1 Department of Microbiology, Zhongshan Medical College, Sun Yat-sen University, 74 Zhongshan Road II, Guangzhou 510080, People's Republic of China
2 Medical Laboratory Center, Guangdong Provincial People's Hospital, 106 Zhongshan Road II, Guangzhou 510080, People's Republic of China
Correspondence
Hui-Yong Wei
(at Zhongshan Medical College)
hywei2002{at}yahoo.com.cn
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ABSTRACT |
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MAIN TEXT |
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The capability of DEN to infect Fc receptor-negative human endothelial cells suggests the presence of other DEN receptors, but how DEN binds to the endothelial cell surface and the nature, number and distribution of specific host cell receptors for DEN have not been elucidated. Similarly, cell surface receptors for DEN type 1 (DEN-1) on Vero and Hep G2 cell lines (Marianneau et al., 1996) have not been characterized definitely. As previous reports suggest that the ECV304 cell line (an human umbilical cord-derived endothelial cell line) is a useful model to study DEN-2 infection (Killen & O'Sullivan, 1993
; Bonner & O'Sullivan, 1998
), ECV304 cells were used in this study to characterize putative virus-binding proteins or host cell receptors.
The New Guinea C strain of DEN-2 was grown in Aedes albopictus C6/36 cells and virus titres were determined using a modified micro-plaque assay. ECV304 cells were cultured in complete H 1640 medium [HEPES (Merck) plus RPMI 1640 (Gibco)] and susceptibility of this cell line to DEN-2 infection was analysed. Approximately 3x105 ECV304 cells were inoculated in duplicate with DEN-2 at an m.o.i. of 1, 10 and 100. After unbound virus was removed, supernatants from infected cells were collected at various times post-infection (p.i.) (24, 48, 72 and 96 h) to assay virus titre by micro-plaque assay and to chart virus growth. Mock-infected or heat-inactivated virus-infected ECV304 cells were used as controls. Virus titres varied when infected at different m.o.i. and at different times p.i. Aliquots taken from cells infected with an m.o.i. of 10 showed an increase in virus titre at 2472 h p.i. A similar increase in virus titre was found also in ECV304 cells infected with an m.o.i. of 1, but this titre was much lower than that of other groups at the same time p.i. Peak levels of DEN-2 titres were 5·5x106 p.f.u. ml-1 at 72 h p.i. (initial m.o.i. of 10) and 4·2x106 p.f.u. ml-1 at 48 h p.i. (initial m.o.i. of 100) (Table 1). Meanwhile, ECV304-infected cells were scraped off culture dishes and collected every 24 h after infection (m.o.i. of 1100) to calculate the percentage of DEN-2 antigen-containing cells by indirect immunofluorescence assay (IFA). In contrast to uninfected cells, which were consistently negative, the number of fluorescent-positive cells increased with increasing duration of infection, although the percentages of fluorescent cells varied among the three groups of infected cells (initial m.o.i. of 1, 10 and 100) (Fig. 1
a). The most intense fluorescence staining was observed at 72 h p.i. These data showed that DEN-2 could replicate efficiently and mature in ECV304 cells and that this cell line could be used as a model cell line for DEN-binding assays.
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A modified VOPBA was carried out to study whether rEgp could block DEN-2 binding with the three ECV304 cell surface proteins identified. After SDS-PAGE and electrotransfer of proteins, the resultant membranes were incubated for 2 h at 4 °C with 0·1, 1 and 10 µg rEgp. PBS without rEgp was used as control. The membranes were then incubated with 35S-labelled DEN-2 and auto-radiographed under the same conditions as described above. No virus-binding bands could be visualized when ECV304 cell membrane preparations (collected by trypsin treatment or by scraping) were pre-incubated with 10 µg rEgp. When the same membranes were pre-incubated with 1·0 µg rEgp, the single virus-binding band identified (29 kDa) could not be visualized from the trypsin-treated preparations but the three binding bands identified could be visualized, albeit indistinctly, from the scraped cell preparations (Fig. 2b). These results suggest that rEgp could inhibit virus infection by competitively blocking the virus-binding proteins on the surface of ECV304 cells.
Human vascular endothelial cells were found to support DEN replication in vitro but early studies demonstrated (Anderson et al., 1992) that these cells were less susceptible to DEN-4 in vivo. An increasing number of reports have indicated that DEN can infect human endothelial cells efficiently and induce cytokine production, and may play an important role in regulating vascular permeability and maintaining haemostasis (Huang et al., 2002
). Recent studies indicate that vascular endothelial cells can be targets for DEN infection but different serotypes of DEN and different strains of the same serotype might exhibit different infectivity to human endothelial cells (Bielefeldt-Ohmann et al., 2001
; Huang et al., 2002
). In the present study, an umbilical cord-derived human endothelial cell line (ECV304) was used to analyse susceptibility to DEN-2 infection. Virus growth curves and virus antigen detection (assessed by IFA staining at various times p.i.) indicated that DEN-2 can reproduce efficiently in ECV304 cells and that this cell line could be used as a model for DEN-binding assays.
Although considerable progress has been made in the identification of host cell receptors for viruses (Shieh et al., 1992), little is known about the nature of DEN receptors on the surface of host cells; no host cell receptors for DEN have been identified in human endothelial cells. Previous reports demonstrated that two membrane proteins served as specific receptors for DEN-4 on the surface of C6/36 cells (Salas-Benito & del Angel, 1997
). Current studies showed that multiple cell surface molecules, including glycosaminoglycans, HS and lipopolysaccharide-binding CD14-associated molecules, might be involved in DEN binding and subsequent virus infection in target cells (Chen et al., 1997
, 1999
; Bielefeldt-Ohmann et al., 2001
). In this study, the ECV304 cell line was chosen to study how DEN-2 binds to endothelial cells and to identify virus binding-associated proteins on the host cell surface. The results of VOPBAs using labelled DEN-2 as probe show that three virus-binding proteins of 29, 34 and 43 kDa were present in the membrane preparations of this human endothelial cell line. Our results also indicate that the 34 and 43 kDa virus-binding proteins were sensitive to trypsin, as trypsin treatment could prevent recognition of these two proteins by labelled DEN-2.
Anderson et al. (1992) observed that binding of the envelope glycoprotein of DEN-4 to cell surface receptor(s) correlated with cell susceptibility to virus infection. The envelope glycoprotein of Flaviviruses presumably plays a role in cellular tropism by being involved in virus binding to host cell receptors and has been identified as the virus attachment protein (Chen et al., 1996
, 1997
). By pre-incubating live ECV304 cells or transferred membranes with rEgp in the present study, we demonstrated that DEN-2 infects host cells and virus binding to the three ECV304 cell surface proteins identified (29, 34 and 43 kDa) could be inhibited in a dose-dependent manner. The result indicated that rEgp might compete with DEN-2 for the same receptor on the surface of the target cell through receptor saturation and steric hindrance.
In this paper, we report (i) that DEN-2 bound specifically to three proteins of 29, 34 and 43 kDa on the surface of human endothelial cells and (ii) that rEgp, a full-length recombinant DEN-2 envelope glycoprotein, inhibited infection of ECV304 cells by DEN-2, suggesting the existence of a specific interaction between these cell surface membrane molecules with the viral envelope glycoprotein. Although the nature of the three virus-binding proteins identified remains to be characterized, isolation, purification and characterization of these protein molecules will be of fundamental importance to identify further the host cell receptor for DEN-2 attachment to and penetration of human vascular endothelial cells.
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Received 22 April 2003;
accepted 24 July 2003.