1 Department of Microbiology, Saitama Medical School, 38 Moroyama, Saitama 350-0495, Japan
2 Department of Virology 1, National Institute of Infectious Diseases, 1-23-1, Toyama, Shinjyuku, Tokyo 162-8640, Japan
Correspondence
Masaru Nawa
mnawa{at}saitama-med.ac.jp
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ABSTRACT |
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MAIN TEXT |
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Vero cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10 % foetal calf serum (FCS). JEV, JaGAr-01 strain, was propagated in Aedes albopictus cell clone C6/36, and purified from culture supernatants of infected cells according to the method described previously (Nawa, 1996). The titre of JEV was 3·4x1010 p.f.u. ml-1. Entry of JEV into Vero cells in the infection process was analysed by detecting viral antigens in the cell. Vero cells growing on Lab-Tek II chamber slides (2·1x105 cells per well) were treated with chlorpromazine (10 µg ml-1), nystatin (10 µg ml-1) and cytochalasin D (5 µg ml-1) for 1 h at 37 °C. Cells were then inoculated with JEV (6·7x107 p.f.u.) in the presence of the drug, and incubated at 37 °C for 4 h. After the first 4 h of the incubation period, cells were washed with Hanks' balanced salt solution (HBSS), pH 7·4, and incubated for a further 3 days in 10 % FCS/DMEM in the presence of JEV-neutralizing, envelope (E) glycoprotein-specific mouse mAb 503 (Kimura-Kuroda & Yasui, 1986
; 200 µg IgG per well, 144 ND50) or normal mouse IgG (200 µg IgG per well). We selected mAb 503 because the JEV-neutralizing activity of mAb 503 is greater than the other JEV E protein-specific mAbs and the polyclonal antibody (Butrapet et al., 1998
). On day 4, cells were washed with PBS and then fixed with 4 % paraformaldehyde in PBS. Virions and non-structural viral antigens present in the cells were detected by flavivirus cross-reactive human IgG purified from dengue patients' sera (Hu-IgG), and then with FITC-conjugated F(ab')2 fragment of goat anti-human immunoglobulins (Zymed).
The percentage of JEV antigen-positive cells was 60 % in the control cell culture, whereas in the cultures treated with chlorpromazine, nystatin and cytochalasin D it was 11, 35 and 42 %, respectively (Fig. 1a). The percentages of viral antigen-positive cells were lower (2 to 5 %) in the cultures treated with mixtures of chlorpromazine and the other two drugs. In the cell cultures treated with a mixture of nystatin and cytochalasin D, 35 % of the cells were JEV antigen-positive.
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Endocytic activity was analysed in Vero cells by examining the uptake of JEV with human transferrin as a reference. Human transferrin is known to be taken up by the cells through clathrin-dependent endocytosis, but it does not enter lysosomes (van Renswoude et al., 1982). Subcellular distribution of transferrin (Fig. 2
a) and JEV (Fig. 2b
) was analysed by Percoll density gradient centrifugation, according to the method described previously (Nawa, 1997
). The FluoReporter cell-surface biotinylation kit (Molecular Probes) was employed to label cell-surface proteins, and used to identify the membrane fraction in the post-nuclear cell homogenate (Fig. 2a
).
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Peaks of HRPtransferrin and biotin activity were detected in fraction nos 4 and 10, respectively (Fig. 2a). The viral antigen was mainly detected in fraction nos 4 to 6, corresponding to the endosome fractions in untreated cells (Fig. 2b
). In chlorpromazine-treated cells, the viral antigen was detected in fraction nos 10 to 12, corresponding to the membrane fractions. The results suggest that JEV bound to the cell surface, but did not translocate to the endosomes during the 15 min chase period in chlorpromazine-treated cells. Chlorpromazine did not inhibit the binding of JEV to Vero cells in the concentration range 2·5 to 20 µg ml-1 (data not shown).
We analysed the effects of chlorpromazine on distribution of the clathrin and viral antigens in the cells by indirect immunofluorescence (Fig. 3). Chlorpromazine-treated and control cells growing on Lab-Tek II chamber slides were inoculated with purified JE virions (ca 3·4x109 p.f.u.) in the presence or absence of chlorpromazine at 37 °C for 30 min. Cells were washed three times with ice-cold HBSS, and fixed with 90 % (v/v) ethanol for 15 min. Then, the cells were reacted with flavivirus-cross-reactive monoclonal antibody D1-4G2 or monoclonal antibody to clathrin heavy chain (Progen). Next the cells were reacted with the FITC-conjugated F(ab')2 fraction of goat anti-mouse IgG (H+L) (Zymed). Viral antigens were detected as small punctate clusters in the control cells (Fig. 3a
) but were absent from the chlorpromazine-treated cells (Fig. 3b
). The clathrin heavy chain antigens were detected as a typical punctate pattern in the control cells (Fig. 3c
), while only small numbers of faint fluorescent clusters were detected at the perinuclear region in the chlorpromazine-treated cells (Fig. 3d
). Fig. 3(e, f)
shows JEV antigens detected in the control (e) and chlorpromazine-treated (f) cells on day 4 after infection. The number of antigen-positive cells was less in the drug-treated cells than in the control cells; however, viral antigens were detected at the same level in both drug-treated and non-drug-treated cells. The results demonstrated that chlorpromazine affected the intracellular distribution of clathrin (Fig. 3c, d
), and inhibited uptake of JEV in Vero cells (Fig. 3a, b
). However, the presence of chlorpromazine at the early stage of infection did not inhibit the synthesis of progeny viral proteins once infection was established (Fig. 3e, f
).
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ACKNOWLEDGEMENTS |
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Received 8 October 2002;
accepted 3 February 2003.