MRC Virology Unit, Institute of Virology, Church Street, G11 5JR, Glasgow, UK1
Division of Virology, University of Glasgow, Institute of Virology, Church Street, G11 5JR, Glasgow, UK2
Author for correspondence: Richard Sugrue. Fax +44 141 337 2236. e-mail r.sugrue{at}vir.gla.ac.uk
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Antibodies.
The F protein monoclonal antibody, MAb19, was provided by Geraldine Taylor. The anti-RSV monoclonal antibody (NCL-RSV3) was purchased from Novocastra Laboratories.
Radiolabelling.
Cell monolayers were infected with RSV at a multiplicity of 2 and following adsorption at 33 °C for 2 h, were incubated at 33 °C for 18 h. At 20 h post-infection, the medium was removed and the cells were incubated in DMEM minus methionine for 1 h prior to radiolabelling with 100 µCi/ml [35S]methionine. In pulsechase experiments, the chase was carried out using DMEM supplemented with 1 mM methionine.
Immunoprecipitation.
Mock-infected or RSV-infected monolayers (60 mm) were extracted at 4 °C for 10 min with 500 µl lysis buffer (1% NP-40, 0·1% SDS, 150 mM NaCl, 1 mM EDTA, 2 mM PMSF, 20 mM TrisHCl, pH 7·5) and clarified by centrifugation. Clarified lysate (100 µl) and 1 µl of MAb19 were added to 600 µl binding buffer (0·5% NP-40, 150 mM NaCl, 1 mM EDTA, 0·25% BSA, 20 mM TrisHCl, pH 8) and incubated overnight at 4 °C. The immune complexes were isolated by adding protein ASepharose for 2 h at 4 °C. The protein ASepharose was washed six times with high salt buffer (1% Triton X-100, 650 mM NaCl, 1 mM EDTA, 10 mM sodium phosphate, pH 7·0) and once with low salt buffer (1% Triton X-100, 150 mM NaCl, 1 mM EDTA, 10 mM sodium phosphate, pH 7·0). The protein ASepharose-bound immune complexes were resuspended in 40 µl boiling mix (1% SDS, 15% glycerol, 1% -mercaptoethanol, 60 mM sodium phosphate, pH 6·8) and heated at 100 °C for 2 min. After removing the protein ASepharose by centrifugation, the samples were analysed by SDSPAGE. The labelled protein bands were detected using a Bio-Rad personal Fx phosphorimager and quantified by density volume analysis using Quantity one software (Bio-Rad, ver. 4). Apparent molecular masses were estimated using 14C-methylated proteins (Amersham) in the molecular mass range 14·3220 kDa: lysozyme (14·3 kDa), soyabean trypsin inhibitor (21·5 kDa), carbonic anhydrase (30 kDa), ovalbumin (46 kDa), serum albumin (66 kDa), phosphorylase b (97·5 kDa) and myosin ( 220 kDa).
Endoglycosidase digestion.
Immunoprecipitates were incubated at 100 °C for 10 min in 0·5% SDS, 1% mercaptoethanol. The samples were then made up to a final concentration of either 50 mM sodium phosphate, 1% NP-40, pH 7·5 or 50 mM sodium citrate, pH 5·5 and incubated at 37 °C for 14 h with 1000 u PNGase F (NEB) or 1000 u Endo H (NEB) respectively.
Dec-RVKR-cmk.
The furin inhibitor decanoyl-Arg-Val-Lys-Arg-chloromethyl ketone (dec-RVKR-cmk) was purchased from Bachem (UK) Ltd. A 10 mM stock solution was prepared in DMSO and diluted into tissue culture medium to give the required final concentration.
Cell surface biotinylation.
The cell surface biotinylation procedure has been described previously (Altin & Pagler, 1995 ). Briefly, cell monolayers were washed three times with ice-cold PBS, pH 8·0, and treated with 500 µg/ml sulfo-NHS-LC-biotin (Pierce) in PBS, pH 8·0, for 30 min at 4 °C. The monolayers were then washed three times with ice-cold PBS, 20 mM glycine, pH 8·0, to remove the unreacted biotin. Cell extracts were prepared using lysis buffer supplemented with 20 mM glycine and the F protein was isolated by immunoprecipitation. The immunoprecipitates were separated by SDSPAGE and transferred by Western blotting onto a PVDF membrane. After transfer, the membranes were washed with PBS and blocked for 60 min in PBS containing 1% Marvel dried milk powder and 0·05% Tween 20. The membrane was washed four times (5 min per wash) in PBS containing 0·05% Tween 20 and then incubated with streptavidinHRP (1/1000 dilution, Amersham) for 60 min. The membrane was washed using PBS and the protein bands were detected using the ECL protein detection system (Amersham).
Immunofluorescence.
Cells were seeded onto 13 mm glass coverslips and incubated overnight at 37 °C. Following infection with RSV for 18 h, the cells were fixed using 3% paraformaldehyde for 30 min at 4 °C. The fixative was removed and the cells were washed five times with PBS+1 mM glycine and once with PBS. The cells were incubated at 25 °C for 1 h with MAb19 (1/700 dilution) after which they were washed and incubated for a further 1 h with anti-mouse IgG (whole molecule) FITC conjugate (1/100 dilution). The stained cells were mounted on slides using Citifluor and visualized using a Zeiss Axioplan 2 confocal microscope. The images were processed using LSM 510 v2.01 software.
Quantification of the surface-expressed F protein was performed by FACS analysis. Aliquots of Vero cells (1x105 cells) were resuspended in PBS+0·5 mM EDTA and incubated with MAb19 (1/500 dilution) at 4 °C in the dark for 25 min. The cells were washed (using PBS+0·1% sodium azide) and incubated with anti-mouse IgG (whole molecule) FITC conjugate (1/100 dilution prepared in PBS+0·1% sodium azide) at 4 °C for a further 25 min. The cells were then washed as above and resuspended in 1% formaldehyde in PBS. The surface fluorescence was measured using a Becton Dickinson FACSCalibur and the data were analysed using Cellquest software (Becton Dickinson) with appropriate gating parameters.
Scanning electron microscopy (SEM).
Cells were seeded onto 10 mm glass coverslips and incubated overnight at 37 °C. Following infection with RSV for 20 h, the samples were processed using a previously published procedure (Parry et al., 1979 ). Briefly, the cells were fixed in 2·5% glutaraldehyde for 30 min at 4 °C after which they were washed five times with PBS+1 mM glycine and once with PBS. The cells were post-fixed in 1% osmium tetroxide for 1 h and washed with PBS. The fixed cells were dehydrated by transfer through a gradient of 30, 50, 70, 90 and 100% ethanol. The samples were finally placed in 100% acetone and critical point dried in a critical point drying apparatus (Polaron CPD). The dried coverslips were mounted on aluminium stubs and gold coated in an Edwards sputter coater device prior to examination in a JEOL T300 scanning electron microscope.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
RSV fusion protein is processed by furin
The LoVo cell line is derived from a human colon adenocarcinoma which has a specific genetic defect resulting in the expression of an inactive form of furin (Takahashi et al., 1993 ). It is recognized that furin is only one of a family of proteases, which include PC2, PC1/PC3, PC4, PC5/6, PC7 and PACE4, and which constitute the mammalian subtilisin-like pro-protein convertases (reviewed in Nakayama, 1997
). Although LoVo cells synthesize an inactive form of furin, they do express a variety of other pro-protein convertases (Bolt & Pedersen, 1998
). Processing of the F protein in RSV-infected Vero and LoVo cells was compared to assess the importance of furin in the activation of the fusion protein.
Vero and LoVo cell monolayers were infected with RSV, pulsechase labelled with [35S]methionine and the F protein detected by RIP and SDSPAGE (Fig. 2A). In this analysis, the amount of labelled F protein that was immunoprecipitated from each cell line was equivalent, but the pattern of the labelled F protein differed. As expected, in RSV-infected Vero cells, both F0 and F2ssF1 were detected after 60 min pulse-label but only the latter was detected following the 60 min chase, due to cleavage of F0. In addition, a 60 kDa protein was detected in RSV-infected Vero cells during the pulse-label which was not present in either mock-infected cells or in RSV-infected Vero cells following the long chase. In addition to F1 and F2, the 60 kDa protein could be efficiently labelled using [3H]mannose, indicating that it is modified by glycosylation (R. J. Sugrue & C. Brown, unpublished observations). It can be speculated that this 60 kDa glycoprotein (gp60) may represent a specific intermediate in the processing pathway of the F protein.
|
|
The above data suggest that furin is the most important host cell protease involved in activating the RSV fusion protein. To strengthen this conclusion, the recombinant vaccinia virus vacc:hfur, which expresses the human form of furin, was used to restore furin protease activity in LoVo cells. The use of vacc:hfur to assay the furin dependence of viral glycoproteins has been described previously (Gotoh et al., 1992 ). RSV-infected LoVo cells were infected with either vacc:hfur or a wild-type parental vaccinia virus strain (WR) and the F protein detected by RIP and SDSPAGE (Fig. 2C
). Cleavage of the F protein was observed in LoVo cells co-infected with both RSV and vacc:hfur, as evidenced by the appearance of the F1 subunit and the absence of F0* (Fig. 2C
, lane 3). In contrast, proteolytic processing of the F protein was not observed in RSV-infected LoVo cells co-infected with WR (Fig. 2C
, lane 4).
Temporal aspects of F0* formation in RSV-infected LoVo cells were examined using pulsechase labelling (Fig. 3). This experiment showed that in LoVo cells, the F protein is initially expressed as F0 but that this is subsequently converted into F0*, approximately 3040 min post synthesis (Fig. 3A
). During the chase, the F protein was assayed for its sensitivity to deglycosylation by treatment with Endo H and PNGase F (Fig. 3B
). The data showed that the onset of Endo H resistance occurred at approximately 3040 min after the initiation of the chase and coincided with the appearance of F0*.
|
|
In a separate experiment, temporal aspects of F protein processing in the presence of dec-RVKR-cmk were investigated. In the presence of 80 µM dec-RVKR-cmk, F0 was initially expressed as F0EHs which was subsequently processed into F0EHr approximately 3040 min after the initiation of the chase (Fig. 5A). This was similar to the time that F2ssF1 is detected, following the cleavage of F protein, in mock-treated cells (Fig. 5B
). As these two events are accompanied by the onset of resistance to Endo H digestion, both F0EHr and F2ssF1 appear to be transported through the trans-Golgi at a similar rate in Vero cells.
|
Cell surface biotinylation of RSV-infected cells was performed using sulfo-NHS-LC-biotin. This N-hydroxysuccimate ester is unable to cross the plasma membrane and specifically couples biotin to cell surface proteins. RSV-infected monolayers were treated with sulfo-NHS-LC-biotin and the surface-labelled F protein transferred by Western blotting onto PVDF membranes, which were subsequently probed using streptavidinHRP (Fig. 6). In the absence of inhibitor F2ssF1 was the predominant F protein species detected on the surface of virus-infected Vero cells (Fig. 6A
, lane 2). In contrast, in the presence of inhibitor, the vast majority of the F protein population detected on the surface of infected cells corresponded in size to F0EHr. Similarly, in RSV-infected LoVo cells, a significant level of biotinylated F0* was detected (Fig. 6B
, lane 2) on the cell surface. In this assay, LoVo cells exhibited a 2- to 3-fold reduction in the level of biotinylated F protein when compared with Vero cells. This presumably reflects differences in the surface expression of non-cleaved F protein in the two cell lines.
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Our observations differ slightly from those presented previously (Bolt et al., 2000 ) which suggested that the cleavage of F0 is a requirement for its surface expression. However, Bolt et al. reported erroneous processing of F0 when cleavage of the fusion protein was inhibited, presumably a consequence of cellular protease activity. We did not observe any such aberrant processing of the non-cleaved fusion protein in our experiments, which may account for differences in the surface detection of this protein.
In this study, we observed the presence of a 60 kDa protein whose detection was dependent upon cleavage of the F protein. The identity of this transient species is at present unclear. However, examination of the F protein amino acid sequence from a variety of different virus isolates reveals an additional potential furin cleavage site (Collins et al., 1984 ; Baybutt & Pringle, 1987
; Lopez et al., 1988
; Lerch et al., 1991
). In addition to KKRKRR, which is located between the F2 and F1 sequences, a second sequence, RARR, is located within the C-terminal half of the F2 amino acid sequence (aa 106109) of the RSV A2 strain. This conforms to the general consensus sequence for a furin recognition site (RxK/RR, where x is any amino acid). It is interesting to note that this second consensus sequence is conserved among human A and B serotypes, in addition to isolates of bovine RSV. In addition, the sequence RARR is present in the putative cleavage site of several other viral glycoproteins (Wunsch et al., 1983
; Keller et al., 1986
; Perez & Hunter, 1987
). However, at present it is not clear if this additional sequence in the RSV F protein is cleaved by furin and this is currently under investigation.
Two different forms of the non-cleaved F protein have been identified in this study, distinguished by differences in their electrophoretic migration during SDSPAGE and susceptibility to deglycosylation by Endo H. These were designated F0EHs and F0EHr, and they are both intermediates in the processing pathway that leads to the formation of F2ssF1. F0EHs, which is located in the endoplasmic reticulum, is subsequently modified by the addition of complex carbohydrates in the trans-Golgi to form F0EHr. The latter is transiently detected in pulsechase experiments and its appearance coincides with cleavage of the fusion protein. However, when cleavage of the fusion protein is inhibited, F0EHr can assemble into an oligomeric structure that is transported to the cell surface. It is not clear to what extent cell surface expression of the immature F protein occurs during natural infection. Although the immature F protein is not present in virions, it has been detected on the surface of virus-infected cells by some workers (Gruber & Levine, 1985 ), while others have noted its absence (Collins & Mottet, 1991
). We were able to detect a small proportion of a non-cleaved F protein species, identical in size to F0EHr, on the surface of RSV-infected Vero cells in the absence of inhibitor (Fig. 6A
, lane 2). A recent report has suggested that the immune response to the immature form of the F protein during natural infection may provide a mechanism by which the virus can evade the hosts immune system (Sakurai et al., 1999
). It was suggested that this may arise from the liberation of immature F protein as a result of cell lysis. However, our data provide an alternative mechanism by which the immature F protein can interact with the hosts immune system, namely its presentation on the surface of virus-infected cells.
The data presented in this report provide clear evidence that furin is the major pro-protein convertase that is involved in the post-translational cleavage of the F protein during virus maturation. However, our data suggest that an additional protease activity, distinct from furin, is able to process the F protein. This unidentified protease activity appears to be less efficient than furin and its functional significance is unclear. An increasing body of information suggests that other members of the pro-protein convertases, in addition to furin, can process viral glycoproteins. It was previously demonstrated that PC1/3 was able to partially process the Newcastle disease virus fusion protein in the absence of furin, but with a vastly reduced efficiency (Gotoh et al., 1992 ). Recent studies have shown that PC5/6 and PC7, in addition to furin, can process gp160 into gp120/gp41 (Ohnishi et al., 1994
; Decroly et al., 1996
; Hallenberger et al., 1997
). In many cases, the cleavability of a fusion protein is an important factor both in viral transmission and tissue tropism (reviewed in Nagai, 1993
; Klenk & Garten, 1994
). In the case of RSV, the infection is usually restricted to the superficial layers of the respiratory tract, resulting in the familiar acute infection. However, under certain conditions, e.g. immunosuppressive therapy, it has been reported that the virus can infect tissues other that those of the respiratory epithelium (Fishaut et al., 1980
; Johnson et al., 1982
; Padman et al., 1985
; Milner et al., 1985
). It is likely that cleavage of the fusion protein by furin, which is distributed ubiquitously throughout different tissues, is a major factor in the disease progression.
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Anderson, K., Stott, E. J. & Wertz, G. W. (1992). Intracellular processing of the human respiratory syncytial virus fusion glycoprotein: amino acid substitutions affecting folding, transport and cleavage. Journal of General Virology 73, 1177-1188.[Abstract]
Baybutt, H. N. & Pringle, C. R. (1987). Molecular cloning and sequencing of the F and 22K membrane protein genes of the RSS-2 strain of respiratory syncytial virus. Journal of General Virology 68, 2789-2796.[Abstract]
Bolt, G. & Pedersen, I. R. (1998). The role of subtilisin-like proprotein convertases for cleavage of the measles virus fusion glycoprotein in different cell types. Virology 252, 387-398.[Medline]
Bolt, G., Pedersen, L. O. & Birkeslund, H. H. (2000). Cleavage of the respiratory syncytial virus fusion protein is required for its surface expression: role of furin. Virus Research 68, 25-33.[Medline]
Cannon, J. M. (1987). Microplaque immunoperoxidase detection of infectious respiratory syncytial virus in the lungs of infected mice. Journal of Virological Methods 16, 293-301.[Medline]
Collins, P. L. & Mottet, G. (1991). Post-translational processing and oligomerization of the fusion glycoprotein of human respiratory syncytial virus. Journal of General Virology 72, 3095-3101.[Abstract]
Collins, P. L., Huang, Y. T. & Wertz, G. W. (1984). Nucleotide sequence of the gene encoding the fusion (F) glycoprotein of human respiratory syncytial virus. Proceedings of the National Academy of Sciences, USA 81, 7683-7687.[Abstract]
Decroly, E., Wouters, S., Di Bello, C., Lazure, C., Ruysschaert, J.-M. & Seidah, N. G. (1996). Identification of the paired basic convertases implicated in HIV gp160 processing based on in vitro assays and expression in CD4+ cell lines. Journal of Biological Chemistry 271, 30442-30450.
Faulkner, G. P., Shirodaria, P. V., Follett, E. A. & Pringle, C. R. (1976). Respiratory syncytial virus ts mutants and nuclear immunofluorescence. Journal of Virology 20, 487-500.[Medline]
Fishaut, M., Tubergen, D. & McIntosh, K. (1980). Cellular response to respiratory viruses with particular reference to children with disorders of cell-mediated immunity. Journal of Pediatrics 96, 179-186.[Medline]
Gotoh, B. Y., Ohnishi, O., Inocencio, N. M., Esaki, E., Nakayama, K., Barr, P. J., Thomas, G. & Nagai, Y. (1992). Mammalian subtilisin-related proteinases in cleavage activation of the paramyxovirus fusion glycoprotein: superiority of furin/PACE to PC2 or PC1/PC3. Journal of Virology 66, 6391-6397.[Abstract]
Gruber, C. & Levine, S. (1985). Respiratory syncytial virus polypeptides. V. The kinetics of glycoprotein synthesis. Journal of General Virology 66, 1241-1247.[Abstract]
Guo, H. G., Veronese, F. M., Tschachler, E., Pal, R., Kalyanaraman, V. S., Gallo, R. C. & Reitz, M. S. (1990). Characterization of an HIV-1 point mutant blocked in envelope glycoprotein cleavage. Virology 174, 217-224.[Medline]
Hallenberger, S., Moulard, M., Sordel, M., Klenk, H.-D. & Garten, W. (1997). The role of eukaryotic subtilisin-like endoproteases for the activation of human immunodeficiency virus glycoproteins in natural host cells. Journal of Virology 71, 1036-1045.[Abstract]
Johnson, R. A., Prince, G. A., Suffin, S. C., Horswood, R. L. & Chanock, R. M. (1982). Respiratory syncytial virus infection in cyclophosphamide-treated cotton rats. Infection and Immunity 37, 369-373.[Medline]
Keller, P. M., Davidson, A. J., Lowe, R. S., Bennett, C. D. & Ellis, R. W. (1986). Identification and structure of the gene encoding gpII, a major glycoprotein of varicella-zoster virus. Virology 152, 181-191.[Medline]
Klenk, H.-D. & Garten, W. (1994). Activation cleavage of viral spike proteins by host proteases. In Cellular Receptors for Animal Viruses , pp. 241-280. Edited by E. Wimmer. Cold Spring Harbor, NY:Cold Spring Harbor Laboratory.
Lerch, R. A., Anderson, K., Amann, V. L. & Wertz, G. W. (1991). Nucleotide sequence analysis of the bovine respiratory syncytial virus fusion protein mRNA and expression from a recombinant vaccinia virus. Virology 181, 118-131.[Medline]
Lopez, J. A., Villanueva, N., Melero, J. A. & Portela, A. (1988). Nucleotide sequence of the fusion and phosphoprotein genes of human respiratory syncytial (RS) virus long strain: evidence of sub-type genetic heterogeneity. Virus Research 10, 249-262.[Medline]
Milner, M. E., de la Monte, S. M. & Hutchins, G. M. (1985). Fatal respiratory syncytial virus infection in severe combined immunodeficiency syndrome. American Journal of Disease in Children 135, 1111-1114.
Moulard, M., Hallenberger, S., Garten, W. & Klenk, H.-D. (1999). Processing and routage of HIV glycoproteins by furin to the cell surface. Virus Research 60, 55-65.[Medline]
Nagai, Y. (1993). Protease-dependent virus tropism and pathogenicity. Trends in Microbiology 1, 81-87.[Medline]
Nakayama, K. (1997). Furin: a mammalian subtilisin/Kex2p-like endoprotease involved in processing of a wide variety of precursor proteins. Biochemical Journal 327, 625-635.[Medline]
Ohnishi, Y., Shioda, T., Nakayama, K., Iwata, S., Gotoh, B., Hamaguchi, M. & Nagai, Y. (1994). A furin-defective cell line is able to process correctly the gp160 of human immunodeficiency virus type 1. Journal of Virology 68, 4075-4079.[Abstract]
Ortmann, D., Ohuchi, M., Angliker, H., Shaw, E., Garten, W. & Klenk, H.-D. (1994). Proteolytic cleavage of wild type and mutants of the F protein of human parainfluenza virus type 3 by two subtilisin-like endoprotease, furin and Kex2. Journal of Virology 68, 2772-2776.[Abstract]
Padman, R., Bye, M. R., Schidlow, D. V. & Zaeri, N. (1985). Severe RSV bronchiolitis in an immunocompromised child. Clinical Pediatrics 24, 719-721.[Medline]
Parry, J. E., Shirodaria, P. V. & Pringle, C. R. (1979). Pneumoviruses: the cell surface of lytically and persistently infected cells. Journal of General Virology 44, 479-491.[Abstract]
Pastey, M. K. & Samal, S. K. (1997). Role of individual N-linked oligosaccharide chains and different regions of bovine respiratory syncytial virus fusion protein in cell surface transport. Archives of Virology 142, 2309-2320.[Medline]
Perez, L. G. & Hunter, E. (1987). Mutations within the proteolytic cleavage site of the Rous sarcoma virus glycoprotein that block processing to gp85 and gp37. Journal of Virology 61, 1609-1614.[Medline]
Roberts, R. R., Compans, R. W. & Wertz, G. A. (1995). Respiratory syncytial virus matures at the apical surfaces of polarized epithelial cells. Journal of Virology 69, 2667-2673.[Abstract]
Sakurai, H., Williamson, R. A., Crowe, J. E., Beeler, J. A., Poignard, P., Bastidas, R. B., Chanock, R. M. & Burton, D. R. (1999). Human antibody responses to mature and immature forms of viral envelope in respiratory syncytial virus infection: significance for subunit vaccines. Journal of Virology 73, 2956-2962.
Scheid, A. & Choppin, P. W. (1977). Two disulfide-linked polypeptide chains constitute the active F protein of paramyxoviruses. Virology 80, 54-66.[Medline]
Stadler, K., Allison, S. L., Schalich, J. & Heinz, F. X. (1997). Proteolytic activation of tick-borne encephalitis virus by furin. Journal of Virology 71, 8475-8481.[Abstract]
Takahashi, S., Kasai, K., Hatsuzawa, K., Kitamura, N., Misumi, Y., Ikehara, Y., Murakami, K. & Nakayama, K. (1993). A mutation of furin causes the lack of precursor-processing activity in human colon carcinoma LoVo cells. Biochemical and Biophysical Research Communications 195, 1019-1026.[Medline]
Taylor, G., Stott, E. J., Furze, J., Ford, J. & Sopp, P. (1992). Protective epitopes on the fusion protein of respiratory syncytial virus recognized by murine and bovine monoclonal antibodies. Journal of General Virology 73, 2217-2223.[Abstract]
Vey, M., Schafer, W., Reis, B., Ohuchi, R., Britt, W., Garten, W., Klenk, H.-D. & Radsak, K. (1995). Proteolytic processing of human cytomegalovirus glycoprotein B (gpUL55) is mediated by the human endoprotease furin. Virology 206, 746-749.[Medline]
Volchkov, V. E., Feldmann, H., Volchkova, V. A. & Klenk, H.-D. (1998). Processing of the Ebola virus glycoprotein by the proprotein convertase furin. Proceedings of the National Academy of Sciences, USA 95, 5762-5767.
Wunsch, M., Schultz, A. S., Koch, W., Friedrich, R. & Hunsmann, G. (1983). Sequence analysis of GardnerArnstein feline leukemia virus envelope gene reveals common structural properties of mammalian retroviral envelope gene. EMBO Journal 2, 2239-2246.[Medline]
Received 28 November 2000;
accepted 16 February 2001.