Laboratory of Virology, Research Institute for Disease Mechanism and Control, Nagoya University School of Medicine, Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan1
Author for correspondence: Yukihiro Nishiyama. Fax +81 52 744 2452. e-mail ynishiya{at}med.nagoya-u.ac.jp
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Abstract |
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In addition to its function as an entry mediator, gD elicits strong humoral and cell-mediated immunological responses in vivo (Zarling et al., 1986 ; Rooney et al., 1989
). Furthermore, Zhou et al. (2000)
showed that gD is involved in prevention of apoptotic cell death.
Like other glycoproteins, gD is synthesized in the rough endoplasmic reticulum (ER), where it immediately acquires high-mannose oligosaccharides, N-linked to the asparagine residues of the consensus sequence. After trimming of the oligosaccharides in the Golgi apparatus, the glycoprotein is transported to the plasma membrane (Serafini-Cessi & Campadelli-Fiume, 1981 ; Johnson & Spear, 1982
).
In spite of a large number of studies on HSV proteins, little is known about proteins secreted from infected cells (Randall et al., 1980 ). We have examined whether any proteins are selectively released into the medium of HSV-infected cells. HSV- or mock-infected HEp2 cells were washed at 3 h post-infection (p.i.) and incubated with methionine-free MEM containing 0·2 mCi/ml of [35S]methionine and cysteine (redivue Pro-mix L-[35S] in vitro cell-labelling mix; Amersham Pharmacia) from 312 h p.i. At 12 h.p.i., the medium was harvested and the cell debris was discarded by centrifugation at 3000 r.p.m. for 5 min and 15000 r.p.m. for 5 min. Proteins in the medium were precipitated with 10% trichloroacetic acid (TCA) and subjected to SDSPAGE as described previously (Murata et al., 2000
), followed by autoradiography using a Fujix Bio-Imaging Analyser BAS2000 System (Fuji). Whole-cell extracts were also electrophoresed in order to confirm successful production of virus proteins. As shown in Fig. 1(A)
, at least one strong signal at about 50 kDa was detected in the medium from HSV-2 (186)-infected cells. When cells were infected with HSV-1 (KOS), a band at about 70 kDa was predominant, but the 50 kDa protein was not found.
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Judging from its molecular mass and the broad pattern of the band, we presumed the 50 kDa protein to be gD. We thus compared the molecular mass of the intracellular gD and the protein in the medium of the HSV-2-infected culture (Fig. 1D). The figure shows that the electrophoretic mobility of the 50 kDa band in the medium was almost the same as that of premature gD (pgD) from infected cells, although the amount of gD in the medium was much less than that of cellular gD. We also found that the 50 kDa protein in the medium could be immunoprecipitated with an anti-gD monoclonal antibody (Fig. 1E
). These results indicated that the 50 kDa protein secreted into the HSV-2-infected culture medium was of gD origin. The electrophoretic mobility of the proteins suggested that the protein in the medium might be pgD (Fig. 1D
, F
). However, the protein formed a broader band than cellular pgD, suggesting that the gD in the medium was a truncated form of fully glycosylated mature gD (Fig. 1F
).
In order to clarify which kind of gD was excreted into the medium, peptide footprinting analysis of the radio-labelled proteins was performed. HEp2 cells were infected with HSV-2 (186) and labelled with [35S]methionine and cysteine. At 10 h.p.i., proteins in the medium and in the cells were harvested separately and subjected to SDSPAGE. Cellular and medium gDs were excised from the gel and again subjected to SDSPAGE. While the second electrophoresis was running, the proteins were digested in-gel with S. aureus V8 protease. Although there were several peptide bands that were not detected in the counterpart (Fig. 2A, arrowheads), the profile of the 50 kDa protein was very similar to that of fully glycosylated mature gD, confirming the previous conclusion that the protein released into the medium is of gD origin.
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To investigate the route of gD secretion into the medium, the effect of tunicamycin (TM) and brefeldin A (BFA) was examined. TM inhibits the transfer of N-acetylglucosamine 1-phosphate and thereby blocks the formation of protein N-glycosidic linkages in the ER (Mahoney & Duksin, 1979 ). BFA blocks protein translocation from the ER to the Golgi apparatus (Strous et al., 1993
). Fig. 3(B)
shows that addition of either TM or BFA blocked the secretion of gD. It is clear in Fig. 3(A)
that the drugs were effective; TM inhibited the production of pgD, which carries high-mannose oligosaccharides, by blocking the linkage of the oligosaccharides, and BFA affected the maturation of N-linked oligosaccharides. These results suggested that gD is secreted through the ER and Golgi apparatus.
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The glycoprotein was secreted from human HEp2 cells or monkey Cos-1 cells when infected with HSV-2, but not from Vero cells (not shown). This result strongly suggests that gD is cleaved by a cellular factor that is missing in Vero cells. However, we cannot exclude the possibility that gD is truncated by a viral factor, because many viruses, including HSV (Liu & Roizman, 1992 ), have proteases. Generally speaking, mammalian proteases comprise both exoproteases (also referred to as exopeptidases), which act at the N- or C-terminal ends of polypeptides, and endoproteases (endopeptidases), which are capable of cleaving peptide bonds in the central regions of polypeptides (Barrett, 1980
). Most herpesvirus gB homologues are processed during maturation in the Golgi apparatus (Hampl et al., 1984
; Montalvo & Grose, 1987
; Spaete et al., 1988
), although HSV gB is not (Claesson-Welsh & Spear, 1986
). These gB homologues, except for HSV gB, carry the specific cleavage motif RXK/RR, which is the recognition motif for proteolytic processing by the cellular endoprotease furin, and they are processed by this enzyme (Vey et al., 1995
). Other viruses, such as respiratory syncytial virus (Bolt et al., 2000
) and influenza virus (Jankovics, 1996
), also require furin for the processing of fusion or haemagglutinin proteins. The HSV gD, however, does not carry the specific motif or a similar sequence for the endoprotease, suggesting that gD is not processed by furin. A surface glycoprotein of Lassa virus is post-translationally cleaved in the ER by the cellular subtilase SKI-1/S1P at the recognition motif R-L-L (Lenz et al., 2001
), but HSV gD does not have this motif. To characterize the endoprotease involved in gD cleavage, several protease inhibitors were examined (leupeptin; serine cysteine protease inhibitor, pepstatin; acidic protease inhibitor, phosphoramidon; metalloprotease inhibitor). However, none prevented secretion of gD (not shown). Since the C-terminal part of HSV gD is rich in basic residues and such sequences are favoured by trypsin-like proteases, this type of protease should be a candidate.
Randall et al. (1980) reported that several proteins were released when BHK 21 cells were infected with HSV. They were referred to as infected-cell-released polypeptides (ICRPs) af, and among them, ICRPc was highly glycosylated with its molecular mass being estimated to be 5054 kDa. However, the authors could not identify the protein. Taking our experiments into consideration, it seems reasonable to assume that ICRPc is a fully glycosylated, cleaved form of gD. According to the above report, ICRPc was found in either HSV-1- or -2-infected cell medium. However, we could not detect the protein in the medium of HSV-1-infected cells. The discrepancy may be due to a difference in cell line, virus strain or the method used to collect the proteins released into the medium.
What is the physiological significance of excretion of gD into the medium? One possibility is that the protein functions as a decoy for immune responses. Extracellular gD was immunoprecipitated with monoclonal gD-specific antibody (Fig. 1). Inactivation of antibodies against gD by binding to extracellular gD would be beneficial for the virus in vivo. First, extracellular virions could evade neutralization by gD-specific antibodies. Secondly, cells infected with HSV, which express viral proteins, could escape binding of antibodies, thereby eluding complement-dependent cytolysis, and escape from killer cells that have Fc receptors, such as macrophages or natural killer cells.
HSVs have evolved several ingenious mechanisms to evade immune systems. Viral glycoproteins gE and gI bind to IgG Fc and protect infected cells from antibody-dependent cell cytotoxicity (Johnson et al., 1988 ). gC plays a role in blocking complement-dependent cell lysis by binding to C3b and factor H (Huemer et al., 1993
). ICP47 inhibits surface expression of class I MHC antigens together with antigenic peptides by binding to TAP (transporter associated with antigen processing; Hill et al., 1995
). Excretion of gD could be another example of evasion of host immune systems.
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Acknowledgments |
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References |
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Received 3 July 2002;
accepted 17 July 2002.