors, together with vectors expressing EBV gB, were co-transfected into CHO-K1 cells and surface expression of gH was measured by flow cytometry of non-permeabilized cells that had been stained either with mAb E1D1, which could detect any combination that included EBV gL, or mAb CL40, which could detect EBV gH and Rh-LCV gH in the absence of gL or in the presence of CalHV3 gL. Rh-LCV gL was not as efficient as EBV gL at facilitating transport of either EBV gH or its own gH to the cell surface, although in each case expression levels were at least 60 % of that of an EBV gHgL complex (Fig. 1). In contrast, CalHV3 gL was unable to increase levels of either EBV gH or Rh-LCV gH at the cell surface over the low levels seen when the gL expression plasmid was replaced by empty vector. We were unable to determine the expression of CalHV3 gH in the presence of CalHV3 gL, since none of our antibodies recognized this combination, but, surprisingly, EBV gL resulted in surface expression of CalHV3 gH at levels very similar to those of EBV gH.
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). EBV gH and gL together with EBV gB induce fusion in approximately 50 % of transfected cells (Wu et al., 2005). A combination of EBV gH and Rh-LCV gL, which was expressed at close to 90 % of the levels of EBV gH and gL, also mediated approximately 90 % of the levels of fusion. However, CalHV3 gL, which had facilitated expression of Rh-LCV gH at levels no higher than those of Rh-LCV gH in the absence of gL, supported less than 20 % of the levels of fusion.
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). CalHV3 gH in combination with EBV gL, which was expressed at the cell surface at levels close to those of EBV gHgL mediated close to 50 % of that supported by EBV gHgL. However, substitution of CalHV3 gL for EBV gL reduced this level of fusion further. To determine if this might reflect an inability of the CalHV3 gH and gL to function fully with EBV gB, we repeated the experiments with CalHV3 gH, gL and gB, including a vector that expressed GFP to mark those cells that had been successfully transfected. Although we were unable to determine the expression levels of the CalHV3 proteins, this combination increased fusion levels to at least 60 % of those supported by the combination of all EBV proteins.
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), plasmids expressing virus proteins together with a plasmid expressing the luciferase gene under the control of a T7 promoter were transfected into CHO-K1 cells, which, unlike AGS cells, do not fuse with each other (McShane & Longnecker, 2004). The transfected cells were overlaid with Daudi 29 cells expressing the T7 polymerase and fusion was measured in terms of an increase in luciferase activity. EBV gH together with Rh-LCV gL mediated between 60 and 70 % of fusion mediated by EBV gH and EBV gL (Fig. 4). Rh-LCV gH together with CalHV3 gL also supported a mean of 50 % of the fusion supported by EBV gH and EBV gL, despite the fact that the Rh-LCV and CalHV3 combination had mediated only very low levels of epithelial cell fusion. Most surprising, however, was the observation that Rh-LCV gH and Rh-LCV gL mediated only 20 % of fusion mediated by EBV gH and gL. This could not simply represent a failure to interact with gp42, which binds directly to gH (Wu & Hutt-Fletcher, 2007) because the combination of Rh-LCV gH, EBV gL and gp42, mediated as much as four times the level of fusion as did the combination of EBV gH, EBV gL and EBV gp42. These high levels of B-cell fusion were reminiscent of those mediated by an EBV gH construct in which an alanine substitution was made for a glutamic acid residue at position 595 in the full-length protein (Wu & Hutt-Fletcher, 2007). This construct could even mediate modest, but significant levels of B cell, though not epithelial cell fusion in the absence of gL. To determine if this were also true for Rh-LCV gH, B-cell fusion was measured in the absence of Rh-LCV gL. The combination of Rh-LCV gH, EBV gp42 and EBV gB mediated a variable but significant level of B-cell fusion (Fig. 5), although, as the EBV gH mutant, Rh-LCV gH mediated no epithelial cell fusion in the absence of gL (data not shown).
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). The only combination that mediated low levels of fusion, close to 20 % of EBV gH, gL, gB and gp42, was a combination of CalHV3 gH, CalHV3 gL, EBV gp42 and CalHV3 gB. To confirm that EBV gp42 could interact with CalHV3 gH, combinations of CalHV3 gH and EBV gH were expressed for radiolabelling in the pTM1 vector together with either EBV gL or EBV gp42. The CalHV3 gH in this vector still retained the premature stop codon and thus was of a similar size to EBV gH. Both EBV and CalHV3 gH, the only proteins among gH, gL and gp42 that contain methionine residues for labelling beside the start residue, could be immunoprecipitated with either antibody to EBV gL or EBV gp42 (Fig. 7).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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None of the proteins used was epitope tagged and no antibodies to Rh-LCV or CalHV3 homologues were available. However, all combinations that included EBV gL could be recognized by mAb E1D1, an antibody that fails to react with any gH homologue expressed alone. Although it is still formally possible that the E1D1 epitope is formed by a particular conformation of EBV gH, Rh-LCV gH or CalHV3 gH that can only be attained in the presence of EBV gL, the observation that E1D1 could recognize Rh-LCV gL in concert with EBV gH or CalHV3 gH, but not CalHV3 gL in combination with any of the three gH homologues does also raise the possibility that the epitope recognized by this antibody includes sequences contributed by both gH and gL. This is of interest since E1D1 can partially block binding of EBV virions and soluble forms of gHgL to the epithelial cell receptor/co-receptor gHgLR (Borza et al., 2004; Molesworth et al., 2000). Maruo et al., (2001) have reported that EBV gL alone can bind, although less robustly, to epithelial cells and this would be consistent with the binding site of gHgLR recognizing a combination of the two proteins.
EBV gH and Rh-LCV gH could both be expressed at the cell surface with their opposite gL homologues at levels close to those of the arbitrary standard of EBV gH in the presence of EBV gL. CalHV3 gL was, however, a poor chaperone for either EBV gH or Rh-LCV gH. This was something of a surprise given the reciprocal ability of EBV gL to mediate high levels of expression of CalHV3 gH and given the ability of EBV gL and varicella-zoster virus gL, much more distantly related viruses, to substitute for each other, at least in terms of intracellular transport (Li et al., 1997a).
Of the three gH homologues Rh-LCV gH was the most fusogenic for epithelial cells and EBV and Rh-LCV proteins in any combination were as, or more fusogenic for epithelial cells than CalHV3 gH. Only with its own gL and gB could CalHV3 gH mediate fusion at levels close to the Rh-LCV and EBV combinations. Beyond this, no CalHV3 gH combination could support more than 20 % fusion with a B cell. It is possible that CalHV3 gp42 fails to interact with HLA class II, or at least the alleles that are expressed on Daudi 29 B cells, but even the presence of EBV gp42, which could clearly interact with CalHV3 gH, could not rescue B-cell fusion to levels equivalent to those seen with epithelial cells. We recognize that the assays used for B-cell and epithelial cell fusion may not be measuring identical events. It is formally possible that the luciferase assay is in some cases measuring pore formation in the absence of the pore expansion that would be necessary for production of multinucleate cells that were scored as epithelial fusion (Wu et al., 2005). However, if anything this might overestimate levels of B-cell fusion relative to that of epithelial cells. Thus, either EBV gp42 is unable to transmit a triggering signal effectively to CalHV3 gH, gL and gB, the three proteins that have been referred to as the ‘core fusion machinery’ of herpeviruses (Spear & Longnecker, 2003), or there are differences in the way in which the core machinery interacts with B cells and epithelial cells that extend beyond differences in the way it is activated. The failure of combinations of CalHV3 proteins to mediate B-cell fusion is, however, consistent with reports that the virus is unable to transform human B cells (Jenson et al., 2002). There is no information with respect to the ability of the virus to infect a human epithelial cell.
Most surprising, perhaps, were the very high levels of B-cell fusion supported by Rh-LCV gH expressed with EBV gL and the low levels that were supported by Rh-LCV gH and its own gL. This again would point to a difficulty in transmission of a signal from EBV gp42 to the Rh-LCV complex since the robust epithelial cell fusion mediated by Rh-LCV gH and Rh-LCV gL confirms its basic integrity. In the absence of Rh-LCV gL a direct interaction of Rh-LCV gH could mediate variable, but significant levels of fusion with a B cell even though its levels of expression at the cell surface were less than half of those in the presence of Rh-LCV gL. This phenotype of Rh-LCV gH is similar, but not identical to that of a point mutant of EBV gH in which a glutamic acid residue at position 595 is replaced with an alanine (Wu & Hutt-Fletcher, 2007). Both proteins mediated higher levels of B-cell fusion than wild-type EBV gH and both mediated some B-cell fusion in the absence of gL. However, Rh-LCV gH also supported significantly higher levels of epithelial cell fusion than EBV gH, whereas the point mutant of EBV gH was significantly inhibited for epithelial cell fusion. The sequences of Rh-LCV gH and EBV gH are identical from residues 592 to 612 so this region is not likely to account for the enhanced activity of Rh-LCV gH.
In view of the limited sequence identity between EBV and Rh-LCV gH homologues and CalHV3 gH it is perhaps not surprising that they do not complement each other well. However, Rh-LCV and EBV proteins do work well together and we have previously shown that chimeras of Rh-LCV gH and EBV gH are viable (Wu et al., 2005). This will provide opportunities to map those regions of the protein that contribute to enhanced B-cell fusion and explore the possibility that differences between support of B-cell and epithelial cell fusion extend beyond differences in activation of a core fusion machinery.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 24 February 2007;
accepted 12 April 2007.
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