Bovine Viral Diarrhea Virus Entry Is Dependent on
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病菌学杂志 2005年第16期
CNRS-UPR2511, Institut de Biologie de Lille and Institut Pasteur de Lille, Lille, France
ABSTRACT
Cellular mechanisms of bovine viral diarrhea virus (BVDV) entry in MDBK cells were investigated. Chloroquine, bafilomycin A1, or ammonium chloride inhibited BVDV infection, indicating that an acidic endosomal pH is required for BVDV entry. The tyrosine kinase inhibitor genistein partially inhibited BVDV infection at a postentry step, whereas BVDV entry was strongly inhibited by chlorpromazine or by the overexpression of a dominant-negative form of EPS15, a protein essential for the formation of clathrin-coated vesicles at the plasma membrane. Together, these data indicate that BVDV infection requires an active clathrin-dependent endocytic pathway.
TEXT
Bovine viral diarrhea virus (BVDV) is a small, enveloped, positive-stranded RNA virus which is the etiological agent of a variety of pathologies in cattle, including fatal mucosal disease (21, 31). BVDV is classified in the Pestivirus genus within the Flaviviridae family, which also contains hepatitis C virus (HCV) and viruses of the Flavivirus genus (21). Little is known about cellular mechanisms leading to the entry of BVDV and other pestiviruses. Their binding to target cells involves envelope glycoproteins Erns and E2 (16, 20, 27, 33) through interactions with glycosaminoglycans (17, 18) and membrane proteins (28, 35), respectively. BVDV receptors include CD46 (22) and low-density-lipoprotein receptor (1). Because of their similarity to flaviviruses, pestiviruses are thought to enter target cells by receptor-mediated endocytosis and acid-triggered fusion of their envelope with an endosomal membrane (1, 22). However, BVDV is also known to be resistant to acidic treatments (9, 19), a condition often found in viruses with pH-independent entry.
In order to assess if BVDV entry is pH-dependent, we first sought to determine if BVDV infection indeed requires low endosomal pH. The importance of endosome acidification was studied with chloroquine and ammonium chloride, two lysosomotropic agents, and with bafilomycin A1, a specific inhibitor of endosomal proton-ATP pumps. MDBK cells were preincubated for 30 min with inhibitors, infected for 1 h at 37°C with BVDV (NADL strain) (23), and cultured for 15 h in the presence of the inhibitors. The virus was diluted such that 30 to 40% of the cells were infected in control experiments with no inhibitor. The infected cells were detected by indirect immunofluorescence microscopy by using a monoclonal antibody to NS3 (5). The nuclei were stained with Hoechst dye. The infections were scored as the ratio of infected cells to total cells. For comparison, we used bovine herpesvirus 1 (BHV-1), which is known to enter cells by a pH-independent mechanism (34). Each drug inhibited BVDV infection in a dose-dependent manner (Fig. 1). In contrast, BHV-1 infection was not inhibited by chloroquine or bafilomycin A1, and ammonium chloride treatment resulted in a partial inhibition of BHV-1 infection. To verify that these drugs interfered with BVDV entry, we asked whether they could act on an early step of the infection. Bafilomycin was added during early or late steps of infection. The drug strongly inhibited BVDV infection when present during the infection and up to 4 h postinfection but was without effect when added later (Fig. 1). Similar results were obtained with chloroquine (data not shown). Taken together, the results obtained with these inhibitors indicate that BVDV infection is sensitive to the low pH of endosomes. Similar results were recently reported by others (12, 19).
The pH-dependent entry of BVDV suggests that the virus is internalized from the cell surface by receptor-mediated endocytosis and reaches an endosomal compartment, where the fusion occurs. Two well-defined endocytic pathways appear to be clathrin-mediated endocytosis and caveolae internalization (25, 29). To determine if BVDV enters cells through a clathrin-mediated or a caveolae-mediated pathway, we first tested the effects of chlorpromazine (32) and genistein (8, 24), respectively. For comparison, the effects of these inhibitors were also assessed on the infection of Sindbis virus, which enters by clathrin-mediated endocytosis (6). Chlorpromazine inhibited BVDV infection in a dose-dependent manner (Fig. 2A). The inhibition was almost complete at a concentration of 10 mM, which is known to block clathrin-coated pit assembly at the plasma membrane (32). As expected, Sindbis virus infection was inhibited by chlorpromazine and BHV-1 infection was not affected, suggesting that the observed effects were not due to the toxicity of the drug (Fig. 2A).
Genistein also inhibited BVDV infection in a dose-dependent manner (Fig. 2B). We observed about 50% inhibition with 50 to 100 μg/ml genistein. Similar concentrations of genistein inhibit simian virus 40 internalization (8) but are without effect on influenza virus infection (30). Sindbis virus infection was not affected in the presence of 50 μg/ml genistein (Fig. 2B). This suggests that the inhibition of BVDV infection by genistein was not due to toxicity on MDBK cells. Taken together, these data show that BVDV infection is sensitive to both chlorpromazine and genistein.
We next verified if these drugs inhibit an early step of BVDV infection. As shown in Fig. 2C, the inhibition of BVDV infection was strong when chlorpromazine was added to the cells for only 4 h at the beginning of the infection but was very weak when the drug was added onto the cells four hours after infection. This action of chlorpromazine during early steps of BVDV infection is consistent with an inhibition of endocytosis. On the other hand, the inhibition by genistein was weaker when the inhibitor was added to the cells during the early steps than during the late steps of infection (Fig. 2C). Therefore, it is unlikely that genistein had an effect on BVDV endocytosis. These results indicate that BVDV entry is inhibited by chlorpromazine, suggesting that it might proceed through clathrin-mediated endocytosis.
This finding is consistent with the recent report that a dominant-negative form of dynamin blocks BVDV infection (19). However, dynamin regulates clathrin-mediated endocytosis, as well as other clathrin-independent pathways of internalization (14). Likewise, none of the drugs that we used are specific endocytosis inhibitors. To confirm that clathrin-mediated endocytosis was involved in BVDV entry, we made use of E95-295, a dominant-negative form of EPS15, which specifically interferes with clathrin-coated vesicle formation at the plasma membrane (3). A recombinant adenoviral vector (rAd:E95-295) was generated to express this mutant, and its functionality was verified on MDBK cells (data not shown). For a control, we used DIII2, another form of EPS15 with no dominant-negative effect on clathrin-mediated endocytosis (4). MDBK cells were transduced with rAd:E95-295 or rAd:DIII2 and subsequently infected with BVDV or control viruses 24 h later. The number of infected cells was dramatically reduced in cells transduced with rAd:E95-295, compared to cells transduced with rAd:DIII2 (Fig. 3A). The infection was scored separately in cells expressing low, moderate, or high levels of E95-295. As expected, E95-295 did not inhibit BHV-1 infection. In contrast, both BVDV and Sindbis virus infections were affected and the inhibition was positively correlated to E95-295 expression levels (Fig. 3B). These data confirm that a functional clathrin-mediated pathway of endocytosis is required for BVDV infection.
A clathrin-mediated entry route has been reported previously for flaviviruses. Both electron microscopy studies and E95-295-mediated inhibition indicated that West Nile Virus enters cells through clathrin-coated vesicles (7, 11). Since clathrin-coated vesicles deliver their cargo in acidic early endosomes, this is consistent with numerous studies which have shown that the envelope glycoprotein E of flaviviruses undergoes a conformational transition under acidic conditions from a native dimeric form to a fusogenic trimeric form (13). Such an acid-triggered conformational transition has not been reported at the present time for the envelope proteins of BVDV or other pestiviruses. In contrast, BVDV is resistant to acidic treatments (9, 19). Such a discrepancy between pH-dependent entry and resistance of the virion to acidic treatments has also been observed for vesicular stomatitis virus (26). It has recently been suggested that BVDV envelope glycoproteins must be primed by disulfide bridge reduction in the endocytotic pathway before an acid-sensitive fusogenic conformation can be reached (19).
In conclusion, our study suggests that, like flaviviruses, pestiviruses enter cells through clathrin-mediated endocytosis and fusion from within an acidic endosomal compartment. It would be interesting to determine if this pathway of endocytosis is also used by other pestiviruses and by HCV and other nonclassified viruses of the Flaviviridae family, such as GB viruses. Recent data suggest that retroviral particles pseudotyped with HCV envelope glycoproteins E1 and E2 are sensitive to agents that neutralize the pH of endosomes (2, 15), but data on clathrin requirement have not yet been reported.
ACKNOWLEDGMENTS
We are grateful to C. M. Rice, E. Thiry, G. Chappuis, and P. P. Pastoret for kindly providing viruses and antibodies. Data presented in this paper were obtained with the help of the Campus Calmette imaging core facility.
This work was funded by the Centre National de la Recherche Scientifique and by European Union grant QLRT-2000-01120.
REFERENCES
Agnello, V., G. ábel, M. Elfahal, G. B. Knight, and Q. X. Zhang. 1999. Hepatitis C virus and other Flaviviridae viruses enter cells via low density lipoprotein receptor. Proc. Natl. Acad. Sci. USA 96:12766-12771.
Bartosch, B., A. Vitelli, C. Granier, C. Goujon, J. Dubuisson, S. Pascale, E. Scarselli, R. Cortese, A. Nicosia, and F.-L. Cosset. 2003. Cell entry of hepatitis C virus requires a set of co-receptors that include the CD81 tetraspanin and the SR-B1 scavenger receptor. J. Biol. Chem. 278:41624-41630.
Benmerah, A., M. Bayrou, N. Cerf-Bensussan, and A. Dautry-Varsat. 1999. Inhibition of clathrin-coated pit assembly by an Eps15 mutant. J. Cell Sci. 112:1303-1311.
Benmerah, A., C. Lamaze, B. Bègue, S. L. Schmid, A. Dautry-Varsat, and N. Cerf-Bensussan. 1998. AP-2/Eps15 interaction is required for receptor-mediated endocytosis. J. Cell Biol. 140:1055-1062.
Boulanger, D., S. Waxweiler, L. Karelle, M. Loncar, B. Mignon, J. Dubuisson, E. Thiry, and P. P. Pastoret. 1991. Characterization of monoclonal antibodies to bovine viral diarrhoea virus: evidence of a neutralizing activity against gp48 in the presence of goat anti-mouse immunoglobulin serum. J. Gen. Virol. 72:1195-1198.
Carbone, R., S. Fre, G. Iannolo, F. Belleudi, P. Mancini, P. G. Pelicci, M. R. Torrisi, and P. P. Di Fiore. 1997. eps15 and eps15R are essential components of the endocytic pathway. Cancer Res. 57:5498-5504.
Chu, J. J. H., and M. L. Ng. 2004. Infectious entry of West Nile virus occurs through a clathrin-mediated endocytic pathway. J. Virol. 78:10543-10555.
Dangoria, N. S., W. C. Breau, H. A. Anderson, D. M. Cishek, and L. C. Norkin. 1996. Extracellular simian virus 40 induces an ERK/MAP kinase-independent signalling pathway that activates primary response genes and promotes virus entry. J. Gen. Virol. 77:2173-2182.
Depner, K., T. Bauer, and B. Liess. 1992. Thermal and pH stability of pestiviruses. Rev. Sci. Tech. 11:885-893.
Duvet, S., F. Chirat, A. M. Mir, A. Verbert, J. Dubuisson, and R. Cacan. 2000. Reciprocal relationship between alpha1,2 mannosidase processing and reglucosylation in the rough endoplasmic reticulum of Man-P-Dol deficient cells. Eur. J. Biochem. 267:1146-1152.
Gollins, S. W., and J. S. Porterfield. 1985. Flavivirus infection enhancement in macrophages: an electron microscopic study of viral cellular entry. J. Gen. Virol. 66:1969-1982.
Grummer, B., S. Grotha, and I. Greiser-Wilke. 2004. Bovine viral diarrhoea virus is internalized by clathrin-dependent receptor-mediated endocytosis. J. Vet. Med. B Infect. Dis. Vet. Public Health 51:427-432.
Heinz, F. X., and S. L. Allison. 2000. Structures and mechanisms in flavivirus fusion. Adv. Virus Res. 55:231-269.
Hinshaw, J. E. 2000. Dynamin and its role in membrane fission. Annu. Rev. Cell Dev. Biol. 16:483-519.
Hsu, M., J. Zhang, M. Flint, C. Logvinoff, C. Cheng-Mayer, C. M. Rice, and J. A. McKeating. 2003. Hepatitis C virus glycoproteins mediate pH-dependent cell entry of pseudotyped retroviral particles. Proc. Natl. Acad. Sci. USA 100:7271-7276.
Hulst, M. M., and R. J. M. Moormann. 1997. Inhibition of pestivirus infection in cell culture by envelope proteins E(rns) and E2 of classical swine fever virus: E(rns) and E2 interact with different receptors. J. Gen. Virol. 78:2779-2787.
Hulst, M. M., H. G. P. van Gennip, and R. J. M. Moormann. 2000. Passage of classical swine fever virus in cultured swine kidney cells selects virus variants that bind to heparan sulfate due to a single amino acid change in envelope protein Erns. J. Virol. 74:9553-9561.
Iqbal, M., H. Flick-Smith, and J. W. McCauley. 2000. Interactions of bovine viral diarrhoea virus glycoprotein E(rns) with cell surface glycosaminoglycans. J. Gen. Virol. 81:451-459.
Krey, T., H. J. Thiel, and T. Rümenapf. 2005. Acid-resistant bovine pestivirus requires activation for pH-triggered fusion during entry. J. Virol. 79:4191-4200.
Liang, D., I. F. Sainz, I. H. Ansari, L. H. V. G. Gil, V. Vassilev, and R. O. Donis. 2003. The envelope glycoprotein E2 is a determinant of cell culture tropism in ruminant pestiviruses. J. Gen. Virol. 84:1269-1274.
Lindenbach, B. D., and C. M. Rice. 2001. Flaviviridae: the viruses and their replication, p. 991-1042. In D. M. Knipe and P. M. Howley (ed.), Fields virology, 4th ed., vol. 1. Lippincott Williams & Wilkins, Philadelphia, Pa.
Maurer, K., T. Krey, V. Moennig, H. J. Thiel, and T. Rümenapf. 2004. CD46 is a cellular receptor for bovine viral diarrhea virus. J. Virol. 78:1792-1799.
Mendez, E., N. Ruggli, M. S. Collett, and C. M. Rice. 1998. Infectious bovine viral diarrhea virus (strain NADL) RNA from stable cDNA clones: a cellular insert determines NS3 production and viral cytopathogenicity. J. Virol. 72:4737-4745.
Parton, R. G., B. Joggerst, and K. Simons. 1994. Regulated internalization of caveolae. J. Cell Biol. 127:1199-1215.
Pelkmans, L., and A. Helenius. 2003. Insider information: what viruses tell us about endocytosis. Curr. Opin. Cell Biol. 15:414-422.
Puri, A., J. Winick, R. J. Lowy, D. Covell, O. Eidelman, A. Walter, and R. Blumenthal. 1988. Activation of vesicular stomatitis virus fusion with cells by pretreatment at low pH. J. Biol. Chem. 263:4749-4753.
Reimann, I., K. Depner, S. Trapp, and M. Beer. 2004. An avirulent chimeric Pestivirus with altered cell tropism protects pigs against lethal infection with classical swine fever virus. Virology 322:143-157.
Schelp, C., I. Greiser-Wilke, G. Wolf, M. Beer, V. Moennig, and B. Liess. 1995. Identification of cell membrane proteins linked to susceptibility to bovine viral diarrhoea virus infection. Arch. Virol. 140:1997-2009.
Sieczkarski, S. B., and G. R. Whittaker. 2002. Dissecting virus entry via endocytosis. J. Gen. Virol. 83:1535-1545.
Sieczkarski, S. B., and G. R. Whittaker. 2002. Influenza virus can enter and infect cells in the absence of clathrin-mediated endocytosis. J. Virol. 76:10455-10464.
Tautz, N., G. Meyers, and H. J. Thiel. 1998. Pathogenesis of mucosal disease, a deadly disease of cattle caused by a pestivirus. Clin. Diagn. Virol. 10:121-127.
Wang, L. H., K. G. Rothberg, and R. G. Anderson. 1993. Mis-assembly of clathrin lattices on endosomes reveals a regulatory switch for coated pit formation. J. Cell Biol. 123:1107-1117.
Wang, Z., Y. Nie, P. Wang, M. Ding, and H. Deng. 2004. Characterization of classical swine fever virus entry by using pseudotyped viruses: E1 and E2 are sufficient to mediate viral entry. Virology 330:332-341.
Wild, P., E. M. Schraner, J. Peter, E. Loepfe, and M. Engels. 1998. Novel entry pathway of bovine herpesvirus 1 and 5. J. Virol. 72:9561-9566.
Xue, W., and H. C. Minocha. 1993. Identification of the cell surface receptor for bovine viral diarrhoea virus by using anti-idiotypic antibodies. J. Gen. Virol. 74:73-79.(Steve Lecot, Sandrine Bel)
ABSTRACT
Cellular mechanisms of bovine viral diarrhea virus (BVDV) entry in MDBK cells were investigated. Chloroquine, bafilomycin A1, or ammonium chloride inhibited BVDV infection, indicating that an acidic endosomal pH is required for BVDV entry. The tyrosine kinase inhibitor genistein partially inhibited BVDV infection at a postentry step, whereas BVDV entry was strongly inhibited by chlorpromazine or by the overexpression of a dominant-negative form of EPS15, a protein essential for the formation of clathrin-coated vesicles at the plasma membrane. Together, these data indicate that BVDV infection requires an active clathrin-dependent endocytic pathway.
TEXT
Bovine viral diarrhea virus (BVDV) is a small, enveloped, positive-stranded RNA virus which is the etiological agent of a variety of pathologies in cattle, including fatal mucosal disease (21, 31). BVDV is classified in the Pestivirus genus within the Flaviviridae family, which also contains hepatitis C virus (HCV) and viruses of the Flavivirus genus (21). Little is known about cellular mechanisms leading to the entry of BVDV and other pestiviruses. Their binding to target cells involves envelope glycoproteins Erns and E2 (16, 20, 27, 33) through interactions with glycosaminoglycans (17, 18) and membrane proteins (28, 35), respectively. BVDV receptors include CD46 (22) and low-density-lipoprotein receptor (1). Because of their similarity to flaviviruses, pestiviruses are thought to enter target cells by receptor-mediated endocytosis and acid-triggered fusion of their envelope with an endosomal membrane (1, 22). However, BVDV is also known to be resistant to acidic treatments (9, 19), a condition often found in viruses with pH-independent entry.
In order to assess if BVDV entry is pH-dependent, we first sought to determine if BVDV infection indeed requires low endosomal pH. The importance of endosome acidification was studied with chloroquine and ammonium chloride, two lysosomotropic agents, and with bafilomycin A1, a specific inhibitor of endosomal proton-ATP pumps. MDBK cells were preincubated for 30 min with inhibitors, infected for 1 h at 37°C with BVDV (NADL strain) (23), and cultured for 15 h in the presence of the inhibitors. The virus was diluted such that 30 to 40% of the cells were infected in control experiments with no inhibitor. The infected cells were detected by indirect immunofluorescence microscopy by using a monoclonal antibody to NS3 (5). The nuclei were stained with Hoechst dye. The infections were scored as the ratio of infected cells to total cells. For comparison, we used bovine herpesvirus 1 (BHV-1), which is known to enter cells by a pH-independent mechanism (34). Each drug inhibited BVDV infection in a dose-dependent manner (Fig. 1). In contrast, BHV-1 infection was not inhibited by chloroquine or bafilomycin A1, and ammonium chloride treatment resulted in a partial inhibition of BHV-1 infection. To verify that these drugs interfered with BVDV entry, we asked whether they could act on an early step of the infection. Bafilomycin was added during early or late steps of infection. The drug strongly inhibited BVDV infection when present during the infection and up to 4 h postinfection but was without effect when added later (Fig. 1). Similar results were obtained with chloroquine (data not shown). Taken together, the results obtained with these inhibitors indicate that BVDV infection is sensitive to the low pH of endosomes. Similar results were recently reported by others (12, 19).
The pH-dependent entry of BVDV suggests that the virus is internalized from the cell surface by receptor-mediated endocytosis and reaches an endosomal compartment, where the fusion occurs. Two well-defined endocytic pathways appear to be clathrin-mediated endocytosis and caveolae internalization (25, 29). To determine if BVDV enters cells through a clathrin-mediated or a caveolae-mediated pathway, we first tested the effects of chlorpromazine (32) and genistein (8, 24), respectively. For comparison, the effects of these inhibitors were also assessed on the infection of Sindbis virus, which enters by clathrin-mediated endocytosis (6). Chlorpromazine inhibited BVDV infection in a dose-dependent manner (Fig. 2A). The inhibition was almost complete at a concentration of 10 mM, which is known to block clathrin-coated pit assembly at the plasma membrane (32). As expected, Sindbis virus infection was inhibited by chlorpromazine and BHV-1 infection was not affected, suggesting that the observed effects were not due to the toxicity of the drug (Fig. 2A).
Genistein also inhibited BVDV infection in a dose-dependent manner (Fig. 2B). We observed about 50% inhibition with 50 to 100 μg/ml genistein. Similar concentrations of genistein inhibit simian virus 40 internalization (8) but are without effect on influenza virus infection (30). Sindbis virus infection was not affected in the presence of 50 μg/ml genistein (Fig. 2B). This suggests that the inhibition of BVDV infection by genistein was not due to toxicity on MDBK cells. Taken together, these data show that BVDV infection is sensitive to both chlorpromazine and genistein.
We next verified if these drugs inhibit an early step of BVDV infection. As shown in Fig. 2C, the inhibition of BVDV infection was strong when chlorpromazine was added to the cells for only 4 h at the beginning of the infection but was very weak when the drug was added onto the cells four hours after infection. This action of chlorpromazine during early steps of BVDV infection is consistent with an inhibition of endocytosis. On the other hand, the inhibition by genistein was weaker when the inhibitor was added to the cells during the early steps than during the late steps of infection (Fig. 2C). Therefore, it is unlikely that genistein had an effect on BVDV endocytosis. These results indicate that BVDV entry is inhibited by chlorpromazine, suggesting that it might proceed through clathrin-mediated endocytosis.
This finding is consistent with the recent report that a dominant-negative form of dynamin blocks BVDV infection (19). However, dynamin regulates clathrin-mediated endocytosis, as well as other clathrin-independent pathways of internalization (14). Likewise, none of the drugs that we used are specific endocytosis inhibitors. To confirm that clathrin-mediated endocytosis was involved in BVDV entry, we made use of E95-295, a dominant-negative form of EPS15, which specifically interferes with clathrin-coated vesicle formation at the plasma membrane (3). A recombinant adenoviral vector (rAd:E95-295) was generated to express this mutant, and its functionality was verified on MDBK cells (data not shown). For a control, we used DIII2, another form of EPS15 with no dominant-negative effect on clathrin-mediated endocytosis (4). MDBK cells were transduced with rAd:E95-295 or rAd:DIII2 and subsequently infected with BVDV or control viruses 24 h later. The number of infected cells was dramatically reduced in cells transduced with rAd:E95-295, compared to cells transduced with rAd:DIII2 (Fig. 3A). The infection was scored separately in cells expressing low, moderate, or high levels of E95-295. As expected, E95-295 did not inhibit BHV-1 infection. In contrast, both BVDV and Sindbis virus infections were affected and the inhibition was positively correlated to E95-295 expression levels (Fig. 3B). These data confirm that a functional clathrin-mediated pathway of endocytosis is required for BVDV infection.
A clathrin-mediated entry route has been reported previously for flaviviruses. Both electron microscopy studies and E95-295-mediated inhibition indicated that West Nile Virus enters cells through clathrin-coated vesicles (7, 11). Since clathrin-coated vesicles deliver their cargo in acidic early endosomes, this is consistent with numerous studies which have shown that the envelope glycoprotein E of flaviviruses undergoes a conformational transition under acidic conditions from a native dimeric form to a fusogenic trimeric form (13). Such an acid-triggered conformational transition has not been reported at the present time for the envelope proteins of BVDV or other pestiviruses. In contrast, BVDV is resistant to acidic treatments (9, 19). Such a discrepancy between pH-dependent entry and resistance of the virion to acidic treatments has also been observed for vesicular stomatitis virus (26). It has recently been suggested that BVDV envelope glycoproteins must be primed by disulfide bridge reduction in the endocytotic pathway before an acid-sensitive fusogenic conformation can be reached (19).
In conclusion, our study suggests that, like flaviviruses, pestiviruses enter cells through clathrin-mediated endocytosis and fusion from within an acidic endosomal compartment. It would be interesting to determine if this pathway of endocytosis is also used by other pestiviruses and by HCV and other nonclassified viruses of the Flaviviridae family, such as GB viruses. Recent data suggest that retroviral particles pseudotyped with HCV envelope glycoproteins E1 and E2 are sensitive to agents that neutralize the pH of endosomes (2, 15), but data on clathrin requirement have not yet been reported.
ACKNOWLEDGMENTS
We are grateful to C. M. Rice, E. Thiry, G. Chappuis, and P. P. Pastoret for kindly providing viruses and antibodies. Data presented in this paper were obtained with the help of the Campus Calmette imaging core facility.
This work was funded by the Centre National de la Recherche Scientifique and by European Union grant QLRT-2000-01120.
REFERENCES
Agnello, V., G. ábel, M. Elfahal, G. B. Knight, and Q. X. Zhang. 1999. Hepatitis C virus and other Flaviviridae viruses enter cells via low density lipoprotein receptor. Proc. Natl. Acad. Sci. USA 96:12766-12771.
Bartosch, B., A. Vitelli, C. Granier, C. Goujon, J. Dubuisson, S. Pascale, E. Scarselli, R. Cortese, A. Nicosia, and F.-L. Cosset. 2003. Cell entry of hepatitis C virus requires a set of co-receptors that include the CD81 tetraspanin and the SR-B1 scavenger receptor. J. Biol. Chem. 278:41624-41630.
Benmerah, A., M. Bayrou, N. Cerf-Bensussan, and A. Dautry-Varsat. 1999. Inhibition of clathrin-coated pit assembly by an Eps15 mutant. J. Cell Sci. 112:1303-1311.
Benmerah, A., C. Lamaze, B. Bègue, S. L. Schmid, A. Dautry-Varsat, and N. Cerf-Bensussan. 1998. AP-2/Eps15 interaction is required for receptor-mediated endocytosis. J. Cell Biol. 140:1055-1062.
Boulanger, D., S. Waxweiler, L. Karelle, M. Loncar, B. Mignon, J. Dubuisson, E. Thiry, and P. P. Pastoret. 1991. Characterization of monoclonal antibodies to bovine viral diarrhoea virus: evidence of a neutralizing activity against gp48 in the presence of goat anti-mouse immunoglobulin serum. J. Gen. Virol. 72:1195-1198.
Carbone, R., S. Fre, G. Iannolo, F. Belleudi, P. Mancini, P. G. Pelicci, M. R. Torrisi, and P. P. Di Fiore. 1997. eps15 and eps15R are essential components of the endocytic pathway. Cancer Res. 57:5498-5504.
Chu, J. J. H., and M. L. Ng. 2004. Infectious entry of West Nile virus occurs through a clathrin-mediated endocytic pathway. J. Virol. 78:10543-10555.
Dangoria, N. S., W. C. Breau, H. A. Anderson, D. M. Cishek, and L. C. Norkin. 1996. Extracellular simian virus 40 induces an ERK/MAP kinase-independent signalling pathway that activates primary response genes and promotes virus entry. J. Gen. Virol. 77:2173-2182.
Depner, K., T. Bauer, and B. Liess. 1992. Thermal and pH stability of pestiviruses. Rev. Sci. Tech. 11:885-893.
Duvet, S., F. Chirat, A. M. Mir, A. Verbert, J. Dubuisson, and R. Cacan. 2000. Reciprocal relationship between alpha1,2 mannosidase processing and reglucosylation in the rough endoplasmic reticulum of Man-P-Dol deficient cells. Eur. J. Biochem. 267:1146-1152.
Gollins, S. W., and J. S. Porterfield. 1985. Flavivirus infection enhancement in macrophages: an electron microscopic study of viral cellular entry. J. Gen. Virol. 66:1969-1982.
Grummer, B., S. Grotha, and I. Greiser-Wilke. 2004. Bovine viral diarrhoea virus is internalized by clathrin-dependent receptor-mediated endocytosis. J. Vet. Med. B Infect. Dis. Vet. Public Health 51:427-432.
Heinz, F. X., and S. L. Allison. 2000. Structures and mechanisms in flavivirus fusion. Adv. Virus Res. 55:231-269.
Hinshaw, J. E. 2000. Dynamin and its role in membrane fission. Annu. Rev. Cell Dev. Biol. 16:483-519.
Hsu, M., J. Zhang, M. Flint, C. Logvinoff, C. Cheng-Mayer, C. M. Rice, and J. A. McKeating. 2003. Hepatitis C virus glycoproteins mediate pH-dependent cell entry of pseudotyped retroviral particles. Proc. Natl. Acad. Sci. USA 100:7271-7276.
Hulst, M. M., and R. J. M. Moormann. 1997. Inhibition of pestivirus infection in cell culture by envelope proteins E(rns) and E2 of classical swine fever virus: E(rns) and E2 interact with different receptors. J. Gen. Virol. 78:2779-2787.
Hulst, M. M., H. G. P. van Gennip, and R. J. M. Moormann. 2000. Passage of classical swine fever virus in cultured swine kidney cells selects virus variants that bind to heparan sulfate due to a single amino acid change in envelope protein Erns. J. Virol. 74:9553-9561.
Iqbal, M., H. Flick-Smith, and J. W. McCauley. 2000. Interactions of bovine viral diarrhoea virus glycoprotein E(rns) with cell surface glycosaminoglycans. J. Gen. Virol. 81:451-459.
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