Institute of Virology, University of Vienna, Kinderspitalgasse 15, A-1095 Vienna, Austria
Correspondence
Christian Mandl
christian.mandl{at}univie.ac.at
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Published ahead of print on 1 October 2002 as DOI 10.1099/vir.0.18723-0.
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Activation cleavages are often carried out by the cellular protease furin, an enzyme that is concentrated in the trans-Golgi network (TGN) but also cycles between endosomes and the plasma membrane (Molloy et al., 1999; Plaimauer et al., 2001
). In naturally occurring substrates, including notonly viral surface proteins but also a large number of cellular proproteins, furin cleaves after the conserved sequence motif RXK/RR (X can be any amino acid), but mutagenesis studies have revealed that the minimum consensus sequence recognized by this enzyme is RXXR (Molloy et al., 1992
; Nakayama, 1997
).
Studies on various virus systems have indicated that the endoproteolytic activation of viral surface proteins (e.g. from alphaviruses or paramyxoviruses) is frequently an essential prerequisite for obtaining infectious virions (Heidner et al., 1994; Li et al., 1998
; Lobigs & Garoff, 1990
; Maisner et al., 2000;
Salminen et al., 1992
), whereas in other cases it appears that infectivity is enhanced by, but does not fully depend on, the cleavage of the precursor protein (Kopp et al., 1994
; Wool-Lewis & Bates, 1999
). In this study the importance of furin-mediated cleavage activation of a flavivirus, tick-borne encephalitis (TBE) virus, for the infectivity of this virus in cell culture is assessed.
The genus Flavivirus, family Flaviviridae, includes, in addition to TBE virus, several other important human pathogens, such as yellow fever virus, Japanese encephalitis virus, West Nile virus and dengue virus. Flavivirus particles consist of a nucleocapsid containing the positive-stranded, non-segmented RNA genome of approximately 11 kb length surrounded by a lipid envelope in which the two surface proteins E (approx. 54 kDa) and M (approx. 8 kDa) are anchored (Lindenbach & Rice, 2001). Protein E mediates both virus attachment and fusion. Structural studies, including the solution of the three-dimensional structure of the TBE virus protein E by X-ray crystallography, have shown that in mature, infectious particles protein E forms head-to-tail dimers, which are orientated parallel to the viral surface in a regular lattice with icosahedral symmetry, thus forming a relatively smooth outer surface of the virus (Kuhn et al., 2002
; Rey et al., 1995
). Incubation of such mature particles at mildly acidic pH as is naturally encountered by the virus in the endosome of the host cell in the course of the virus entry process triggers substantial conformational changes in protein E, including its rearrangement from dimers into trimers (Allison et al., 1995
). In the course of this process, an internal fusion peptide becomes exposed and interacts with cellular membranes, initiating the process of membrane fusion, which is necessary for the release of the nucleocapsid into the host cell cytoplasm (Allison et al., 2001
; Stiasny et al., 2002
).
Flavivirus particles are synthesized intracellularly in an immature form in which protein E forms heterodimeric complexes with protein prM, the approximately 27 kDa precursor of the small protein M (prM) (Chambers et al., 1990; Lindenbach & Rice, 2001
). Cleavage of protein prM is apparently mediated by furin in the TGN shortly before the release of virus particles from the cell and it has been demonstrated that this cleavage activates the membrane fusion capacity of protein E (Stadler et al., 1997
). Immature particles have been purified directly from infected cells (Wengler & Wengler, 1989
) or have been produced in secreted form by growing virus in furin-deficient LoVo cells, by preventing cleavage by means of specific furin inhibitors (Stadler et al., 1997
) or by raising the pH in the TGN by the addition of acidotropic reagents such as ammonium chloride or bafilomycin A1 (Allison et al., 1995
; Guirakhoo et al., 1991
, 1992
; Heinz et al., 1994
; Randolph & Stollar, 1990
; Randolph et al., 1990
). Immature particles prepared in any of these ways were consistently found to be resistant to acidic pH and did not undergo the structural and oligomeric changes in protein E that are required for fusion activity, suggesting that the presence of prM might physically block these changes. They also did not exhibit any haemagglutination activity, which in the case of flaviviruses also depends on low pH-induced conformational changes. Nevertheless, the infectivity of these preparations was not completely abolished and specific infectivities were found to be reduced only between 20- and 50-fold compared to mature virions (Guirakhoo et al., 1992
; Heinz et al., 1994
; Randolph et al., 1990
; Stadler et al., 1997
). Furthermore, some flaviviruses, especially dengue virus, are released from certain cells with a high proportion of uncleaved prM, but are still infectious (He et al., 1995
; Iacono-Connors et al., 1996
; Murray et al., 1993
; Randolph & Stollar, 1990
). These observations raised the question of whether cleavage of protein prM was absolutely essential for infectivity.
In this study, we took a mutagenesis approach to address this question. We demonstrate that a mutant of TBE virus carrying a single amino acid deletion within its prM furin cleavage site (RTRR was changed into RTR) produces intact immature virions that are completely non-infectious for BHK-21 cells. Infectivity of these mutant particles, however, could be restored by the addition of exogenous trypsin, which is apparently able to cleave at the mutated site within protein prM. Our results indicate that cleavage of prM is indeed essential for infectivity of TBE virus in cell culture.
![]() |
Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cloning, sequencing and RNA transcription.
The desired mutations were introduced into the genome of TBE virus strain Neudoerfl using the infectious cDNA clone system described in detail elsewhere (Mandl et al., 1997). The mutations were first introduced into plasmid pTNd/5', which carries cDNA corresponding to the 5'-terminal one-third (approximately) of the TBE virus genome. Using the primers listed in Table 1
, two PCR fragments were prepared, one of which carried the desired deletion of codon 88 of protein prM (coding for the amino acid arginine). In addition, nucleotide changes were introduced that did not cause alterations at the amino acid level but created a unique recognition sequence for the restriction endonuclease XbaI. This newly created restriction site was then used to ligate the two PCR fragments together and the combined fragment was swapped for the corresponding wild-type sequence of plasmid pTNd/5' by taking advantage of restriction sites for the enzymes MluI and AgeI at positions 208 and 960 of the TBE virus genomic sequence, respectively, which are unique in this plasmid. Subsequently, the mutated sequence was introduced into the full-length cDNA clone pTNd/c by swapping a fragment obtained by cutting with the enzymes SalI/SnaBI, which have unique recognition sequences in this plasmid upstream of the TBE 5' end and at position 1883 of the TBE genome sequence, respectively (Mandl et al., 1997
). The resulting full-length mutant plasmid, designated pTNd/prM(
R88), was amplified in Escherichia coli strain HB101 and purified using commercially available systems (Qiagen).
|
Full-length RNA was transcribed from the wild-type plasmid pTNd/c or the mutant plasmid pTNd/prM(R88) using T7 RNA polymerase (Ambion), as reported previously (Mandl et al., 1997
).
Cell cultures, virus passaging and trypsin activation.
BHK-21 cells were grown under standard conditions (minimal essential medium supplemented with 5 % FCS, 1 % neomycin and 1 % glutamine). In vitro-transcribed RNA was introduced into these cells by electroporation using a Bio-Rad Gene Pulser (two subsequent pulses; setup values: 1·8 kV, 25 µF and 200 ), as described in detail in a previous study (Mandl et al., 1997
). At approximately 12 h post-transfection, the medium was replaced and the concentration of FCS was reduced to 0·5 %. Intracellular expression of protein E was visualized 3 days post-transfection by indirect immunofluorescence staining after fixation of cells with acetone/methanol (1 : 1) using a polyclonal rabbit anti-protein E serum and FITC-conjugated anti-rabbit antibody (Jackson Immune Research Laboratory). At the same time, protein E released into the supernatant was detected by a four-layer enzyme-linked immunosorbent assay (ELISA) (Heinz et al., 1986
). For passaging experiments, 200 µl aliquots of supernatants cleared from cell debris and insoluble material by low-speed centrifugation were transferred onto fresh BHK-21 cells in 24-well cluster plates. After 1 h, the cells were washed twice and then new medium containing 0·5 % FCS was added. Infection of cells was determined 3 days post-inoculation by immunofluorescence and ELISA, as before.
For a quantitative analysis of infectivity, supernatants were harvested 2 days post-transfection and the amount of protein E was measured by a previously described quantitative ELISA after denaturing the samples with SDS (SDS-ELISA) (Heinz et al., 1994). Then a log10 dilution series starting from a standardized concentration of protein E was prepared and used to infect fresh BHK-21 cells as described above. Virus infectivity titres were derived by determining the limiting dilution that scored positive when tested for infection 3 days post-inoculation, as before.
In trypsin-activation experiments, the protease (Trypsin 1 : 250 from porcine pancreas, Sigma; in initial experiments at concentrations ranging between 0·625 and 50 µg ml-1; then generally used at 25 µg ml-1) was added to the cell culture medium 12 h post-electroporation. Passaging experiments were performed exactly as described above but a constant concentration of trypsin was maintained in the medium.
Particle characterization.
For the preparation of purified mutant virus particles, supernatants from BHK-21 cells transfected with the mutant RNA were harvested 48 h post-transfection. Typically, supernatants from 20 to 24 tissue culture flasks (175 cm2) were collected for one preparation. Particles were pelleted by ultracentrifugation at 44 000 r.p.m. (Ti45 rotor; Beckman) for 2 h at 4 °C and then purified by rate zonal centrifugation at 38 000 r.p.m. (SW40 rotor; Beckman) for 3 h at 4 °C in a 1050 % sucrose gradient. For determination of the buoyant density, virus was harvested and pelleted as described above. The pellet was then subjected to rate zonal centrifugation at 38 000 r.p.m. (SW40 rotor; Beckman) for 70 min at 4 °C and subsequent equilibrium density gradient centrifugation at 38 000 r.p.m. (SW40 rotor; Beckman) in 2050 % sucrose for 24 h at 4 °C. All gradients were fractionated with an ISCO 640 gradient fractionator and the concentration of protein E in each fraction was determined by SDS-ELISA (Heinz et al., 1994). The sucrose density of the peak fraction of the equilibrium density gradient was measured in an Abbé refractometer (Atago) with corrections for temperature using standard tables (ISCO).
The antigenic structure of virions was analysed essentially as described previously (Schalich et al., 1996) using a set of monoclonal antibodies (mAbs) directed against protein E or, in the case of one mAb, against protein prM. Purified virus preparations at a concentration of 1 µg ml-1 and a single dilution of each mAb were tested in a four-layer ELISA (Heinz et al., 1986
).
Haemagglutination activity was determined using goose erythrocytes at pH 6·4 by the method of Clarke & Casals (1958).
For gel electrophoresis, purified virions were precipitated with deoxycholate and trichloroacetic acid and separated under SDS-denaturing conditions on 15 % polyacrylamide gels, as described elsewhere (Laemmli & Favre, 1973). Protein bands were visualized using Coomassie PhastGel Blue R (Pharmacia) or by immunoblotting onto a PVDF membrane using the Bio-Rad Trans-Blot Semidry Transfer Cell and Immunoenzymatic Detection kit, as described previously (Schalich et al., 1996
).
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Viability in BHK-21 cells
In a first experiment we wanted to test the viability of genome-length RNA carrying the prM mutation in BHK-21 cells. Equal amounts of either the mutant, pTNd/prM(R88), or the wild-type, pTNd/c, cDNA were transcribed in vitro into RNA and then introduced into BHK-21 cells by electroporation. At 3 days post-transfection, the cells were tested for protein E expression by immunofluorescence staining and the cell culture medium was monitored for the presence of secreted viral antigen using an ELISA (Heinz et al., 1986
). As shown in Fig. 2
(a, c), viral protein E could be detected both in cells transfected with the wild-type and in cells transfected with the mutant RNA. In each case, secreted protein E was also detected in the cell culture supernatants. Quantification of exported protein E 24 h post-transfection (a time at which the effect of secondary infections is still negligible) by SDS-ELISA (Heinz et al., 1994
) yielded concentrations between 0·3 and 0·4 µg ml-1 in both cases. This indicated that the efficiency of RNA transfection, protein expression and protein export of mutant prM(
R88) were not significantly impaired.
|
A comparison of samples standardized to contain equal amounts of protein E (25 ng) obtained from supernatants of BHK-21 cells transfected with wild-type or mutant RNA revealed an infectivity titre of 104 in the case of the wild-type control, whereas no infectivity was detected with the mutant particles in any of several independent experiments. Even when samples containing more protein E (up to 250 ng) were tested, the mutant was found to be unable to infect new BHK-21 cells. These results indicated that even if the mutant prM(R88) possessed a low level of infectivity that was below the detection limit of our assays, its specific infectivity could be no more than 1/10 000 of that of the wild-type control.
Physical characterization of mutant particles
In order to characterize the mutant virus particles, supernatant from BHK-21 cells transfected with prM(R88) RNA was precleared by low-speed centrifugation and then subjected to ultracentrifugation to collect pelletable material. The pellet fraction was then purified by rate zonal centrifugation followed by equilibrium density sucrose gradient analysis. As shown in Fig. 3
, the mutant particles banded at the same position as the wild-type virus control in the equilibrium gradient and both species were found to have a buoyant density of 1·19 g cm-3, which is the same as has been reported previously for the wild-type virus (Heinz & Kunz, 1979
; Schalich et al., 1996
). As expected for an immature particle (Stadler et al., 1997
), we were not able to detect any haemagglutination activity with the mutant.
|
|
|
In a preliminary experiment, we determined that BHK-21 cells suffered a severe CPE after cultivating them for 2 days with medium containing 30 µg trypsin ml-1 and 0·5 % FCS, but were able to tolerate the presence of 25 µg trypsin ml-1 under otherwise identical conditions.
BHK-21 cells were then transfected with prM(R88) in vitro RNA transcripts but this time propagated in medium containing 25 µg trypsin ml-1. At 3 days after transfection, cells and supernatant were tested for protein E production, as described in the earlier experiments. Immunofluorescence analysis (Fig. 6
a) confirmed that the cells could be transfected efficiently and protein E was also detected in the supernatant by ELISA (data not shown). A portion of the supernatant was then transferred to fresh BHK-21 cells. When trypsin was continuously present in the medium before and after washing of the cells, infection was possible and virus could be passaged at least three times (Fig. 6b, c
). Taking the supernatant of one of these passages and attempting to infect cells without further addition of trypsin to the medium after washing still resulted in some positive immunofluorescence staining (Fig. 6d
f) and the supernatants from these cells also tested positive for protein E. However, virus particles in these supernatants were unable to initiate a further round of infection in the absence of trypsin (Fig. 6g
i). These results indicate that the mutant prM(
R88) is potentially infectious but requires exogenous cleavage in order to be able to induce a single round of infection.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In light of our current data, we believe that residual infectivity of immature virions observed in earlier studies was due to inefficient suppression of prM cleavage by ammonium chloride, bafilomycin A1, and other treatments, rather than any inherent infectivity of the immature particles themselves. The amount of processed protein M in these preparations may have been too small to be detected in biochemical tests but sufficiently large to provide these preparations with residual infectivity. The deletion introduced into protein prM in this study destroyed the minimal sequence required for furin-mediated cleavage (arginine residues at positions -1 and -4) (Molloy et al., 1992; Nakayama, 1997
) and this was apparently more successful in completely suppressing the processing of protein prM than previous approaches.
The observation that cleavage of protein prM is essential for infectivity of flaviviruses corresponds well to results obtained in analogous studies using alphaviruses. Flaviviruses and alphaviruses share a great deal of structural and functional similarity. The solution of the atomic structures of their fusion proteins (protein E in flaviviruses and protein E1 in alphaviruses) has revealed a striking resemblance, including the position of an internal fusion peptide (Lescar et al., 2001; Rey et al., 1995
). The fusion capacity of these so-called class II fusion proteins is activated by the furin-mediated cleavage of an auxiliary protein, i.e. protein prM in flaviviruses and protein PE2 (or p62) in alphaviruses (Heinz & Allison, 2000
; Kielian et al., 2000
). Mutagenesis studies on protein PE2 that abolished cleavage either by a substitution of the arginine at position -1 of the furin site (Davis et al., 1991
; Lobigs & Garoff, 1990
; Salminen et al., 1992
) or by introducing a signal for N-linked glycosylation (Heidner et al., 1994
) immediately downstream of the cleavage sequence yielded completely non-infectious virus mutants. However, in the alphavirus system, the spontaneous emergence of infectious revertants carrying second-site mutations in proteins E1, E2 or E3 has been observed (Davis et al., 1991
; Heidner et al., 1994
). So far, this has not been detected with the TBE virus mutant described in this study. However, our results certainly do not exclude the possibility that such revertants may arise under different growth conditions, such as prolonged growth periods or other host cell systems.
Infectivity of the TBE virus mutant prM(R88) could be restored by the addition of trypsin to the cell culture medium. This finding indicated that the processing and assembly of the immature particles had been correct except for the missing cleavage step, which was then presumably carried out by trypsin after the particles had been released from the cells. Activation by exogenous proteases has also been achieved with mutants of other viruses in which the natural furin cleavage site had been destroyed. For instance, it was demonstrated that the fusion activity and infectivity of furin-cleavage deficient mutants of Semliki Forest virus, measles virus and Newcastle disease virus could be restored by the addition of trypsin (Li et al., 1998
; Lobigs & Garoff, 1990
; Maisner et al., 2000
). The trypsin concentrations applied in these studies (15, 1 and 5 µg ml-1, respectively) were considerably lower than in this study (25 µg ml-1), but this is probably due to the fact that in our experiments the cell culture medium contained 0·5 % FCS, which may have partially inhibited trypsin activity.
The ability to activate the furin-deficient mutant by the addition of exogenous protease provides a system that allows the generation of single-round infectious flavivirus particles. These could be useful in the development of vaccines or RNA-based gene delivery systems. It was not possible, however, to restore infectivity to wild-type levels using trypsin, which was expected because trypsin is known to preferentially cleave protein E (Heinz et al., 1991), resulting in the loss of entry functions. This problem could be circumvented by replacing the furin cleavage site by a recognition sequence for a protease that is more specific than trypsin. In studies with Semliki Forest virus and human immunodeficiency virus, the furin cleavage site was changed into a chymotrypsin recognition site and it was demonstrated that the fusogenic activity of these mutants (measured in these studies as syncytium formation) could be activated by this protease (Jain et al., 1991
; McCune et al., 1988
). It has also been shown that viruses with altered protease specificities can be generated by selection in the presence of the corresponding protease (Hsu et al., 1987
). Finally, the virus mutant prM(
R88) may be a useful tool for studying the structure of immature flavivirus particles.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Allison, S. L., Schalich, J., Stiasny, K., Mandl, C. W. & Heinz, F. X. (2001). Mutational evidence for an internal fusion peptide in flavivirus envelope protein E. J Virol 75, 42684275.
Chambers, T. J., Hahn, C. S., Galler, R. & Rice, C. M. (1990). Flavivirus genome organization, expression, and replication. Annu Rev Microbiol 44, 649688.[CrossRef][Medline]
Clarke, D. H. & Casals, J. (1958). Techniques for hemagglutination and hemagglutination inhibition with arthropod-borne viruses by antibody absorption. Am J Trop Med Hyg 7, 561573.
Davis, N. L., Powell, N., Greenwald, G. F., Willis, L. V., Johnson, B. J., Smith, J. F. & Johnston, R. E. (1991). Attenuating mutations in the E2 glycoprotein gene of Venezuelan equine encephalitis virus: construction of single and multiple mutants in a full-length cDNA clone. Virology 183, 2031.[CrossRef][Medline]
Guirakhoo, F., Heinz, F. X., Mandl, C. W., Holzmann, H. & Kunz, C. (1991). Fusion activity of flaviviruses: comparison of mature and immature (prM-containing) tick-borne encephalitis virions. J Gen Virol 72, 13231329.[Abstract]
Guirakhoo, F., Bolin, R. A. & Roehrig, J. T. (1992). The Murray Valley encephalitis virus prM protein confers acid resistance to virus particles and alters the expression of epitopes within the R2 domain of E glycoprotein. Virology 191, 921931.[Medline]
He, R. T., Innis, B. L., Nisalak, A., Usawattanakul, W., Wang, S., Kalayanarooj, S. & Anderson, R. (1995). Antibodies that block virus attachment to Vero cells are a major component of the human neutralizing antibody response against dengue virus type 2. J Med Virol 45, 451461.[Medline]
Heidner, H. W., McKnight, K. L., Davis, N. L. & Johnston, R. E. (1994). Lethality of PE2 incorporation into Sindbis virus can be suppressed by second-site mutations in E3 and E2. J Virol 68, 26832692.[Abstract]
Heinz, F. X. & Kunz, C. (1979). Protease treatment and chemical crosslinking of a flavivirus: tick borne encephalitis virus. Arch Virol 60, 207216.[Medline]
Heinz, F. X. & Kunz, C. (1981). Homogeneity of the structural glycoprotein from European isolates of tick-borne encephalitis virus: comparison with other flaviviruses. J Gen Virol 57, 263274.[Abstract]
Heinz, F. X. & Allison, S. L. (2000). Structures and mechanisms in flavivirus fusion. Adv Virus Res 55, 231269.[Medline]
Heinz, F. X., Tuma, W., Guirakhoo, F. & Kunz, C. (1986). A model study of the use of monoclonal antibodies in capture enzyme immunoassays for antigen quantification exploiting the epitope map of tick-borne encephalitis virus. J Biol Stand 14, 133141.[Medline]
Heinz, F. X., Mandl, C. W., Holzmann, H., Kunz, C., Harris, B. A., Rey, F. & Harrison, S. C. (1991). The flavivirus envelope protein E: isolation of a soluble form from tick-borne encephalitis virus and its crystallization. J Virol 65, 55795583.[Medline]
Heinz, F. X., Stiasny, K., Puschner Auer, G., Holzmann, H., Allison, S. L., Mandl, C. W. & Kunz, C. (1994). Structural changes and functional control of the tick-borne encephalitis virus glycoprotein E by the heterodimeric association with protein prM. Virology 198, 109117.[CrossRef][Medline]
Hernandez, L. D., Hoffman, L. R., Wolfsberg, T. G. & White, J. M. (1996). Viruscell and cellcell fusion. Annu Rev Cell Dev Biol 12, 627661.[CrossRef][Medline]
Hsu, M. C., Scheid, A. & Choppin, P. W. (1987). Protease activation mutants of Sendai virus: sequence analysis of the mRNA of the fusion protein (F) gene and direct identification of the cleavage-activation site. Virology 156, 8490.[CrossRef][Medline]
Iacono-Connors, L. C., Smith, J. F., Ksiazek, T. G., Kelley, C. L. & Schmaljohn, C. S. (1996). Characterization of Langat virus antigenic determinants defined by monoclonal antibodies to E, NS1 and preM and identification of a protective, non-neutralizing preM-specific monoclonal antibody. Virus Res 43, 125136.[CrossRef][Medline]
Jain, S. K., DeCandido, S. & Kielian, M. (1991). Processing of the p62 envelope precursor protein of Semliki Forest virus. J Biol Chem 266, 57565761.
Kielian, M., Chatterjee, P. K., Gibbons, D. L. & Lu, Y. E. (2000). Specific roles for lipids in virus fusion and exit. Examples from the alphaviruses. Subcell Biochem 34, 409455.[Medline]
Klenk, H. D. & Garten, W. (1994). Activation cleavage of viral spike proteins by host proteases. In Cellular Receptors for Animal Viruses, pp. 241279. Cold Spring Harbour, NY: Cold Spring Harbour Laboratory Press.
Kofler, R. M., Heinz, F. X. & Mandl, C. W. (2002). Capsid protein C of tick-borne encephalitis virus tolerates large internal deletions and is a favorable target for attenuation of virulence. J Virol 76, 35343543.
Kopp, A., Blewett, E., Misra, V. & Mettenleiter, T. C. (1994). Proteolytic cleavage of bovine herpesvirus 1 (BHV-1) glycoprotein gB is not necessary for its function in BHV-1 or pseudorabies virus. J Virol 68, 16671674.[Abstract]
Kuhn, R. J., Zhang, W., Rossmann, M. G. & 9 other authors (2002). Structure of dengue virus: implications for flavivirus organization, maturation, and fusion. Cell 108, 717725.[Medline]
Laemmli, U. K. & Favre, M. (1973). Maturation of the head of bacteriophage T4. I. DNA packaging events. J Mol Biol 80, 575599.[Medline]
Lescar, J., Roussel, A., Wien, M. W., Navaza, J., Fuller, S. D., Wengler, G., Wengler, G. & Rey, F. A. (2001). The fusion glycoprotein shell of Semliki Forest virus: an icosahedral assembly primed for fusogenic activation at endosomal pH. Cell 105, 137148.[Medline]
Li, Z., Sergel, T., Razvi, E. & Morrison, T. (1998). Effect of cleavage mutants on syncytium formation directed by the wild-type fusion protein of Newcastle disease virus. J Virol 72, 37893795.
Lindenbach, B. D. & Rice, C. M. (2001). Flaviviridae: The viruses and their replication. In Fields Virology, 4th edn, pp. 9911041. Edited by D. M. Knipe & P. M. Howley. Philadelphia: Lippincott Williams & Wilkins.
Lobigs, M. & Garoff, H. (1990). Fusion function of the Semliki Forest virus spike is activated by proteolytic cleavage of the envelope glycoprotein precursor p62. J Virol 64, 12331240.[Medline]
McCune, J. M., Rabin, L. B., Feinberg, M. B., Lieberman, M., Kosek, J. C., Reyes, G. R. & Weissman, I. L. (1988). Endoproteolytic cleavage of gp160 is required for the activation of human immunodeficiency virus. Cell 53, 5567.[Medline]
Maisner, A., Mrkic, B., Herrler, G., Moll, M., Billeter, M. A., Cattaneo, R. & Klenk, H. D. (2000). Recombinant measles virus requiring an exogenous protease for activation of infectivity. J Gen Virol 81, 441449.
Mandl, C. W., Heinz, F. X. & Kunz, C. (1988). Sequence of the structural proteins of tick-borne encephalitis virus (Western subtype) and comparative analysis with other flaviviruses. Virology 166, 197205.[Medline]
Mandl, C. W., Heinz, F. X., Stockl, E. & Kunz, C. (1989). Genome sequence of tick-borne encephalitis virus (Western subtype) and comparative analysis of nonstructural proteins with other flaviviruses. Virology 173, 291301.[CrossRef][Medline]
Mandl, C. W., Ecker, M., Holzmann, H., Kunz, C. & Heinz, F. X. (1997). Infectious cDNA clones of tick-borne encephalitis virus European subtype prototypic strain Neudoerfl and high virulence strain Hypr. J Gen Virol 78, 10491057.[Abstract]
Mandl, C. W., Kroschewski, H., Allison, S. L., Kofler, R., Holzmann, H., Meixner, T. & Heinz, F. X. (2001). Adaptation of tick-borne encephalitis virus to BHK-21 cells results in the formation of multiple heparan sulfate binding sites in the envelope protein and attenuation in vivo. J Virol 75, 56275637.
Molloy, S. S., Bresnahan, P. A., Leppla, S. H., Klimpel, K. R. & Thomas, G. (1992). Human furin is a calcium-dependent serine endoprotease that recognizes the sequence ArgXXArg and efficiently cleaves anthrax toxin protective antigen. J Biol Chem 267, 1639616402.
Molloy, S. S., Anderson, E. D., Jean, F. & Thomas, G. (1999). Bi-cycling the furin pathway: from TGN localization to pathogen activation and embryogenesis. Trends Cell Biol 9, 2835.[CrossRef][Medline]
Murray, J. M., Aaskov, J. G. & Wright, P. J. (1993). Processing of the dengue virus type 2 proteins prM and C-prM. J Gen Virol 74, 175182.[Abstract]
Nakayama, K. (1997). Furin: a mammalian subtilisin/Kex2p-like endoprotease involved in processing of a wide variety of precursor proteins. Biochem J 27, 625635.
Plaimauer, B., Mohr, G., Wernhart, W., Himmelspach, M., Dorner, F. & Schlokat, U. (2001). Shed furin: mapping of the cleavage determinants and identification of its C-terminus. Biochem J 354, 689695.[CrossRef][Medline]
Randolph, V. B. & Stollar, V. (1990). Low pH-induced cell fusion in flavivirus-infected Aedes albopictus cell cultures. J Gen Virol 71, 18451850.[Abstract]
Randolph, V. B., Winkler, G. & Stollar, V. (1990). Acidotropic amines inhibit proteolytic processing of flavivirus prM protein. Virology 174, 450458.[Medline]
Rey, F. A., Heinz, F. X., Mandl, C., Kunz, C. & Harrison, S. C. (1995). The envelope glycoprotein from tick-borne encephalitis virus at 2 Å resolution. Nature 375, 291298.[CrossRef][Medline]
Salminen, A., Wahlberg, J. M., Lobigs, M., Liljestrom, P. & Garoff, H. (1992). Membrane fusion process of Semliki Forest virus. II. Cleavage-dependent reorganization of the spike protein complex controls virus entry. J Cell Biol 116, 349357.[Abstract]
Schalich, J., Allison, S. L., Stiasny, K., Mandl, C. W., Kunz, C. & Heinz, F. X. (1996). Recombinant subviral particles from tick-borne encephalitis virus are fusogenic and provide a model system for studying flavivirus envelope glycoprotein functions. J Virol 70, 45494557.[Abstract]
Stadler, K., Allison, S. L., Schalich, J. & Heinz, F. X. (1997). Proteolytic activation of tick-borne encephalitis virus by furin. J Virol 71, 84758481.[Abstract]
Stiasny, K., Allison, S. L., Schalich, J. & Heinz, F. X. (2002). Membrane interactions of the tick-borne encephalitis virus fusion protein E at low pH. J Virol 76, 37843790.
Wengler, G. & Wengler, G. (1989). Cell-associated West Nile flavivirus is covered with E+pre-M protein heterodimers which are destroyed and reorganized by proteolytic cleavage during virus release. J Virol 63, 25212526.[Medline]
White, J. M. (1992). Membrane fusion. Science 258, 917924.[Medline]
Wool-Lewis, R. J. & Bates, P. (1999). Endoproteolytic processing of the Ebola virus envelope glycoprotein: cleavage is not required for function. J Virol 73, 14191426.
Received 24 July 2002;
accepted 16 September 2002.