1 Department of Neurology, Evanston Hospital, 2650 Ridge Avenue, Evanston, IL 60201, USA
2 Departments of Neurology, Northwestern University, Chicago, IL, USA
3 Department of MicrobiologyImmunology, Northwestern University, Chicago, IL, USA
4 Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Chicago, IL, USA
Correspondence
Howard Lipton
hllipton{at}merle.acns.nwu.edu
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ABSTRACT |
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MAIN TEXT |
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Virus binding to the surface of permissive cells is a major determinant of virus host range and tissue tropism. In the case of non-enveloped viruses such as picornaviruses, binding involves the recognition of unique structural features on the virion coat by host cell-surface protein(s). A prominent virion depression or receptor site, termed the pit, is present on the capsid, with VP1 loops I and II blocking lateral extension of this depression (Grant et al., 1992; Luo et al., 1992
). Observations have indicated that the cardiovirus pit is involved in receptor recognition (Kim et al., 1990
) and mutations in selected BeAn virus pit residues support this observation (Hertzler et al., 2000
). The pit in cardioviruses is thought to be analogous to the canyon that circulates around the fivefold axis of polioviruses (Hogle et al., 1985
), rhinoviruses (Rossmann et al., 1985
) and coxsackie B viruses (Muckelbauer et al., 1999
). These and other members of the Picornaviridae family have been demonstrated to use protein entry receptors (Evans & Almond, 1998
; Rieder & Wimmer, 2002
).
The protein entry receptor for TMEV is not yet known. A 34 kDa unidentified protein was found by virus overlay protein binding assay to be bound by both low- and high-neurovirulence TMEV (Kilpatrick & Lipton, 1991). Libbey et al. (2001)
have shown that the peripheral nerve protein, PO, could function as a TMEV receptor in some cells, but this observation requires further confirmation. It has been postulated that TMEV uses at least two molecules for attachment and entry (Jnaoui & Michiels, 1999
). It is possible that both neurovirulence groups use a common protein entry receptor but different attachment factors, or maybe even different protein entry receptors. Recently, Hertzler et al. (2001)
demonstrated that TMEV needs the UDP-galactose transporter (UGT) for binding and infection, by using a BHK-21 receptor negative cell line (R26), selected through resistance to BeAn virus infection. These results were supported using a Chinese hamster ovary (CHO) mutant cell line, Lec-8, which is deficient in the UGT.
UGT is a member of the trans-Golgi network nucleotide-sugar transporter proteins associated with the biosynthesis of complex carbohydrates. Although UGT is a trans-Golgi-associated protein, some cycling to the cell surface may occur (Ladinsky & Howell, 1993; Shukla et al., 1999
). The requirement of UGT for TMEV binding and entry in BHK-21 and CHO cells originally suggested that the transporter itself might function as a TMEV receptor (Hertzler et al., 2001
). To examine a role for UGT as a receptor, a rabbit anti-human UGT antibody was used to block virus binding and infection. However, we first needed to demonstrate that rabbit anti-human UGT recognized hamster UGT on the surface of BHK-21 cells. Since Aoki et al. (1999)
were only able to detect human UGT after its overexpression, pCMVSPORT hamster UGT (Hertzler et al., 2001
) was transfected into BHK-21 cells, which were examined by flow cytometry. As shown in Fig. 1
(A), the human UGT antibody detected hamster UGT on the cell surface. Next, BHK-21 cells (1x106) were incubated with 0·1 and 1 µg of polyclonal rabbit antiserum prior to binding and infection with the high-neurovirulence GDVII and low-neurovirulence BeAn viruses. [35S]Methionine-labelled virus (2x104 particles per cell) was incubated with UGT antibody-bound cells for 30 min at 4 °C and the supernatant and cell-associated radioactivity was determined for triplicate samples in a Beckman LS5000TD scintillation counter and plotted as the percentage of cell-associated counts. Neither antibody concentration inhibited binding of either virus to BHK-21 cells (Fig. 1B
). UGT antibody-treated BHK-21 cells were also infected with both viruses at an m.o.i. of 10 and cell viability measured by MTT assay (Denizot & Lang, 1986
). The percentage of lysed cells, i.e. cytopathology, was plotted at 16 h post-infection (p.i.) with uninfected cells as controls. As shown in Fig. 1(C)
, neither BeAn nor GDVII virus infection was inhibited by UGT antibody. These results indicated that UGT itself is not a TMEV receptor.
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High-neurovirulence GDVII and FA strains use heparan sulfate as an attachment factor to mediate cell entry (Reddi & Lipton, 2002). Heparan sulfate is linked to the protein core by a tetra-saccharide linker containing two molecules of galactose (Perrimon & Bernfield, 2000
). A CHO cell proteoglycan-deficient mutant, pgsD-677, has been found to be resistant to infection by GDVII (Reddi & Lipton, 2002
). D-677 expresses the protein core of the proteoglycan with only a part of the tetra-saccharide linker needed to add glycosaminoglycans the part that has only xylose followed by two galactose molecules (Lidholt et al., 1992
). Despite expression of galactose on the surface of the protein core, the resistance of these cells to GDVII infection also indicates that GDVII does not bind directly to galactose, but rather needs another moiety. Taken together, these data not only confirm that TMEV does not bind directly to galactose or use it for entry, but also indicate that TMEV uses glycosylated molecules for entry.
It is possible that viruses of both neurovirulence groups use a common receptor with different attachment factors. Recent studies demonstrating the use of sialic acid and heparan sulfate (molecules that need galactose for assembly) as attachment factors by BeAn and GDVII viruses, respectively, highlight the importance of both molecules. The present data eliminating the involvement of UGT or galactose in viral binding and entry provide further insight into the interaction of TMEV with host cells and should help facilitate ongoing studies to identify a TMEV receptor.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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---|
Denizot, F. & Lang, R. (1986). Rapid colorimetric assay for cell growth and survival. Modifications to the tetrazolium dye procedure giving improved sensitivity and reliability. J Immunol Methods 89, 271277.[CrossRef][Medline]
Deutscher, S. L., Nuwayhid, N., Stanley, P. & Briles, E. B. (1984). Translocation across Golgi vesicle membranes: a CHO glycosylation mutant deficient in CMP-sialic acid transport. Cell 39, 295299.[Medline]
Evans, D. J. & Almond, J. W. (1998). Cell receptors for picornaviruses as determinants of cell tropism and pathogenesis. Trends Microbiol 6, 198202.[CrossRef][Medline]
Fotiadis, C., Kilpatrick, D. R. & Lipton, H. L. (1991). Comparison of the binding characteristics to BHK-21 cells of viruses representing the two Theiler's virus neurovirulence groups. Virology 182, 365370.[CrossRef][Medline]
Grant, R. A., Filman, D. J., Fujinami, R. S., Icenogle, J. P. & Hogle, J. M. (1992). Three-dimensional structure of Theiler's virus. Proc Natl Acad Sci U S A 89, 20612065.[Abstract]
Hertzler, S., Luo, M. & Lipton, H. L. (2000). Mutation of predicted virion pit residues alters binding of Theiler's murine encephalomyelitis virus to BHK-21 cells. J Virol 74, 19942004.
Hertzler, S., Kallio, P. & Lipton, H. L. (2001). UDP-galactose transporter is required for Theiler's virus entry into mammalian cells. Virology 286, 336344.[CrossRef][Medline]
Hogle, J. M., Chow, M. & Filman, D. J. (1985). Three-dimensional structure of poliovirus at 2·9 Å resolution. Science 229, 13581365.[Medline]
Jnaoui, K. & Michiels, T. (1999). Analysis of cellular mutants resistant to Theiler's virus infection: differential infection of L929 cells by persistent and neurovirulent strains. J Virol 73, 72487254.
Jolly, C. L., Beisner, B. M. & Holmes, H. H. (2000). Rotavirus infection of MA104 cells is inhibited by Ricinus lectin and separately expressed single binding domains. Virology 275, 8897.[CrossRef]
Kilpatrick, D. R. & Lipton, H. L. (1991). Predominant binding of Theiler's viruses to a 34-kilodalton receptor protein on susceptible cell lines. J Virol 65, 52445249.[Medline]
Kim, S., Boege, U., Krishnaswamy, S., Minor, I., Smith, T. J., Luo, M., Scraba, D. G. & Rossmann, M. G. (1990). Conformational variability of a picornavirus capsid: pH-dependent structural changes of Mengo virus related to its host receptor attachment site and disassembly. Virology 175, 176190.[Medline]
Ladinsky, M. S. & Howell, K. E. (1993). An electron microscopic study of TGN38/41 dynamics. J Cell Sci (Suppl.) 17, 4147.
Libbey, J. E., McCright, I. J., Tsunoda, I., Wada, Y. & Fujinami, R. S. (2001). Peripheral nerve protein, PO, as a potential receptor for Theiler's murine encephalomyelitis virus. J Neurovirol 7, 97104.[CrossRef][Medline]
Lehrich, J. R., Arnason, B. G. W. & Hochberg, F. H. (1976). Demyelinating myelopathy in mice induced by the DA virus. J Neurol Sci 29, 149160.[CrossRef][Medline]
Lidholt, K., Weinke, J. L., Kiser, C. S., Lugemwa, F. N., Bame, K. J., Cheifetz, S., Massague, J., Lindahl, U. & Esko, J. D. (1992). A single mutation affects both N-acetylglucosaminyltransferase and glucuronosyltransferase activities in a Chinese hamster ovary cell mutant defective in heparan sulfate biosynthesis. Proc Natl Acad Sci U S A 89, 22672271.[Abstract]
Lipton, H. L. (1975). Theiler's virus infection in mice: an unusual biphasic disease process leading to demyelination. Infect Immun 11, 11471155.[Medline]
Luo, M., He, C., Toth, K. S., Zhang, C. X. & Lipton, H. L. (1992). Three-dimensional structure of Theiler's murine encephalomyelitis virus (BeAn strain). Proc Natl Acad Sci U S A 89, 24092413.[Abstract]
Muckelbauer, J. K., Kremer, M., Minor, I., Diana, G., Dutko, F. J., Groarke, J., Pevear, D. C. & Rossmann, M. G. (1999). The structure of coxsackievirus B3 at 3·5 Å resolution. Structure 3, 653667.
Perrimon, N. & Bernfield, M. (2000). Specificities of heparan sulphate proteoglycans in developmental processes. Nature 404, 725728.[CrossRef][Medline]
Reddi, H. V. & Lipton, H. L. (2002). Heparan sulfate mediates infection of high-neurovirulence Theiler's viruses. J Virol 76, 84008407.
Rieder, E. & Wimmer, E. (2002). Cellular receptors of picornaviruses: an overview. In Molecular Biology of Picornaviruses, pp. 6170. Edited by B. L. Semler & E. Wimmer. Washington, DC: ASM Press.
Rossmann, M. G., Arnold, E., Erickson, J. W. & 10 other authors (1985). Structure of a human common cold virus and functional relationship to other picornaviruses. Nature 317, 145153.[Medline]
Shah, A. H. & Lipton, H. L. (2002). Low-neurovirulence Theiler's viruses use sialic acid moieties on N-linked oligosaccharide structures for attachment. Virology 304, 443450.[CrossRef][Medline]
Shukla, D., Liu, J., Blaiklock, P., Shworak, N. W., Bai, X., Esko, J. D., Cohen, G. H., Eisenberg, R. D. & Spear, P. G. (1999). A novel role for 3-O-sulfated heparan sulfate in herpes simplex virus 1 entry. Cell 99, 1322.[Medline]
Received 3 August 2002;
accepted 2 December 2002.
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