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Department of Immunology and Microbiology, Faculty of Pharmaceutical Sciences, Health Sciences University of Hokkaido, Ishikari-Tobetsu 061-0293, Japan
Correspondence
Katsunori Okazaki
kokazaki{at}hoku-iryo-u.ac.jp
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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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). The virus produces at least 11 glycoproteins, of which glycoproteins B (gB), gD and gH/gL are essential for virus replication. PRV gB is one of the most abundant proteins in the viral membrane and plays essential roles in the penetration and cell-to-cell spread of the virus (Rauh & Mettenleiter, 1991). The primary translation product of PRV gB consists of 913 aa, while the mature form of the glycoprotein after removal of the signal sequence has 855 aa. The fully glycosylated gB (gBa) is a 120 kDa polypeptide that is cleaved between Arg444 and Ser445 by a cellular protease to yield two subunits, gBb (68 kDa) and gBc (55 kDa) (Hampl et al., 1984; Whealy et al., 1990; Wolfer et al., 1990).
gB homologues exhibit the highest amino acid sequence conservation among herpesvirus glycoproteins, and most of these homologues, including those of alpha-, beta- and gammaherpesviruses, are processed to yield two subunits (Baghian et al., 2000; Ben-Porat & Kaplan, 1985; Britt & Vugler, 1989; Johannsen et al., 2004; Meredith et al., 1989; Okazaki et al., 1986, 1990; Ross et al., 1989; van Drunen Littel-van den Hurk & Babiuk, 1986). It was also reported that the processing of human cytomegalovirus (HCMV) gB is mediated by the ubiquitous protease furin (Vey et al., 1995). Although such proteolytic cleavage is necessary for the activation of other viral fusion proteins, such as influenza virus haemagglutinin and Newcastle disease virus (NDV) fusion (F) protein (Klenk et al., 1975; Nagai et al., 1976), cleavage of bovine herpesvirus 1 (BoHV-1) and HCMV gBs is not required for their functions in vitro (Kopp et al., 1994; Strive et al., 2002). Moreover, the gB of herpes simplex virus (HSV) is not cleaved at all (Claesson-Welsh & Spear, 1986).
In the present study, I found that furin is responsible for the cleavage of PRV gB and investigated the effects of cleavage of the glycoprotein on its functions. The results indicate that the cleavage is responsible for syncytium formation but not involved in cell entry of PRV.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Immunoprecipitation.
A mouse monoclonal antibody, 18/4, specific for PRV gB was kindly provided by Dr A. Takada (Department of Global Epidemiology, Hokkaido University, Japan) and immobilized on protein A–Sepharose (Pharmacia) by dimethylpimelidate as described previously (Harlow & Lane, 1988). Virus-infected cells were treated with lysis buffer (1 % sodium deoxycholate, 1 % Triton X-100, 140 mM NaCl, 10 mM Tris/HCl pH 7.4, 1 mM EDTA) and clarified by centrifugation. The supernatant was incubated with the antibody-coupled Sepharose beads for 1 h at 37 °C, followed by five washes by centrifugation in the lysis buffer. After washing, the beads were boiled in Laemmli sample buffer (Laemmli, 1970) and subjected to SDS-PAGE. The immunoprecipitated proteins were visualized by silver staining (Wako).
Digestion of immunoprecipitated gB by furin.
The gB-bound beads were washed with saline and incubated with 100 U furin (Sigma) ml–1 in 150 mM NaCl, 4 mM CaCl2 and 40 mM Bis-Tris (pH 5.8) for 1 h at 30 °C. After washing with the lysis buffer, the beads were subjected to SDS-PAGE under reducing conditions.
Virus growth in the presence of a furin inhibitor.
A peptide furin inhibitor, decanoyl-Arg–Val–Lys–Arg-chloromethylketone (Vey et al., 1995), was purchased from Wako. MDBK cells were infected with PRV or NDV at an m.o.i. of 0.1 and incubated with different concentrations of the inhibitor. At 60 h post-infection, the supernatants were collected and clarified by centrifugation, followed by determination of the viral titres by plaque assays.
Establishment of LoVo cells stably expressing furin.
The complete coding sequence of human furin was excised from the plasmid pSG5-hfurin (Takahashi et al., 1993), which was kindly provided by Dr Y. Misumi (Department of Cell Biology, School of Medicine, Fukuoka University, Japan). Following complete digestion of the plasmid with EcoRV and partial digestion with BamHI, a 2.7 kb fragment was obtained and cloned into the PvuII/BamHI sites of pCEP4 (Invitrogen). The resultant plasmid was designated pCEP4-hfurin. Transfection of LoVo cells with pCEP4-hfurin was carried out using LipofectAMINE 2000 (Gibco-BRL) as described previously (Okazaki et al., 1993). At 48 h post-transfection, the cells were transferred into fresh medium containing 40 µg hygromycin B ml–1. The hygromycin-resistant cells were screened for furin production by immunofluorescence staining using a rabbit polyclonal IgG against furin (Santa Cruz Biotechnology) as described previously (Okazaki et al., 1987). The positive cells were isolated and subjected to single-cell cloning.
Penetration assay.
The penetration kinetics were measured by inactivation of the extracellular virus with acid solution as described previously (Okazaki et al., 1991). Approximately, 1000 p.f.u. of the virus grown in LoVo or MDBK cells was allowed to adsorb onto monolayers of MDBK cells in 60 mm dishes on ice. After washing with cold MEM, the monolayers were covered with pre-warmed MEM and shifted to 37 °C. At different times after the temperature shift, the cells were treated with acid solution (0.1 M HCl, 0.1 M sodium citrate, pH 2.5) for 5 min, extensively washed and overlaid with agar.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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, gBb and gBc were scarcely detected in LoVo cells, although a sufficient amount of gBa was detected under reducing conditions. In all the other cell lines, gBb and gBc were detected along with differing amounts of gBa. Under non-reducing conditions, proteins co-migrating with gBa, which was a mixture of uncleaved gB and a disulfide-linked complex of gBb and gBc, and/or migrating more slowly, were found in each cell line. These findings indicate that the extent of cleavage may depend on the cell line involved and that, in particular, no gB cleavage occurs in LoVo cells.
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, the gBa on the beads was converted into two subunits by furin digestion. The molecular masses of the subunits obtained from gBa coincided with those obtained from MDBK cells infected with PRV. These findings suggest that the PRV gB from LoVo cells is cleaved by furin.
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, the inhibitor decreased the infectious titre of NDV in the culture fluid in a concentration-dependent manner. At a concentration of 80 µM the titre was decreased to less than 1/10 000 of the control. The virus generated in the presence of the inhibitor at concentrations of more than 20 µM was activated up to 103 p.f.u. ml–1 by trypsin, whereas the enzyme had no effect on the virus generated at lower concentrations of the inhibitor. These findings indicate that the F protein of the virus particles released from the cells was mostly cleaved at the inhibitor concentrations below 20 µM, while the inhibitor interfered with the cleavage and decreased the infectious titres in a concentration-dependent manner between 20 and 80 µM. In contrast, the titres of PRV in the culture fluid were scarcely affected by the inhibitor (Fig. 3b). Even at the inhibitor concentration of 80 µM, the titre was only decreased to 1/10 of the control. Furthermore, no increase in the titre was observed in the presence of trypsin. These results confirm that furin does not play a role in generating infectious particles of PRV.
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). However, it is noteworthy that the cells incubated with the inhibitor only showed rounding CPE, whereas syncytia were formed in the absence of the inhibitor, where gBa was cleaved into gBb and gBc. These findings may indicate that cleavage of gB is responsible for the cell fusion caused by PRV.
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shows that, in the cells expressing furin, gBa was definitely converted into gBb and gBc, while in naïve LoVo cells the glycoprotein was scarcely cleaved. PRV-infected LoVo cells expressing furin also exhibited cell fusion clearly, while the cells not expressing the enzyme showed no such fusion (Fig. 5b). These results demonstrate that cleavage of gB is responsible for syncytium formation by PRV.
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). The virus grown in LoVo cells developed full tolerance to the acid treatment at 20 min after the incubation, and the virus grown in MDBK cells required 30 min to achieve such tolerance. These results may indicate that the penetration efficiency of PRV is separate from the cleavage of gB.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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), although virus-infected cells contain the uncleaved form (Gong et al., 1987). On the other hand, the gBs of BoHV-1 and HCMV do not require cleavage for their functions, as evaluated using genetically engineered viruses (Kopp et al., 1994; Strive et al., 2002). The present study using the naïve virus has demonstrated for the first time that cleavage of gB is not essential for replication of PRV, but is required for the syncytium formation. We also found that furin serves as the cleavage enzyme for the glycoprotein.
PRV was found to replicate in a variety of cell lines accompanied by extensive CPE. With the exception of LoVo cells, all the cells examined generated cleaved gB and formed syncytia, whereas LoVo cells, which lacks furin, neither cleaved the glycoprotein nor formed syncytia. MDBK cells treated with a furin inhibitor also generated the uncleaved form of gB and exhibited only rounding CPE when the cells were infected with PRV. Although gD and gH of PRV contain the proteolytic cleavage consensus sequence, RXR/KR, no cleaved product was observed in either virus-infected cells or virions (Klupp et al., 1992; Whealy et al., 1991). No other glycoproteins in addition to gB, gD and gH possess the consensus sequence. In spite of the possibility that an unknown number of accessory molecules play a role in syncytium formation, the present findings may indicate that cleavage of gB is involved in the function of the glycoprotein. Kopp et al. (1994) described that BoHV-1 carrying uncleavable gB is inferior to the wild-type virus in its ability to spread from infected cells to adjacent uninfected cells. The present results appear to be consistent with this observation, although BoHV-1 scarcely forms syncytia in MDBK cells. In contrast, the cleavability of BoHV-1 gB has no influence on the capacity for direct cell-to-cell spreading of the virus in a PRV background (Kopp et al., 1994).
In the presence of a furin inhibitor, PRV gB remained in the uncleaved form even in MDBK cells. Uncleaved gB isolated from LoVo cells was successfully converted into two subunits after furin digestion. Moreover, constitutional expression of furin allowed LoVo cells to process the glycoprotein. Since pCEP4 is maintained episomally in the nucleus, the endogenous gene expression of the host cells is barely affected. Therefore, expression of furin must solely cause the cleavage of the glycoprotein to form syncytia in LoVo cells after virus infection. The ubiquitous protease furin is responsible for activation of the glycoproteins of many enveloped viruses (de Haan et al., 2004; Ortmann et al., 1994; Volchkov et al., 1998; Zhang et al., 2003), as well as the capsid protein of non-enveloped papillomavirus, for entry into cells (Richards et al., 2006). It was also demonstrated that HCMV gB was cleaved by furin and that the same inhibitor as used in this study had no influence on release of infectious particles carrying uncleaved gB (Vey et al., 1995). On the other hand, Jean et al. (2000) reported that a different furin inhibitor caused mislocalization of gB and blocked generation of infectious particles of HCMV.
Since the virus achieved multistep replication in LoVo cells or in the presence of the inhibitor, cleavage of gB is dispensable for the adsorption, penetration and release of PRV. In particular, the penetration kinetics were unaffected by proteolytic modification of the glycoprotein. The observed reduction in the virus titres in the presence of the inhibitor is probably due to the deficiency in syncytium formation, since no difference in the titre was found between the drug-treated and untreated cells at an m.o.i. of 5 (data not shown). Vey et al. (1995) reported no cytotoxicity of decanoyl-Arg–Val–Lys–Arg-chloromethylketone for human fibroblast cells. Cleavage of PRV gB does not seem to be involved in virus–cell fusion, but does seem to be required for cell–cell fusion. Although gB takes part in membrane fusion events by PRV, different mechanisms must participate in virus–cell and cell–cell fusion. Different regions of gB were reported to determine the penetration kinetics and syncytium formation phenotype of HSV1 (Bzik et al., 1984). Moreover, a synthetic peptide corresponding to a heptad repeat region of gB does not affect the penetration of BoHV-1, but does interfere with its cell-to-cell infection (Okazaki & Kida, 2004).
As shown in Table 1, only viruses belonging to the genus Simplexvirus lack the consensus sequence motif for furin, RXR/KR, in their gB molecules. It is interesting to consider the relationship between the functions of gB and virus evolution. Co-expression of gB, gD, gH and gL is necessary for HSV1 to induce sufficient cell fusion (Turner et al., 1998), whereas gB, gH and gL are sufficient for PRV (Klupp et al., 2000). Furthermore, HSV1 gB does not complement the lethal defect in gB PRV although reciprocal complementation is affected (Mettenleiter & Spear, 1994). Further investigations into this essential glycoprotein may provide insights into the mechanisms of membrane fusion by herpesviruses.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Ben-Porat, T. & Kaplan, A. S. (1985). Molecular biology of pseudorabies virus. In The Herpesviruses, pp. 105–173. Edited by B. Roizman. New York, NY: Plenum Publishing Corp.
Britt, W. J. & Vugler, L. G. (1989). Processing of the gp55/116 envelope glycoprotein complex (gB) of human cytomegalovirus. J Virol 63, 403–410.
Bzik, D. J., Fox, B. A., DeLuca, N. A. & Person, S. (1984). Nucleotide sequence of a region of the herpes simplex virus type 1 gB glycoprotein gene: mutations affecting rate of virus entry and cell fusion. Virology 137, 185–190.[CrossRef][Medline]
Claesson-Welsh, L. & Spear, P. G. (1986). Oligomerization of herpes simplex virus glycoprotein B. J Virol 60, 803–806.
de Haan, C. A., Stadler, K., Godeke, G. J., Bosch, B. J. & Rottier, P. J. (2004). Cleavage inhibition of the murine coronavirus spike protein by a furin-like enzyme affects cell-cell but not virus-cell fusion. J Virol 78, 6048–6054.
Gong, M., Ooka, T., Matsuo, T. & Kieff, E. (1987). Epstein-Barr virus glycoprotein homologous to herpes simplex virus gB. J Virol 61, 499–508.
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.
Harlow, E. & Lane, D. (1988). Immunoaffinity purification. In Antibodies: a Laboratory Manual, pp. 511–552. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Jean, F., Thomas, L., Molloy, S. S., Liu, G., Jarvis, M. A., Nelson, J. A. & Thomas, G. (2000). A protein-based therapeutic for human cytomegalovirus infection. Proc Natl Acad Sci U S A 97, 2864–2869.
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.
Klenk, H. D., Rott, R., Orlich, M. & Blodorn, J. (1975). Activation of influenza A viruses by trypsin treatment. Virology 68, 426–443.[CrossRef][Medline]
Klupp, B. G., Visser, N. & Mettenleiter, T. C. (1992). Identification and characterization of pseudorabies virus glycoprotein H. J Virol 66, 3048–3055.
Klupp, B. G., Nixdorf, R. & Mettenleiter, T. C. (2000). Pseudorabies virus glycoprotein M inhibits membrane fusion. J Virol 74, 6760–6768.
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]
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.
Mettenleiter, T. C. & Spear, P. G. (1994). Glycoprotein gB (gII) of pseudorabies virus can functionally substitute for glycoprotein gB in herpes simplex virus type 1. J Virol 68, 500–504.
Nagai, Y., Ogura, H. & Klenk, H. (1976). Studies on the assembly of the envelope of Newcastle disease virus. Virology 69, 523–538.[CrossRef][Medline]
Okazaki, K. & Kida, H. (2004). A synthetic peptide from a heptad repeat region of herpesvirus glycoprotein B inhibits virus replication. J Gen Virol 85, 2131–2137.
Okazaki, K., Honda, E., Minetoma, T. & Kumagai, T. (1986). Mechanisms of neutralization by monoclonal antibodies to different antigenic sites on the bovine herpesvirus type 1 glycoproteins. Virology 150, 260–264.[CrossRef][Medline]
Okazaki, K., Honda, E., Minetoma, T. & Kumagai, T. (1987). Intracellular localization and transport of three different bovine herpesvirus type 1 glycoproteins involved in neutralization. Arch Virol 92, 17–26.[CrossRef][Medline]
Okazaki, K., Kumagai, T., Honda, E. & Okazaki, K. (1990). The immunodominant glycoprotein complex of equid herpesvirus 1 (EHV-1) and the counterpart of EHV-4. Nippon Juigaku Zasshi 52, 1127–1130.[Medline]
Okazaki, K., Matsuzaki, T., Sugahara, Y., Okada, J., Hasebe, K., Iwamura, Y., Ohnishi, M., Kanno, T., Shimizu, M. & other authors (1991). BHV-1 adsorption is mediated by the interaction of glycoprotein gIII with heparinlike moiety on the cell surface. Virology 181, 666–670.[CrossRef][Medline]
Okazaki, K., Kanno, T., Honda, E. & Kono, Y. (1993). Hemadsorptive activity of transfected COS-7 cells expressing BHV-1 glycoprotein gIII. Virology 193, 1024–1027.[CrossRef][Medline]
Ortmann, D., Ohuchi, M., Angliker, H., Shaw, E., Garten, W. & Klenk, H. D. (1994). Proteolytic cleavage of wild type and mutants of the F protein of human parainfluenza virus type 3 by two subtilisin-like endoproteases, furin and Kex2. J Virol 68, 2772–2776.
Rauh, I. & Mettenleiter, T. C. (1991). Pseudorabies virus glycoproteins gII and gp50 are essential for virus penetration. J Virol 65, 5348–5356.
Richards, R. M., Douglas, L. R., Schiller, J. T. & Day, P. M. (2006). Cleavage of the papillomavirus minor capsid protein, L2, at a furin consensus site is necessary for infection. Proc Natl Acad Sci U S A 103, 1522–1527.
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.
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.
Takahashi, S., Kasai, K., Hatsuzawa, K., Kitamura, N., Misumi, Y., Ikehara, Y., Murakami, K. & Nakayama, K. (1993). A mutation of furin causes the lack of precursor-processing activity in human colon carcinoma LoVo cells. Biochem Biophys Res Commun 195, 1019–1026.[CrossRef][Medline]
Turner, A., Bruun, B., Minson, T. & Browne, H. (1998). Glycoproteins gB, gD, and gH/gL of herpes simplex virus type 1 are necessary and sufficient to mediate membrane fusion in a Cos cell transfection system. J Virol 72, 873–875.
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]
Volchkov, V. E., Feldmann, H., Volchkova, V. A. & Klenk, H. D. (1998). Processing of the Ebola virus glycoprotein by the proprotein convertase furin. Proc Natl Acad Sci U S A 95, 5762–5767.
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.
Whealy, M. E., Card, J. P., Meade, R. P., Robbins, A. K. & Enquist, L. W. (1991). Effect of brefeldin A on alphaherpesvirus membrane protein glycosylation and virus egresss. J Virol 65, 1066–1081.
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.
Zhang, X., Fugere, M., Day, R. & Kielian, M. (2003). Furin processing and proteolytic activation of Semliki Forest virus. J Virol 77, 2981–2989.
Received 2 October 2006;
accepted 1 March 2007.
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