Integrated Graduate Program, Northwestern University Medical School, Chicago, IL, USA1
Department of Neurology, Evanston Hospital, 2650 Ridge Avenue, Evanston, IL 60201, USA2
Departments of Neurology, Microbiology-Immunology, and Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evanston/Chicago, IL, USA3
Author for correspondence: Howard Lipton (at Evanston Hospital). Fax +1 847 570 1568. e-mail hllipton{at}merle.acns.nwu.edu
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Abstract |
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The availability of a cell line lacking a specific virus receptor but otherwise fully susceptible to infection by that virus provides a powerful reagent for the identification of the receptor molecule. In this instance, a clone or clones in a cDNA library expressing the receptor can be detected by its ability to mediate virus entry in the receptor-negative cell line. This is particularly relevant since a TMEV receptor protein has not yet been identified. However, extensive screening of animal and human cell lines has not revealed a TMEV receptor-negative cell line, perhaps because the TMEV receptor is widely expressed in eukaryotic cells. Therefore, we used an alternative strategy to select a TMEV receptor-negative cell line, based on the observation that a small fraction (<0·1%) of BHK-21 cells survive infection with BeAn virus at high m.o.i. The possibility that these cells represent genetic variants that are resistant to virus infection is consistent with several reports of receptor-negative cells derived from cells surviving persistent infections (Borzakian et al., 1992 ; Kaplan et al., 1989
; Kaplan & Racaniello, 1991
). To explore the nature of the resistance of BHK-21 cells to TMEV, clonal populations of resistant cells were derived and further characterized.
The strategy for isolation of BeAn virus-resistant BHK-21 cell lines was as follows. After four cycles of infection of BHK-21 cells with BeAn virus at an m.o.i. of 10, and allowing several weeks for growth of surviving cells after each cycle, a subpopulation arose that showed no sign of CPE at 10 days post-infection (p.i.) with BeAn virus. In contrast, BHK-21 cell monolayers infected in parallel displayed extensive CPE at 24 h. Multiple passages of the resistant cells for more than 6 months did not result in crisis, i.e. the appearance of CPE due to virus persistence and the emergence of susceptible cells. The morphology of the cells was identical to that of parental BHK-21 cells except that there were more rounded cells (see below). 35S-Labelled BeAn and GDVII viruses showed <10% binding to the resistant cells at 40 min compared to 4055% binding to BHK-21 cells (Fig. 1). The binding assay revealed <10% BeAn binding in six TMEV-resistant BHK-21 clones isolated by limiting dilution; one clone, designated R26, was chosen for further characterization (Fig. 1
). The doubling time of R26 cells was the same as that of BHK-21 cells (data not shown), with the morphology identical to that of uncloned resistant cells.
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Although the protein receptor for TMEV on host cells has not been identified, evidence from competitive inhibition of virus binding suggests that viruses of both TMEV neurovirulence groups bind the same protein receptor (Fotiadis et al., 1991 ). The present data support this notion since the resistant R26 cells selected by repeated rounds of infection with BeAn virus were also resistant to infection with GDVII virus. On the other hand, it is well-established that the low- but not the high-neurovirulence TMEV strains also bind sialic acid which may facilitate transfer of the virus to the protein receptor (Fotiadis et al., 1991
; Zhou et al., 1997
, 2000
). Thus, the high-neurovirulence TMEV, such as GDVII, appear to bind the protein receptor directly, whereas the low-neurovirulence TMEV bind first to sialic acid and then to the protein receptor.
Several studies have shown that passage of persistently infected cells leads to the co-evolution of the virus and cells, resulting in stable cell lines with a block in virus binding (Borzakian et al., 1992 ; Kaplan et al., 1989
; Kaplan & Racaniello, 1991
). Kaplan et al. (1989)
, for example derived a stable HeLa cell line lacking functional virus receptors after co-transfection with poliovirus RNA and R1, a poliovirus subgenomic RNA deleted of nearly all of the capsid region. Southern blot analysis of genomic DNA of these cells revealed no gross alterations, Northern blotting revealed no expression of poliovirus receptor (PVR) transcripts, and transcription of PVR-specific RNA was reduced in an in vitro nuclear run-on assay (Kaplan & Racaniello, 1991
). Treatment of these cells with 5-azacytidine restored susceptibility to poliovirus and the appearance of poliovirus receptors on the cell surface, and a role for methylation in regulating the PVR gene was suggested (Kaplan & Racaniello, 1991
). Another study demonstrated a correlation between the maintenance of persistent poliovirus variants in HEp-2 cells and selection of mutant cell populations of various phenotypes (Borzakian et al., 1992
). In one phenotype, the PVR was not detected by cytofluorimetry, although the mechanism involved in the loss of the cell surface receptor was not further investigated. Other studies have used either physical or chemical mutagenesis followed by virus infection to select for resistant cell lines lacking virus receptors. For example, Hara et al. (1989)
derived a mutant murine cell line, designated Had-1, which was deficient in Newcastle disease virus receptors. The genetic defect in Had-1 cells was complemented by UDP-galactose transporter (UGT) cDNA; UGT is necessary for sialylation of the cell surface required for Newcastle disease virus attachment (Ishida et al., 1996
; Yoshioka et al., 1997
). Jnaoui & Michels (1999)
recently derived an L cell line partially resistant to entry by GDVII but not DA virus. This pattern of resistance was verified using TMEV recombinants in which the capsid sequences of GDVII and DA were exchanged. These experiments suggest the existence of a co-receptor specific for GDVII virus.
Our study demonstrates the feasibility of selecting cell variants that do not bind TMEV after several passages at high m.o.i. rather than through co-evolution of virus variants during persistent infection or mutagenesis. To our knowledge this is the first example of the selection of a virus receptor-negative cell line by merely passaging the virus in susceptible cells.
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Acknowledgments |
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References |
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Received 30 March 2000;
accepted 29 June 2000.
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