Laboratory of Microbiology, Department of Disease Control, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan
Correspondence
Katsunori Okazaki
ko{at}vetmed.hokudai.ac.jp
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ABSTRACT |
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INTRODUCTION |
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Herpesviruses initially attach to heparan sulfate moieties on a host cell and enter the cytoplasm by fusion of the viral envelope with the plasma membrane (Mettenleiter et al., 1990; Okazaki et al., 1991
; Spear et al., 2000
; WuDunn & Spear, 1989
). Glycoprotein B (gB), the most conserved herpesvirus structural component, plays a role in the fusion process (DeLuca et al., 1982
; Fitzpatrick et al., 1990
; Li et al., 1997
; Navarro et al., 1993
; Rauh & Mettenleiter, 1991
). Bovine herpesvirus type 1 (BoHV-1) gB is synthesized primarily as a 932 aa translation product and, after removal of the signal sequence, the mature form of the glycoprotein has 865 aa (Whitbeck et al., 1988
). Fully glycosylated gB (gBa) is a 130 kDa polypeptide and is partially cleaved between Arg-438 and Ala-439 by a cellular protease to yield two subunits, gBb (75 kDa) and gBc (55 kDa), which are covalently linked via disulfide bonds (van Drunen Little-van den Hurk et al., 1984
, 1992
; Marshall et al., 1986
; Okazaki et al., 1986
). Despite compelling evidence for the activity of gB as a fusion protein (Fitzpatrick et al., 1990
; van Drunen Little-van den Hurk et al., 1992
), no hydrophobic fusion domain has been identified near the cleavage site of the glycoprotein.
Here, we report the identification of HR regions within gBs of different herpesviruses and demonstrate that a synthetic peptide derived from one of the regions within BoHV-1 gBc bound to the glycoprotein and inhibited the replication of BoHV-1. The peptide did not affect virus entry but interfered with cell-to-cell spread to reduce virus production. The peptide inhibited the replication not only of pseudorabies virus (PRV) and herpes simplex virus (HSV) but also that of Newcastle disease virus (NDV), although to a lesser degree.
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METHODS |
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Peptide.
Peptides B477510 and B120, which correspond to the HR1 region and the N-terminal end of the mature form of BoHV-1 gB, respectively, were purchased from Sigma Genosys. The amino acid sequences were those from the Cooper strain of BoHV-1 (Whitbeck et al., 1988). The purities of the peptides were 99·1 % (B477510) and 98·2 % (B120). A human 33 aa peptide, S182 (aa 345377), was purchased from Phoenix Pharmaceuticals and its purity was 99·4 %.
Virus growth in the presence of the peptide.
Cell monolayers were infected with the viruses at an m.o.i. of 0·01 in the presence of different concentrations of the peptide and incubated at 37 °C for 48 h. In the one-step growth experiment, the cells were infected at an m.o.i. of 10 and incubated for 24 h. The supernatants were clarified by centrifugation and viral titres were determined by plaque assays on appropriate cell monolayers.
Fluorescent antibody staining.
MDBK cell monolayers were infected with BoHV-1 in the presence (100 µM) or absence of the peptide and incubated at 37 °C for 18 h. After fixation with methanol, the cells were incubated with monoclonal antibody (mAb) specific for BoHV-1 gD (Okazaki et al., 1986), washed with PBS and exposed to FITC-conjugated anti-mouse IgG.
Expression of BoHV-1 gB.
The complete coding sequence of gB was excised from the plasmid pVA97, containing the HindIIIA fragment of the Los Angeles strain genome, by digestion with SalI and KpnI and cloned into the SalI/KpnI sites of pUC19. The resulting plasmid was completely digested with EcoRI and partially with PstI and a 3·0 kb fragment was cloned into the EcoRI/PstI sites of pUCBM21 (Boehringer), which contained an in-frame deletion in the leader sequence but retained the authentic signal sequence of gB. The altered coding sequence of gB was excised by digestion with HindIII and EcoRI and cloned into the HindIII/EcoRI sites of pcDNAI/Amp (Invitrogen) to express the full-length mature form of gB.
Transfection of 293T cells was carried out as described previously (Okazaki et al., 1991). At 4860 h post-transfection, cells were harvested and lysed with 1 % Triton X-100/1 % sodium deoxycholate, followed by centrifugation to remove debris.
Surface plasmon resonance (SPR) spectrometer analysis.
The interaction of the synthetic peptide with gB was evaluated by SPR spectroscopy using SPR-670M (Nippon Laser & Electronics Laboratory). mAb 185/2 (Okazaki et al., 1986), specific for BoHV-1 gB, was immobilized via 4,4'-dithiodibutyric acid on to the gold film in the SPR cells standing in a row in the SPR instrument. After blocking with 1 mg casein ml1, the cell lysate containing gB was injected and washed with PBS at the rate of 15 µl min1. After baseline stabilization, 50 µl of the peptide B477510 or B120 was injected in parallel and washed with PBS. The changes in the SPR signal were monitored at the rate of 15 µl min1.
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RESULTS |
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DISCUSSION |
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Antiviral activity of a synthetic peptide corresponding to the HR region of a glycoprotein was first described for HIV (Wild et al., 1992). DP-107 is a 38 aa peptide representing the HR1 region of HIV gp41 and contains a leucine zipper motif. The peptide inhibits HIV infection by affecting both the cell-free virus and cell-to-cell fusion, although it has little or no effect on cell-free virions. DP-178, an amphipathic 36 aa peptide corresponding to the HR2 region of HIV gp41, inhibits viral entry and cell-to-cell fusion in vitro, with an EC50 that is much lower than that of DP-107 (Wild et al., 1994
), and virus replication in vivo (Kilby et al., 1998
). Although peptides derived from the HR1 region of measles virus and Sendai virus F proteins show no inhibition (Rapaport et al., 1995
; Wild & Buckland, 1997
), those derived from the HR1 region of NDV F protein are as inhibitory as, or more inhibitory than, peptides from the HR2 region (San Roman et al., 2002
; Young et al., 1997
, 1999
). These HR regions are located near the fusion peptide and the TM domains of the glycoproteins. During fusion, they interact with each other and refold into helical bundles to place the fusion peptide near the TM domains, resulting in the close proximity of the attaching and target membranes (Kliger et al., 2001
; Chen et al., 2001
). Peptides with sequences from either region are supposed to block this interaction (Rimsky et al., 1998
). Peptide B477510 may also bind to the HR2 region of gB of BoHV-1, PRV or HSV-1 and prevent it from interacting with the corresponding HR1 region. Despite there being no fusion peptide within the sequence of gB, the approach of the virus-infected cell membrane to the adjacent cell membrane may be inhibited. Interestingly, the peptide corresponding to the HR1 region of BoHV-1 gB partly inhibited the replication of NDV. It is postulated that not only the sequence but also the secondary structure or hydrophobicity profile of the peptide is important for the interaction. Hecate, a 23 aa amphipathic
-helical peptide analogue of melittin, has been reported to inhibit HSV-1-induced cell fusion and virus spread, although the mechanism is unknown (Baghian et al., 1997
).
BoHV-1 gB has three continuous hydrophobic segments in its C terminus. It has been demonstrated that the second segment, aa 717736, is involved in the fusogenic activity of the glycoprotein and that the third functions as the membrane anchor (Li et al., 1997). The HR2 region of BoHV-1 gB overlaps the first and second segments. A hydrophobic stretch of 34 aa near the TM domain of human cytomegalovirus gB, containing the entire HR2 region of the glycoprotein, has been proposed as a fusogenic domain (Bold et al., 1996
). Some residues within the HR2 region of HSV-1 gB, which are conserved among herpesviruses, have also been shown to be important for virus penetration (Wanas et al., 1999
). The putative interaction between the HR1 and HR2 regions may be essential for fusion by herpesvirus gBs.
Co-expression of gB, gD, gH and gL is necessary for HSV-1 to induce sufficient membrane fusion (Turner et al., 1998), whereas gB, gH and gL are sufficient for PRV (Klupp et al., 2000
). In the case of BoHV-1, expression of gB alone can induce membrane fusion (Fitzpatrick et al., 1990
; van Drunen Little-van den Hurk et al., 1992
). Although the fusion process by herpesviruses is complex and may be modulated by different viral components, it is certain that gB plays a central role in the process. Further studies should give insight into the mechanism of infection by herpesviruses.
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ACKNOWLEDGEMENTS |
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Received 23 February 2004;
accepted 16 April 2004.