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Short Communication |
Department of Microbiology and Immunology, The Feinberg School of Medicine, Northwestern University, Chicago, IL 60611, USA
Correspondence
Richard Longnecker
r-longnecker{at}northwestern.edu
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ABSTRACT |
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furin) was created lacking the motif. This cleavage mutant was expressed well in cell culture but was not cleaved. Experiments examining gB furin in a cell-fusion assay revealed that fusion was reduced by 52 % in epithelial and 28 % in B cells when compared with wild-type EBV gB. This decrease in cell–cell fusion is similar to that observed with multiple alphaherpesvirus gB cleavage mutants and supports a conserved function for cleaved gB. |
MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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; Rickinson & Kieff, 2007). EBV is recognized to infect epithelial cells as well as B lymphocytes during its normal cycle of persistence (Hutt-Fletcher, 2007). EBV is the causative agent of infectious mononucleosis, and has also been associated with a number of human malignancies of epithelial and B-cell origin, including Burkitt's lymphoma and nasopharyngeal carcinoma (Kieff & Rickinson, 2007; Rickinson & Kieff, 2007). Similar to other herpesvirus family members, EBV encodes a number of membrane glycoproteins. Membrane glycoproteins are important in a variety of viral processes including entry of herpesviruses into target cells. Along with membrane glycoproteins H (gH) and gL, herpesvirus gB has been shown to be essential for herpesvirus fusion, and together they form the core virus fusion machinery (Pereira, 1994; Spear & Longnecker, 2003).
Herpesvirus gB homologues are highly conserved, and a number of gB homologues across all three of the herpesvirus subfamilies (alpha-, beta- and gammaherpesviruses) possess a known cleavage motif R-X-K/R-R recognized by the cellular protease furin and are cleaved (Backovic et al., 2007; Baghian et al., 2000; Britt & Vugler, 1989; Fleckenstein et al., 1982; Hampl et al., 1984; Johannsen et al., 2004; Loh, 1991; Meredith et al., 1989; Okazaki, 2007; Ross et al., 1989; Sullivan et al., 1989; van Drunen Littel-van den Hurk & Babiuk, 1986; Vey et al., 1995; Whealy et al., 1990; Wolfer et al., 1990). EBV has been shown to possess the defined cleavage motif and is cleaved at this defined site by a cellular protease (Backovic et al., 2007). In EBV virions, most gB present is in the cleaved form, with only a fraction of total gB present in the uncleaved form (Johannsen et al., 2004). The physiological relevance of this proteolytic processing of gB to its function in infection is not well understood and loss of cleavage has no effect on viral growth of bovine herpes virus 1 (BoHV-1), pseudorabies virus (PRV) or human cytomegalovirus gB (Kopp et al., 1994; Okazaki, 2007; Strive et al., 2002). However, loss of cleavage in BoHV-1 and PRV gB decreases viral cell–cell spread, suggesting that these cleaved herpesvirus gBs may function differently in virus–cell and cell–cell fusion (Kopp et al., 1994; Okazaki, 2007). Previous studies have shown that there is not sufficient homology between the different subfamily gBs to allow complementation in other members of the herpesvirus family (Lee et al., 1997). Unlike alpha- and betaherpesvirus gB homologues, EBV gB is present predominantly in the membranes of the nucleus and endoplasmic reticulum and only in small amounts on the plasma membrane (Emini et al., 1987; Gong & Kieff, 1990; Gong et al., 1987; Qualtiere & Pearson, 1979).
Whilst the function of gB cleavage has been investigated for a number of herpesviruses, the function of EBV gB cleavage has not been examined previously. To investigate the importance of gB cleavage for fusion activity of EBV gB, a group of mutants was constructed. Mutants were generated using a QuikChange site-directed mutagenesis kit (Stratagene), with the plasmid encoding wild-type EBV gB in the Stratagene pSG5 vector used as template (Haan et al., 2001). Positive clones were sequenced, grown in large quantities, isolated using an EndoFree Plasmid Maxi kit (Qiagen) and sequenced again. Three mutants were constructed: one in which the specific cleavage motif (R-R-R-R-R) was deleted and two in which five amino acid stretches near each side of the cleavage motif (Fig. 1) were deleted to serve as controls.
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, immunoprecipitation followed by SDS-PAGE (Laemmli, 1970) and Western blotting (Backovic et al., 2007; Burnette, 1981) were performed on proteins expressed by Chinese hamster ovary (CHO)-K1-transfected cells (Fig. 2). Briefly, following lysis of transfected CHO-K1 cells with 1 % Triton-X lysis buffer, EBV gB was immunoprecipitated with protein G–Sepharose beads (GE Healthcare) bound to anti-gB monoclonal antibody (mAb) CL55 (Backovic et al., 2007; McShane & Longnecker, 2004) before SDS-PAGE and Western blotting. Immunoprecipitates were run on 10 % Tris/HCl Ready Gel pre-cast gels (Bio-Rad) in SDS running buffer at 100 V for 100 min. Proteins were transferred to Immobilon-P membranes in transfer buffer at 100 V for 90 min with cooling. Blots were blocked in Tris-buffered saline with Tween 20 plus 5 % skimmed milk for 1 h at room temperature or overnight at 4 °C and then incubated for an hour at room temperature with a rabbit polyclonal anti-gB antibody (Backovic et al., 2007; McShane & Longnecker, 2004) diluted 1 : 1000 in blocking solution. Blots were washed, and a horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (Cell Signaling) was applied for 45 min at room temperature. Blots were washed again and then mixed in equal volumes of ECL solutions and exposed to hyperfilm (Amersham Biosciences).
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), all three gB mutants were expressed similarly to wild-type gB, indicating that the described mutations did not affect production of the protein. However, the C-terminal cleavage product of gB (Fig. 2a, arrow) was not present in the lane of gB furin, indicating that this mutant was not cleaved in the same way as wild-type gB. The mutants containing the deletions on either side of the cleavage motif were cleaved similarly to wild-type gB, indicating that loss of gB cleavage was due to loss of the specific cleavage motif and was not a non-specific effect of deletion in the region. Under non-reducing conditions (Fig. 2b), it was shown that all three gB mutants were glycosylated and were able to oligomerize similarly to wild-type gB, indicating that these processes were not significantly affected by the described deletions. The gB cleavage product was not visible under these conditions.
To examine cell-surface localization of wild-type gB and the gB mutants, transfected CHO-K1 cell-surface proteins were biotinylated with the membrane-impermeable biotinylation agent sulfosuccinimidyl-6-(biotinamido) hexanoate (Pierce) prior to lysis and SDS-PAGE and Western blot analysis. This established approach (Daniels & Amara, 1998) has been used successfully for detection of surface-expressed EBV gB (Backovic et al., 2007). Following biotinylation, surface-expressed gB was immunoprecipitated with anti-gB antibody as described above, immunoprecipitates were run on a 7.5 % Tris/HCl Ready Gel pre-cast gel (Bio-Rad) in SDS running buffer, proteins were transferred to Immobilon-P membranes as described above and biotinylated gB was detected by avidin conjugated to HRP (Bio-Rad) (Fig. 2c). All three gB mutants showed surface expression comparable to wild-type gB (Fig. 2c), indicating that the deletions did not affect trafficking of the protein to the cell surface. A background band was apparent in all samples including the vector control just under the 250 kDa marker (Fig. 2c).
To determine the ability of gB variants to mediate fusion with the two cell types that EBV infects in vivo – epithelial and B cells – a virus-free cell-based fusion assay was utilized (Backovic et al., 2007; Kirschner et al., 2006; McShane & Longnecker, 2004, 2005). Mammalian B cells were Daudi B lymphocytes (ATCC) stably selected with G418 to express T7 RNA polymerase (Daudi-T7) (Silva et al., 2004). Mammalian epithelial cells were human embryonic kidney (HEK) 293T14 cells that express simian virus 40 large T antigen (ATCC) and have been modified to express T7 RNA polymerase stably under selection of 100 µg zeocin ml–1 (Omerovic et al., 2005). The cells were maintained in culture as described previously (McShane & Longnecker, 2004). Briefly, effector CHO-K1 cells were transfected (Kirschner et al., 2006; McShane & Longnecker, 2004) with plasmids encoding the glycoproteins required for fusion (gB, gH and gL, and gp42 transfected for fusion with both epithelial and B cells), as well as a plasmid encoding luciferase under the control of T7 polymerase. Six hours after transfection, cells were washed and returned to Ham's F-12 complete medium, and 24 h post-transfection cells were washed with PBS and detached with Versene. All cells (CHO-K1, 293T14 and Daudi-T7) were counted with a Beckman Coulter Z1 particle counter, and the effector and target cells were then mixed in equal amounts (2.5x105 cells per sample) and plated in duplicate into a 24-well plate in Ham's F-12 medium. Twenty-four hours later, the cells were washed with PBS and lysed, and luciferase activity was quantified using a Promega Reporter Assay system. Relative luciferase activity was measured on a Perkin-Elmer Victor plate reader.
The ability of each of the three gB mutants to mediate fusion with epithelial and B cells is summarized in Fig. 3. The gB 415–419 and gB 443–447 mutants showed no significant difference in ability to mediate fusion similar to wild-type gB with epithelial or B cells. This similar ability indicated that deletions made in this region of the EBV gB protein near the furin cleavage motif had no significant effect on the ability of gB to mediate fusion. A significant decrease in fusion ability was seen, however, with the gB furin mutant, which exhibited 48 and 72 % of the wild-type gB fusion activity with epithelial and B cells, respectively. This decrease in ability to mediate fusion indicated that loss of gB cleavage had an inhibitory effect on EBV-induced cell–cell fusion, with both epithelial and B cells.
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; Okazaki, 2007). Whilst this function of gB cleavage in these alphaherpesviruses is well characterized, the function of cleavage of other herpesvirus gB homologues containing the identified cleavage motif was unknown. We have shown that, similar to BoHV-1 and PRV gB, cleavage of EBV gB plays a role in cell–cell fusion, indicating a conserved function of cleavage across gB homologues from different herpesvirus subfamilies. Whilst a general decrease in cell–cell spread has been observed previously with cleavage-deficient BoHV-1 and PRV gB mutants, our study is the first to attempt to quantify cell fusion, allowing greater insight into the functional significance of gB cleavage. Interestingly, a greater decrease was seen in EBV-induced fusion with epithelial cells than fusion with B cells. This difference, although small, was statistically significant (P<0.05) and may indicate a difference in gB function during fusion with each cell type. This hypothesis is supported by a previous study showing that epithelial-cell fusion, but not B-cell fusion, is mediated by gB mutants with enhanced cell-surface expression, independent of other viral proteins (McShane & Longnecker, 2004).
It is possible that cell–cell spread of EBV is more efficient with epithelial cells than with B cells. Fusion levels with transiently transfected HEK-293-P (epithelial) cells are increased in relation to similarly transfected Daudi B cells in a virus-free cell-fusion assay (McShane & Longnecker, 2004), and this result was duplicated when the stably transfected cell lines 293T14 (epithelial cells) and Daudi-T7 (B cells) were substituted. Reactivation of EBV from latency is quickly controlled by the immune system in immunocompetent individuals. Efficient cell–cell spread of EBV between oral epithelial cells may improve viral spread to uninfected individuals by allowing production of larger amounts of infectious virions during the small window of virus reactivation before host immune system detection and elimination. EBV infection of some epithelial cell lines is known to occur more efficiently by cell–cell contact (Imai et al., 1998; Speck & Longnecker, 2000). Efficient cell–cell spread of EBV between B lymphocytes may not significantly affect viral spread after EBV reactivation, as virions released from infected B lymphocytes efficiently infect epithelial cells, the cell type from which a majority of viruses shed in saliva are produced (Hutt-Fletcher, 2007). Efficient cell–cell spread of EBV between epithelial cells may confer a survival advantage to EBV. This advantage could help explain both the general difference in quantity of cell–cell fusion observed between epithelial and B cells and the specific difference in effect of gB cleavage motif mutation. Further studies beyond our in vitro fusion assay will be necessary to determine conclusively the efficiency or relevance of cell–cell spread of EBV between epithelial and B cells. The varying levels of cell–cell fusion inhibition caused by eliminating gB cleavage and the previously suggested difference in the function of EBV gB between virus entry and cell–cell fusion highlight the critical and complicated role gB plays in EBV infection and spread.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTMAIN TEXTREFERENCES |
|---|
Baghian, A., Luftig, M., Black, J. B., Meng, Y. X., Pau, C. P., Voss, T., Pellett, P. E. & Kousoulas, K. G. (2000). Glycoprotein B of human herpesvirus 8 is a component of the virion in a cleaved form composed of amino- and carboxyl-terminal fragments. Virology 269, 18–25.[CrossRef][Medline]
Britt, W. J. & Vugler, L. G. (1989). Processing of the gp55–116 envelope glycoprotein complex (gB) of human cytomegalovirus. J Virol 63, 403–410.
Burnette, W. N. (1981). "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate–polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem 112, 195–203.[CrossRef][Medline]
Daniels, G. M. & Amara, S. G. (1998). Selective labeling of neurotransmitter transporters at the cell surface. Methods Enzymol 296, 307–318.[Medline]
Emini, E. A., Luka, J., Armstrong, M. E., Keller, P. M., Ellis, R. W. & Pearson, G. R. (1987). Identification of an Epstein–Barr virus glycoprotein which is antigenically homologous to the varicella-zoster virus glycoprotein II and the herpes simplex virus glycoprotein B. Virology 157, 552–555.[CrossRef][Medline]
Fleckenstein, B., Muller, I. & Collins, J. (1982). Cloning of the complete human cytomegalovirus genome in cosmids. Gene 18, 39–46.[CrossRef][Medline]
Gong, M. & Kieff, E. (1990). Intracellular trafficking of two major Epstein–Barr virus glycoproteins, gp350/220 and gp110. J Virol 64, 1507–1516.
Gong, M., Ooka, T., Matsuo, T. & Kieff, E. (1987). Epstein–Barr virus glycoprotein homologous to herpes simplex virus gB. J Virol 61, 499–508.
Haan, K. M., Lee, S. K. & Longnecker, R. (2001). Different functional domains in the cytoplasmic tail of glycoprotein B are involved in Epstein–Barr virus-induced membrane fusion. Virology 290, 106–114.[CrossRef][Medline]
Hampl, H., Ben-Porat, T., Ehrlicher, L., Habermehl, K. O. & Kaplan, A. S. (1984). Characterization of the envelope proteins of pseudorabies virus. J Virol 52, 583–590.
Hutt-Fletcher, L. M. (2007). Epstein–Barr virus entry. J Virol 81, 7825–7832.
Imai, S., Nishikawa, J. & Takada, K. (1998). Cell-to-cell contact as an efficient mode of Epstein–Barr virus infection of diverse human epithelial cells. J Virol 72, 4371–4378.
Johannsen, E., Luftig, M., Chase, M. R., Weicksel, S., Cahir-McFarland, E., Illanes, D., Sarracino, D. & Kieff, E. (2004). Proteins of purified Epstein–Barr virus. Proc Natl Acad Sci U S A 101, 16286–16291.
Kieff, E. & Rickinson, A. B. (2007). Epstein–Barr virus and its replication. In Fields Virology, 5th edn, vol. 2, pp. 2603–2654. Edited by D. M. Knipe, P. M. Howley, D. E. Griffin, R. A. Lamb, M. A. Martin, B. Roizman & S. E. Straus. Philadelphia, PA: Lippincott Williams & Wilkins.
Kirschner, A. N., Omerovic, J., Popov, B., Longnecker, R. & Jardetzky, T. S. (2006). Soluble Epstein–Barr virus glycoproteins gH, gL, and gp42 form a 1 : 1 : 1 stable complex that acts like soluble gp42 in B-cell fusion but not in epithelial cell fusion. J Virol 80, 9444–9454.
Kopp, A., Blewett, E., Misra, V. & Mettenleiter, T. C. (1994). Proteolytic cleavage of bovine herpesvirus 1 (BHV-1) glycoprotein gB is not necessary for its function in BHV-1 or pseudorabies virus. J Virol 68, 1667–1674.
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.[CrossRef][Medline]
Lee, S. K., Compton, T. & Longnecker, R. (1997). Failure to complement infectivity of EBV and HSV-1 glycoprotein B (gB) deletion mutants with gBs from different human herpesvirus subfamilies. Virology 237, 170–181.[CrossRef][Medline]
Loh, L. C. (1991). Synthesis and processing of the major envelope glycoprotein of murine cytomegalovirus. Virology 180, 239–250.[CrossRef][Medline]
McShane, M. P. & Longnecker, R. (2004). Cell-surface expression of a mutated Epstein–Barr virus glycoprotein B allows fusion independent of other viral proteins. Proc Natl Acad Sci U S A 101, 17474–17479.
McShane, M. P. & Longnecker, R. (2005). Analysis of fusion using a virus-free cell fusion assay. Methods Mol Biol 292, 187–196.[Medline]
Meredith, D. M., Stocks, J. M., Whittaker, G. R., Halliburton, I. W., Snowden, B. W. & Killington, R. A. (1989). Identification of the gB homologues of equine herpesvirus types 1 and 4 as disulphide-linked heterodimers and their characterization using monoclonal antibodies. J Gen Virol 70, 1161–1172.
Okazaki, K. (2007). Proteolytic cleavage of glycoprotein B is dispensable for in vitro replication, but required for syncytium formation of pseudorabies virus. J Gen Virol 88, 1859–1865.
Omerovic, J., Lev, L. & Longnecker, R. (2005). The amino terminus of Epstein–Barr virus glycoprotein gH is important for fusion with epithelial and B cells. J Virol 79, 12408–12415.
Pereira, L. (1994). Function of glycoprotein B homologues of the family Herpesviridae. Infect Agents Dis 3, 9–28.[Medline]
Qualtiere, L. F. & Pearson, G. R. (1979). Epstein–Barr virus-induced membrane antigens: immunochemical characterization of Triton X-100 solubilized viral membrane antigens from EBV-superinfected Raji cells. Int J Cancer 23, 808–817.[Medline]
Rickinson, A. B. & Kieff, E. (2007). Epstein–Barr virus. In Fields Virology, 5th edn, vol. 2, pp. 2655–2700. Edited by D. M. Knipe, P. M. Howley, D. E. Griffin, R. A. Lamb, M. A. Martin, B. Roizman & S. E. Straus. Philadelphia, PA: Lippincott Williams & Wilkins.
Ross, L. J., Sanderson, M., Scott, S. D., Binns, M. M., Doel, T. & Milne, B. (1989). Nucleotide sequence and characterization of the Marek's disease virus homologue of glycoprotein B of herpes simplex virus. J Gen Virol 70, 1789–1804.
Silva, A. L., Omerovic, J., Jardetzky, T. S. & Longnecker, R. (2004). Mutational analyses of Epstein–Barr virus glycoprotein 42 reveal functional domains not involved in receptor binding but required for membrane fusion. J Virol 78, 5946–5956.
Spear, P. G. & Longnecker, R. (2003). Herpesvirus entry: an update. J Virol 77, 10179–10185.
Speck, P. & Longnecker, R. (2000). Infection of breast epithelial cells with Epstein-Barr virus via cell-to-cell contact. J Natl Cancer Inst 92, 1849–1851.
Strive, T., Borst, E., Messerle, M. & Radsak, K. (2002). Proteolytic processing of human cytomegalovirus glycoprotein B is dispensable for viral growth in culture. J Virol 76, 1252–1264.
Sullivan, D. C., Allen, G. P. & O'Callaghan, D. J. (1989). Synthesis and processing of equine herpesvirus type 1 glycoprotein 14. Virology 173, 638–646.[CrossRef][Medline]
van Drunen Littel-van den Hurk, S. & Babiuk, L. A. (1986). Synthesis and processing of bovine herpesvirus 1 glycoproteins. J Virol 59, 401–410.
Vey, M., Schafer, W., Reis, B., Ohuchi, R., Britt, W., Garten, W., Klenk, H. D. & Radsak, K. (1995). Proteolytic processing of human cytomegalovirus glycoprotein B (gpUL55) is mediated by the human endoprotease furin. Virology 206, 746–749.[CrossRef][Medline]
Whealy, M. E., Robbins, A. K. & Enquist, L. W. (1990). The export pathway of the pseudorabies virus gB homolog gII involves oligomer formation in the endoplasmic reticulum and protease processing in the Golgi apparatus. J Virol 64, 1946–1955.
Wolfer, U., Kruft, V., Sawitzky, D., Hampl, H., Wittmann-Liebold, B. & Habermehl, K. O. (1990). Processing of pseudorabies virus glycoprotein gII. J Virol 64, 3122–3125.
Received 9 September 2008;
accepted 25 November 2008.
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