TherapiaGene, 341 Bojung-Ri, Koosung-Eup, Yongin-City, Kyonggi-Do, 449-913, Korea1
Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon, 305-701, Korea2
Author for correspondence: Tae-Yeon Kim at TherapiaGene. Fax +82 31 266 3516. e-mail ettsia{at}greencross.com
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Abstract |
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Introduction |
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In order to multiply, a virus must first infect a cell. The host range of a particular virus defines both the type of tissue that the virus infects and the animal species in which it multiplies. To infect a cell, the virion must attach to the cell surface, penetrate the cell and become sufficiently uncoated to make its genome accessible to the virus or host machinery for transcription and translation. The attachment step constitutes specific binding of a viral protein (anti-receptor) to a constituent of the cell surface (receptor). Cellular receptors play an important role in virus pathogenesis. Specific initial interactions with cells largely determine the host range specificities and tissue tropisms of viruses (Fields & Greene, 1982 ).
The basis for cell and tissue tropism of viruses is often related to the ability of virus attachment proteins (VAPs) to bind a specific cell receptor. Virus binding is followed by membrane fusion, leading to productive infection in susceptible hosts. The envelope glycoproteins (G1 and G2) of members of the family Bunyaviridae (including Hantaan virus) are the major structural proteins exposed on the surface and have been identified as bunyavirus VAPs (Gonzalez et al., 1982 ; Grady et al., 1983
; Kingford et al., 1983
). It is presumed that the corresponding envelope proteins (G1 and G2) of Hantaan virus are involved in attachment, although the counterpart cellular receptor has yet to be identified (Arikawa et al., 1989
; Dantas et al., 1986
). Identification of the receptor is necessary to understand tissue tropism, pathogenesis and virus replication in cellular hosts. Recent reports demonstrating that infectivity of Hantaan virus is inhibited by a
3-specific monoclonal antibody indicate that
3 integrins mediate the cellular entry of hantaviruses, including that of Hantaan virus (Gavrilovskaya et al., 1998
, 1999
). While the entry of Hantaan virus may be mediated by
3 integrins, no currently available data define the virus-binding site or receptor protein on cells.
In this study, we investigate the attachment of Hantaan virus to a permissive animal cell line, Vero-E6. We report a cell surface protein specifically recognized by Hantaan virus. This candidate receptor for Hantaan virus is a cell membrane protein, with a relative molecular mass of 30 kDa. The role of this protein in virus infection remains to be determined.
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Methods |
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Hantaan virus strain 76-118 was propagated in Vero-E6 cells, as described previously (Elliott et al., 1984 ).
Preparation of labelled Hantaan virus.
Radiolabelled Hantaan virus was obtained from infected Vero-E6 cells. Briefly, 1·5x107 cells were infected at an m.o.i. of 0·01 p.f.u. per cell. At day 8 post-infection, the medium was replaced with methionine-free DMEM containing 5 mCi [35S]methionine (Dupont) and 10% FBS. Cells were then incubated at 37 °C. After 2 days, the supernatant was obtained by centrifugation at 200 g for 10 min and layered onto a 20% sucrose cushion for further centrifugation at 80000 g for 2 h. Pellets were suspended in TNE buffer (50 mM TrisHCl, 150 mM NaCl and 5 mM EDTA, pH 7·4) and incubated overnight at 4 °C. The virus suspension was layered onto a 2060% sucrose gradient and centrifuged at 80000 g for 3 h. The opaque band containing virus particles was isolated and dialysed overnight against TNE buffer at 4 °C. Virus material was pelleted at 160000 g for 1 h and stored at -70 °C in TNE buffer. The virus titre was determined with a focus-forming assay using Vero-E6 cells, as described previously (Kariwa et al., 1994 ). As a control, a labelled sample from uninfected cells was prepared using the same protocol as that described above.
Binding assay.
Direct binding assays were performed to characterize the attachment of Hantaan virus to Vero-E6 cells. For these studies, 12-well plates with 1x105 Vero-E6 cells per well were placed at 4 °C for 2 h before incubation with different amounts of radiolabelled Hantaan virus. The experiment was performed at 4 °C to avoid virus penetration of cells. After 2 h, the medium was removed, cells were washed twice with fresh medium and lysed with IP buffer (10 mM PBS, 150 mM NaCl, 1% Triton X-100 and 0·1 % SDS) (Anderson et al., 1992 ). Radioactivity in both the medium and cells was measured in a scintillation counter. Assays were conducted in triplicate. For calculation of non-specific binding in the saturation experiment, cells were preincubated with different concentrations of unlabelled Hantaan virus prior to the addition of 8x104 c.p.m. of labelled Hantaan virus. Binding assays were performed twice. Each point was determined in duplicate and values from independent experiments varied from 3 to 5% from the average.
For trypsin treatment, 5x106 Vero-E6 cells were incubated with a 0·075% solution of trypsin for 30 min at 37 °C (Daughaday et al., 1981 ), followed by incubation for 45 min in fresh medium supplemented with 10% FBS to inactivate the trypsin. Finally, cell monolayers were washed twice with fresh medium and a binding assay was performed, as described above.
Preparation of cell membrane protein.
To obtain cell membrane proteins, phase partitioning with Triton X-114 was performed (Bordier, 1981 ). Vero-E6 cells were washed three times with TBS (10 mM TrisHCl and 150 mM NaCl, pH 7·5). Cells were lysed in 4 cell pellet vols of 1% Triton X-114 in TBS in the presence of the protease inhibitor cocktail (Sigma) at 4 °C and centrifuged at 4000 g> for 30 min. The supernatant was incubated overnight at -20 °C and for 10 min at 37 °C and then centrifuged at 60 g for a further 15 min. The pellet was resuspended in the same volume of TBS in the presence of protease inhibitors and incubated at 4 °C for 30 min. To ensure efficient recovery of membrane proteins, the procedure was repeated, starting with incubation at 37 °C. Acetone precipitation was performed to eliminate detergent and the protein was quantified by the Micro BCA method (Bio-Rad).
Trypsin treatment was performed as described above, with an increased incubation time of 1 h. Following incubation, cells were washed three times with cold PBS and cell extracts were prepared as described above.
Virus overlay protein-binding (VOPB) assay.
To identify cell polypeptides involved in virus binding, a VOPB assay was carried out. This assay was performed as described by Jin et al. (1994) , Ludwig et al. (1996)
, Salas-Benito & del Angel (1997)
and Crane et al. (1991)
, with some modifications. Briefly, 200 µg total or membrane proteins from the cell line were subjected to SDSPAGE and transferred onto nitrocellulose membranes using a semi-dry blotting apparatus in transfer buffer (48 mM Tris, 29 mM glycine and 20% methanol) (Smith & Wright, 1985
). After overnight renaturalization of transferred proteins with 4% BSA in PBS at 4 °C, membranes were blocked for 1 h at room temperature with 5% skimmed milk (Gibco-BRL) in PBS and re-washed three times with PBS. Membranes were incubated overnight with radiolabelled (1x106 c.p.m.) Hantaan virus in MEM supplemented with 10% FBS at room temperature with gentle rocking. After washing four times for 15 min with 2% BSA in PBS and once with 0·1% Nonidet P-40 in PBS at room temperature, membranes were dried and autoradiographed.
To determine the specificity of the viruscell protein interactions, VOPB assays were performed under conditions of high salt concentrations. Briefly, before incubation with virus, membranes were washed once for 5 min with PBS and 1% skimmed milk and once in high-salt washing solution (PBS, 1% skimmed milk and 220 mM NaCl). Incubation with virus was performed under the same conditions as described above but in the presence of high-salt washing solution. Finally, membranes were washed three times with high-salt solution prior to exposure to X-ray film (Marianneau et al., 1996 ).
Protein purification.
Total cell membrane proteins were separated by 120% non-reducing SDSPAGE. A protein with a molecular mass of 30 kDa was electroeluted from the gel at 140 V for 20 min in electroelution apparatus (Whole Gel Eluter, Bio-Rad). Protein concentrations were estimated with the Micro BCA method (Bio-Rad).
Production of polyclonal antibody.
BALB/c mice were immunized five times intraperitoneally with 20 µg soluble 30 kDa (30K) protein obtained by electroelution, emulsified in Freund's complete adjuvant for primary immunization and in Freund's incomplete adjuvant for five other immunizations at 7 day intervals. Mouse sera were obtained 6 days after the final immunization and tested by Western blot analyses.
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Results and Discussion |
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Antibodies against the 30K protein block Hantaan virus binding
To investigate the particular function of the 30K protein in virus infection, a polyclonal antibody obtained after immunization of mice with the electroeluted 30K protein was generated. This antibody was employed to detect the protein in Western blot assays (Fig. 4a, lane 2). No bands were observed after incubation with preimmune serum (Fig. 4a
, lane 1). To determine whether the antibodies recognized the same protein that bound Hantaan virus, a binding assay was performed. Polyclonal serum against the 30K protein blocked the binding of labelled virus to Vero-E6 cells by 50%, while preimmune serum did not affect virus binding (Fig. 4b
). To analyse whether the antibody inhibited virus binding to the 30K protein in cells, a blocking assay with anti-30K protein antibody was performed. Preincubation of Vero-E6 cells with anti-30K protein antibody inhibited Hantaan virus infection of cells by a maximum of 70% (Fig. 4c
), compared to an average of 60%. In contrast, preimmune serum did not inhibit virus infection. These results collectively suggest that the 30K protein plays a role as a Hantaan virus receptor.
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The 30K protein is located on the surface of Vero-E6 cells
To examine the location of the 30K protein within the cell, Vero-E6 cells were preincubated with trypsin and VOPB and binding assays were then performed. VOPB assays with membrane proteins of Vero-E6 cells treated with trypsin demonstrated that trypsinization removed the 30K protein from the surface of Vero-E6 cells (Fig. 5a, lane 2). Trypsin digestion for 30 min reduced binding by 80% after 1 h of incubation with labelled viruses (Fig. 5b
), indicating a peptidic and surface nature of the 30K protein. However, after 3 h of incubation, an increase in binding was observed (up to twofold increase in c.p.m. values), suggesting an active replacement of the 30K protein on the cell membrane. Trypsin treatment of the cells inhibited virus binding. The susceptibility of the virus-binding protein to protease treatment was confirmed in these assays, where the addition of trypsin prevented recognition of the 30K proteins by labelled Hantaan virus. The presence of the 30K protein on the surface of Vero-E6 cells and the observation that electroeluted protein and its antibody inhibit Hantaan virus binding strongly support the theory that the protein is a putative receptor, or part of a receptor complex, for Hantaan virus.
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The cellular entry of hantaviruses constitutes specific interactions between the virus and cells mediated by 3 integrins (Gavrilovskaya et al., 1998
, 1999
). However, there is no direct evidence that Hantaan virus binds to
3 integrins on the cell surface. According to Gavrilovskaya et al. (1999)
, infection of cells by HFRS-causing hantaviruses (including Hantaan virus) was partially inhibited by antibodies to
3 integrin and the integrin ligand vitronectin. As stated above, it is possible that additional interactions exist between the virus and the host cells. It is currently unclear whether
3 integrin is a virus-binding protein (or site) on cells or whether it is involved in the internalization of virus only. Our data indicate that it is unlikely that
3 integrin is related to the 30K protein. The two proteins differ considerably in terms of relative molecular mass (
3 integrin has a molecular mass of about 105 kDa). Moreover, in additional experiments performed in our laboratory, the binding of the 30K protein to its virus counterpart was not inhibited by either vitronectin or fibronectin (ligands for integrin) and did not interact with a monoclonal antibody to
3 integrin. We are currently in the process of sequencing the 30K protein, following which, its relationship with
3 integrin will be ascertained.
A wide variety of cell surface molecules serve as virus attachment receptors. Such receptors range from cell-specific transmembrane proteins of well-defined receptor superfamilies (such as CD4 as a receptor for HIV) to the more ubiquitous cell surface-associated carbohydrate moieties (the common carbohydrate moiety sialic acid for influenza virus). To date, the mechanism by which Hantaan virus attaches to its host cell has not been elucidated. Despite growing interest in the putative receptor for this virus, the events that govern the initial attachment of Hantaan virus remain poorly understood.
We aim to completely characterize the DNA and amino acid sequences of the novel 30K protein to confirm that it is a Hantaan virus receptor. Structural nuclear magnetic resonance or X-ray crystallography studies may be required to facilitate the characterization of proteinprotein interactions between Hantaan virus and the cellular receptor. If the 30K protein is indeed a specific receptor for Hantaan virus, it would be of interest to determine its distribution in susceptible and resistant cell lines. Further characterization and sequencing of the receptor protein will inevitably aid in determining its normal cellular functions and understanding its interactions with Hantaan virus.
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References |
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Received 30 July 2001;
accepted 5 December 2001.