Department of Clinical Veterinary Medicine, University of Cambridge, Madingley Road, Cambridge CB3 0ES, UK1
Author for correspondence: Laurence Tiley. Fax +44 1223 337610. e-mail Lst21{at}cam.ac.uk
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Abstract |
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Introduction |
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Maedivisna virus (MVV) is a lentivirus that infects sheep and goats, and predominantly infects cells from the monocytemacrophage lineage (Gendelman et al., 1986 ). The tropism of MVV is partly due to differential expression of cellular transactivators of the virus LTR (Gabuzda et al., 1989
) but is also due to other factors that have yet to be determined. Co-receptor expression is an important determinant of macrophage tropism for HIV at the level of entry, although it is not entirely predictive (Deng et al., 1996
; Dittmar et al., 1997
; reviewed by Berger et al., 1999
). However, the receptor for MVV is unlikely to be the major determinant of host cell tropism. Evidence from tissue culture experiments showed that MVV readily infects fibroblastic cells from a variety of ovine and caprine sources (Lee et al., 1994
; Da Silva Teixeira et al., 1997
). Experiments using vesicular stomatitis virus (VSV) pseudotyping and early cell culture experiments indicated that the MVV receptor was present on cell lines derived from many non-ungulate species from a variety of tissue origins (MacIntyre et al., 1972
; August & Harter, 1974
; Gilden et al., 1981
).
Efforts to identify the receptor using virus overlay protein blotting assays led to MHC-II being proposed as a possible receptor (Dalziel et al., 1991 ). However, while this protein could be involved in some situations, the known distribution of the receptor is inconsistent with this hypothesis. A second candidate identified using chemical cross-linking and co-immunoprecipitation with the MVV Env protein had a molecular mass of 50 kDa (Crane et al., 1991
). Despite successfully raising antibodies to this protein, it has not been possible to identify, clone or definitively demonstrate its role in virus fusion and entry (Bruett et al., 2000
).
As a prelude to employing an expression cloning strategy to identify the MVV receptor, we have studied its distribution on cell types from a variety of ovine and non-ovine origins. Cell fusion assays employing recombinant vaccinia virus-expressed MVV Env and semi-quantitative PCR assays for virus reverse transcription demonstrated that the receptor was present on a wide range of cell lines from different species. It was sensitive to proteolytic digestion with papain, but was resistant to trypsin. Cell lines of Chinese hamster origin (CHO) do not express the receptor. However, somatic cell hybrid lines carrying various complements of murine chromosomes were permissive for virus entry. This demonstrated that receptor function could be complemented by transfer of genes from a permissive line to a non-permissive line, indicating that a cDNA expression cloning strategy may be feasible. The hybrid cell analysis showed that the receptor was carried on murine chromosome 2 or 4 (although involvement of chromosomes 6 and X could not be definitively excluded). Consequently, several potential candidates for the MVV receptor can be discounted, including MHC-II, Ram-1, and most currently mapped chemokine receptors.
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Methods |
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Plasmid and viruses.
Plasmid pT7P2GFP comprises the T7 late promoter, poliovirus type 2 IRES, green fluorescence protein (GFP) gene and T7 late terminator cloned in sequence into pACYC184 (New England Biolabs). It produces high levels of GFP when transfected into cells in which T7 RNA polymerase is expressed and no detectable GFP in the absence of T7 RNA polymerase. VV-MVV-Env is a recombinant vaccinia virus that expresses the envelope protein from the EV-1 strain of MVV (J. R. Rodriguez, personal communication). Vaccinia virus vTF7.3 constitutively expresses the T7 RNA polymerase gene (Fuerst et al., 1986 ). MVV stocks (strain EV1; Sargan et al., 1991
) were cultured in OSCs. Virus infections were performed using EMEM supplemented with 2·5% FCS for all viruses except where indicated otherwise.
Co-cultivation cell fusion assays.
Preliminary assays using VV-MVV-Env were performed by infecting near-confluent monolayers of cells with an m.o.i. of 0·1 TCID50 of virus and incubating overnight. The monolayers were stained with Giemsas or methylene blue stain prior to microscopic examination for self-fusion. Cell lines that did not show a self-fusing phenotype were co-cultivated for 8 h at a ratio of 5:1 with BHK 21 cells infected with VV-MVV-Env 16 h earlier with an m.o.i. of 3.
Protease digestion of cell surface proteins.
Target LSCC-H32 cells were incubated in EMEM plus 10% FCS and 1 µg/ml brefeldin A for 1 h before infection. The cells were washed twice in PBS and incubated for 5 min in 0·25% papain (21 U/mg) or 0·25% trypsin 250 (Difco). The cells were washed with medium containing antipain (Sigma) and 10% FCS to stop proteolysis. Treated cells were co-cultured at a ratio of 5:1 with BHK 21 cells (infected with VV-MVV-Env, 16 h earlier) in EMEM plus 10% FCS and 1 µg/ml brefeldin A. The cultures were stained 8 h later with methylene blue and were examined for fusion.
Fusion-activated reporter assay.
A modified version of a vaccinia-based fusion assay was developed (Nussbaum et al., 1994 ). Donor CHO cells were transfected with pT7P2GFP using Fugene 6 (Boehringer Mannheim). Transfection efficiencies were estimated by the transfection of pEGFP-C1 (Clontech) and were found to be approximately 2040%. After 24 h culture in EMEM plus 10% FCS cells were infected with VV-MVV-Env at an m.o.i. of 3 and cultured for 16 h in EMEM plus 2·5% FCS. Target cells were infected with vaccinia virus vTF7.3 at an m.o.i. of 3 and cultured for 16 h to allow expression of the T7 RNA polymerase. Donor and target cells were washed three times with PBS and dissociated using non-enzymatic dissociation buffer (Sigma). The two populations of cells were then co-cultured at a ratio of 1:1 for 68 h at 37 °C. Fusion between donor and target cells activates the expression of GFP due to the action of pre-made T7 RNA polymerase in the target cell.
Semi-quantitative PCR to detect MVV reverse transcription products.
Virus inoculum plus 10 mM MgCl2 was treated with 100 U/ml DNase I for 30 min at room temperature and filtered through a 0·2 µm filter to reduce the level of contaminating viral DNA in the inoculum. Cells were infected at an m.o.i. of 0·25 TCID50 in medium containing 2·5% FCS for 1 h at 37 °C with 5% CO2. They were then incubated for 16 h in medium containing 10% FCS. The cells were washed three times in PBS and lysed in 10 mM TrisHCl pH 8·0, 10 mM EDTA, 100 mM NaCl, 0·5% SDS and 100 µg/ml proteinase K for 16 h at 37 °C. The lysates were extracted with phenolchloroform and the DNA content was quantified by the Picogreen fluorescence assay (Molecular Probes). Serial tenfold dilutions were analysed for the presence of viral DNA by nested-PCR. Primary amplification was performed in 20 µl total volumes containing the following: diluted template DNA; 1 µM primer 5' ACT GTC AGG RCA GAG AAC ARA TGC C 3' (nt 89148938 in U3 of the published EV-1 sequence) (Sargan et al., 1991 ); 1 µM primer 5' CTC TCT TAC CTT ACT TCA GG 3' (nt 328309 downstream of U5); 1 U Taq polymerase; 1·5 mM MgCl2; 2 µl 10x Taq polymerase buffer; and 200 µM dNTPs. The samples were denatured at 94 °C for 1·5 min and then for 2 min at 80 °C during which the Taq polymerase was added (hot start). The DNA was amplified with 30 cycles of 94 °C for 30 s, 57 °C for 30 s and 72 °C for 30 s. The primary PCR product (1 µl) was used as the template in a second round of amplification, carried out as above but with the following primer pair: 5' AAG TCA TGT AKC AGC TGA TGC TT 3' (nt 90499071 in U3) and 5' TTG CAC GGA ATT AGT AAC G 3' (nt 129111 in U5). Amplification parameters were as before except that there was no 80 °C hot start and the annealing temperature was 50 °C. Reaction products were resolved by 1·5 or 2% agarose gel electrophoresis and visualized by ethidium bromide fluorescence.
Inhibition of fusion with MVV-specific antisera.
Inhibition of the fusion assay was achieved by preincubating the donor cells with a 1:10 dilution of hyper-immune rabbit serum raised against recombinant MVV Env or normal rabbit serum as a control for 2 h at 4 °C prior to co-cultivation with the target cells. Sera were maintained at the same working concentration throughout the course of the assays.
Digital image manipulation.
Electronic versions of photographic images were produced using a Umax Magiscan II scanner at 600 d.p.i. resolution. Composite images were produced manually or by using Adobe Photoshop 5LE.
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Results |
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Discussion |
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We were searching for a cell line that lacked the MVV receptor that would be suitable for a strategy to identify the receptor by expression cloning. The EV-1 strain of MVV was isolated by co-culture of primary ovine skin fibroblasts with PBMCs from a sheep with acute maedivirus disease. Skin cells are not normally productively infected in the natural host but are highly permissive for field isolates and laboratory strains of MVV in culture. The tissue culture grown EV-1 has been studied extensively both in vitro and in vivo. It maintains its narrow tropism for macrophages and dendritic cells, and causes disease in sheep that is indistinguishable from natural infection (Eriksson et al., 1999 ). Hence, it does not appear that the virus has undergone a major adaptation in its receptor tropism during its short passage history in OSCs. Our primary screens for the presence of receptor were based on a vaccinia virus recombinant that expresses the cloned MVV EV-1 Env protein. Nine, non-ovine, cell lines were tested. We confirmed that the distribution of the receptor was very broad, including cells of avian origin and thus more similar to that of the amphotropic type C retroviruses than to the other members of the lentiviruses. Only Chinese hamster cells (CHO, V79 TOR) were found to lack the receptor. This was confirmed using a sensitive virus-infection assay where virus entry was determined by the appearance of viral DNA. This also demonstrated that all cell types that bore the virus receptor were permissive for reverse transcription.
Having identified a cell line lacking the receptor, it was necessary to determine whether the receptor would be amenable to an expression cloning strategy. To this end, it was important to demonstrate that the receptor was a protein, and that transfer of genes from a receptor-bearing cell line into Chinese hamster cells would confer susceptibility to virus entry or at least Env-mediated fusion.
The case for the virus receptor being a protein was supported by its sensitivity to proteolysis. Fusion was resistant to trypsin but was sensitive to papain. This is consistent with the involvement of one or more proteins on the target cell membrane with the process of fusion but does not rule out the possibility of other non-protein components.
A panel of somatic cell hybrid Chinese hamster cells containing different complements of murine chromosomes was used to demonstrate that Chinese hamster cells could be made susceptible to virus entry. Because the chromosome complement of the hybrids was known, it also allowed the chromosome carrying the receptor to be tentatively identified. Based upon the presence of a particular chromosome in permissive hybrids, and the absence in non-permissive hybrids, the murine homologue of the MVV receptor is carried by chromosome 2 or 4. This conclusion is subject to three caveats: (i) more than one protein on different chromosomes may serve as the receptor; (ii) as in the case of HIV, there may be a requirement for a co-receptor on a different chromosome; and (iii) cell lines may be non-permissive because of transcriptional silencing or DNA rearrangement rather than because they lack the chromosome or gene concerned. Taken together it is apparent that the hybrid lines available cannot provide an absolutely certain location for the murine homologue of the MVV receptor. Chromosome 6, for example, is excluded by only a single negative data point and could still be implicated if there was a requirement for a co-receptor, as could the X chromosome. The experiments presented here do not allow us to address whether there is a requirement for a co-receptor for MVV entry. However, if one does exist, it/they too must be very widely distributed across species.
Despite the caveats listed above, the hybrid data excludes several important potential candidates for the MVV receptor (Table 2). Ram-1 is the receptor for amphotropic murine leukaemia viruses (Miller et al., 1994
). It has a widespread distribution across different species that very closely matches that observed for MVV. CHO cells contain a homologue of Ram-1, but it is non-functional because it is blocked by a specific inhibitor secreted by CHO cells (Miller & Miller, 1992
). Expression of this inhibitor is prevented by treatment with tunicamycin, with concomitant restoration of susceptibility to infection for CHO cells by amphotropic viruses. Tunicamycin has no effect on MVV entry into CHO cells (data not shown). GLVR-1 a close relative of Ram-1 is located on chromosome 2 (Kaelbling et al., 1991
). Initially this protein was excluded because cell hybrid CV4/2/1/1 was believed to lack chromosome 2. However, FISH analysis using mouse chro- mosome-specific paints (Rabbitts et al., 1995
) revealed that chromosome 2 was present in this line (data not shown). Chromosome 2 is present in only a small proportion of MOV11/3/1/2 cells. Since this line was consistently the most permissive for MVV entry (see Fig. 4
) it would seem unlikely that chromosome 2 carries the MVV receptor, but it cannot definitely be excluded at this stage. Thus, GLVR-1 is still a potential candidate for the MVV receptor although, it too would be expected to respond to tunicamycin treatment in the same way as Ram-1. MHC-II has been proposed previously as a candidate for the MVV receptor (Dalziel et al., 1991
). While it is possible that this protein could be involved in entry into macrophages, it is extremely unlikely that it is responsible for entry into the cell lines used in this study. This is reinforced by the cell hybrid data that excludes this protein based on chromosome location. The majority of chemokine receptors that are currently mapped in the mouse lie on chromosome 1 (CXCR) and chromosome 9 (CCR) and are excluded by this analysis. The chemokine receptors belong to a very large super-family of 7-transmembrane domain proteins. It is possible that the receptor for MVV is a member of this group of proteins, but currently there is no evidence supporting this and no a priori reason why this should be so.
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Acknowledgments |
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References |
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---|
Berger, E. A., Murphy, P. M. & Farber, J. M.(1999). Chemokine receptors as HIV-1 coreceptors: roles in viral entry, tropism, and disease.Annual Review of Immunology17, 657-700.[Medline]
Brown, K. E., Anderson, S. M. & Young, N. S.(1993). Erythrocyte P antigen: cellular receptor for B19 parvovirus.Science262, 114-117.[Medline]
Bruett, L., Barber, S. A. & Clements, J. E.(2000). Characterization of a membrane-associated protein implicated in visna virus binding and infection.Virology271, 132-141.[Medline]
Cahan, L. D., Singh, R. & Paulson, J. C.(1983). Sialyloligosaccharide receptors of binding variants of polyoma virus. Virology130, 281-289.[Medline]
Clements, J. E., Gabuzda, D. H. & Gdovin, S. L.(1990). Cell type specific and viral regulation of visna virus gene expression.Virus Research16, 175-183.[Medline]
Crane, S. E., Buzy, J. & Clements, J. E.(1991). Identification of cell-membrane proteins that bind visna virus.Journal of Virology65, 6137-6143.[Medline]
Da Silva Teixeira, M. F., Lambert, V., Mselli-Lakahl, L., Chettab, A., Chebloune, Y. & Mornex, J. F.(1997). Immortalization of caprine fibroblasts permissive for replication of small ruminant lentiviruses.American Journal of Veterinary Research 58, 579-584.[Medline]
Dalgleish, A. G., Beverley, P. C., Clapham, P. R., Crawford, D. H., Greaves, M. F. & Weiss, R. A.(1984). The CD4 (T4) antigen is an essential component of the receptor for the AIDS retrovirus.Nature312, 763-767.[Medline]
Dalziel, R. G., Hopkins, J., Watt, N. J., Dutia, B. M., Clarke, H. A. K. & McConnell, I.(1991). Identification of a putative cellular receptor for the lentivirus visna virus.Journal of General Virology72, 1905-1911.[Abstract]
Deng, H., Liu, R., Ellmeier, W., Choe, S., Unutmaz, D., Burkhart, M., Di Marzio, P., Marmon, S., Sutton, R. E., Hill, C. M., Davis, C. B., Peiper, S. C., Schall, T. J., Littman, D. R. & Landau, N. R.(1996). Identification of a major co-receptor for primary isolates of HIV-1.Nature381, 661-666.[Medline]
Deng, H. K., Unutmaz, D., KewalRamani, V. N. & Littman, D. R.(1997). Expression cloning of new receptors used by simian and human immunodeficiency viruses.Nature388, 296-300.[Medline]
Dittmar, M. T., McKnight, A., Simmons, G., Clapham, P. & Weiss, R. A.(1997). HIV-1 tropism and co-receptor use.Nature385, 495-496.[Medline]
DuBridge, R. B., Tang, P., Hsia, H. C., Leong, P. M., Miller, J. H. & Calos, M. P.(1987). Analysis of mutation in human cells by using an EpsteinBarr virus shuttle system.Molecular and Cellular Biology7, 379-387.[Medline]
Eriksson, K., McInnes, E., Ryan, S., Tonks, P., McConnell, I. & Blacklaws, B.(1999). CD4+ T-cells are required for the establishment of maedi-visna virus infection in macrophages but not dendritic cells in vivo.Virology258, 355-364.[Medline]
Fuerst, T. R., Niles, E. G., Studier, F. W. & Moss, B.(1986). Eukaryotic transient-expression system based on recombinant vaccinia virus that synthesizes bacteriophage T7 RNA polymerase.Proceedings of the National Academy of Sciences, USA83, 8122-8126.[Abstract]
Gabuzda, D. H., Hess, J. L., Small, J. A. & Clements, J. E.(1989). Regulation of the visna virus long terminal repeat in macrophages involves cellular factors that bind sequences containing AP-1 sites.Molecular and Cellular Biology9, 2728-2733.[Medline]
Gendelman, H. E., Narayan, O., Kennedy-Stoskopf, S., Kennedy, P. G., Ghotbi, Z., Clements, J. E., Stanley, J. & Pezeshkpour, G.(1986). Tropism of sheep lentiviruses for monocytes: susceptibility to infection and virus gene expression increase during maturation of monocytes to macrophages. Journal of Virology58, 67-74.[Medline]
Gilden, D. H., Devlin, M. & Wroblewska, Z.(1981). The use of vesicular stomatitis-virus (visna virus) pseudotypes to demonstrate visna virus receptors in cells from different species.Archives of Virology67, 181-185.[Medline]
Harter, D. H., Hsu, K. C. & Rose, H. M.(1968). Multiplication of visna virus in bovine and porcine cell lines.Proceedings of the Society for Experimental Biology and Medicine129, 295-300.
Jackson, T., Ellard, F. M., Ghazaleh, R. A., Brookes, S. M., Blakemore, W. E., Corteyn, A. H., Stuart, D. I., Newman, J. W. & King, A. M.(1996). Efficient infection of cells in culture by type O foot-and-mouth disease virus requires binding to cell surface heparan sulfate.Journal of Virology70, 5282-5287.[Abstract]
Kaelbling, M., Eddy, R., Shows, T. B., Copeland, N. G., Gilbert, D. J., Jenkins, N. A., Klinger, H. P. & OHara, B.(1991). Localization of the human gene allowing infection by gibbon ape leukaemia virus to human chromosome region 2q11-q14 and to the homologous region on mouse chromosome 2.Journal of Virology65, 1743-1747.[Medline]
Klatzmann, D., Champagne, E., Chamaret, S., Gruest, J., Guetard, D., Hercend, T., Gluckman, J. C. & Montagnier, L.(1984). T-lymphocyte T4 molecule behaves as the receptor for human retrovirus LAV.Nature312, 767-768.[Medline]
Lee, W. C., McConnell, I. & Blacklaws, B. A.(1994). Cytotoxic activity against maedi-visna virus-infected macrophages.Journal of Virology68, 8331-8338.[Abstract]
Lerondelle, C., Godet, M. & Mornex, J. F.(1999). Infection of primary cultures of mammary epithelial cells by small ruminant lentiviruses. Veterinary Research30, 467-474.[Medline]
MacIntyre, E. H., Wintersgill, C. J. & Thormar, H.(1972). Morphological transformation of human astrocytes by visna virus with complete virus production.Nature New Biology237, 111-113.[Medline]
Markwell, M. A. & Paulson, J. C.(1980). Sendai virus utilizes specific sialyloligosaccharides as host cell receptor determinants.Proceedings of the National Academy of Sciences, USA77, 5693-5697.[Abstract]
Miller, D. G. & Miller, A. D.(1992). Tunicamycin treatment of CHO cells abrogates multiple blocks to retrovirus infection, one of which is due to a secreted inhibitor.Journal of Virology66, 78-84.[Abstract]
Miller, D. G., Edwards, R. H. & Miller, A. D.(1994). Cloning of the cellular receptor for amphotropic murine retroviruses reveals homology to that for gibbon ape leukemia virus.Proceedings of the National Academy of Sciences, USA91, 78-82.[Abstract]
Morais, R., Desjardins, P., Zinkewich-Peotti, K. & Richer, C. L.(1988). Chicken embryo cell line exhibits Japanese quail markers.In Vitro Cellular & Developmental Biology24, 1061-1063.[Medline]
Nussbaum, O., Broder, C. C. & Berger, E. A.(1994). Fusogenic mechanisms of enveloped-virus glycoproteins analyzed by a novel recombinant vaccinia virus-based assay quantitating cell fusion-dependent reporter gene activation.Journal of Virology68, 5411-5422.[Abstract]
Rabbitts, P., Impey, H., Heppell-Parton, A., Langford, C., Tease, C., Lowe, N., Bailey, D., Ferguson-Smith, M. & Carter, N.(1995). Chromosome specific paints from a high resolution flow karyotype of the mouse.Nature Genetics9, 369-375.[Medline]
Sargan, D. R., Bennet, I. D., Cousens, C., Roy, D. J., Blacklaws, B. A., Dalziel, R. G., Watt, N. J. & McConnell, I.(1991). Nucleotide sequence of EV1, a British isolate of maedi-visna virus.Journal of General Virology72, 1893-1903.[Abstract]
Sattentau, Q. J., Clapham, P. R., Weiss, R. A., Beverley, P. C., Montagnier, L., Alhalabi, M. F., Gluckmann, J. C. & Klatzmann, D.(1988). The human and simian immunodeficiency viruses HIV-1, HIV-2 and SIV interact with similar epitopes on their cellular receptor, the CD4 molecule.AIDS2, 101-105.[Medline]
Schlegel, R., Tralka, T. S., Willingham, M. C. & Pastan, I.(1983). Inhibition of VSV binding and infectivity by phosphatidylserine: is phosphatidylserine a VSV-binding site?Cell32, 639-646.[Medline]
Small, J. A., Bieberich, C., Ghotbi, Z., Hess, J., Scangos, G. A. & Clements, J. E.(1989). The visna virus long terminal repeat directs expression of a reporter gene in activated macrophages, lymphocytes, and the central nervous systems of transgenic mice.Journal of Virology63, 1891-1896.[Medline]
Tsvetkova, I. V., Lipkind, M. A., Zakstslskaia, L., Iusipova, N. A. & Rozenfeld, E. L. (1967). Neuraminic acid as a cell receptor for virus of influenza. Biokhimiia 32, 994999 (in Russian).[Medline]
Weiss, R. A. & Tailor, C. S.(1995). Retrovirus receptors. Cell82, 531-533.[Medline]
Willett, B. J., Picard, L., Hosie, M. J., Turner, J. D., Adema, K. & Clapham, P. R.(1997). Shared usage of the chemokine receptor CXCR4 by the feline and human immunodeficiency viruses.Journal of Virology71, 6407-6415.[Abstract]
Williamson, P., Holt, S., Townsend, S. & Boyd, Y.(1995). A somatic cell hybrid panel for mouse gene mapping characterized by PCR and FISH.Mammalian Genome6, 429-432.[Medline]
Received 15 May 2000;
accepted 24 August 2000.