Flavivirus particles are synthesized in an immature form containing heterodimers of the proteins prM and E. Shortly before release from the cell, prM is cleaved by the host protease furin to yield mature virions. In this study, the furin-mediated cleavage of the tick-borne encephalitis (TBE) virus protein prM was prevented by specific mutagenesis of the cleavage site. This resulted in the production of immature TBE virions, which were shown to be completely non-infectious in BHK-21 cells. This finding contrasted with previous studies in which immature flavivirus particles produced by other techniques were shown to have considerable residual infectivity. The structural integrity of the mutant virus particles was confirmed by the characterization of physical and antigenic properties. Most importantly, infectivity could be restored by the addition of trypsin, which presumably cleaved protein prM at one of the monobasic sites retained in the mutated sequence. In the presence of trypsin, the mutant could be passaged repeatedly in BHK-21 cells, but if the protease was removed, the activated particles could initiate only a single round of infection, which again generated noninfectious virus progeny. These observations provide evidence that the infectivity of flaviviruses depends on the endoproteolytic cleavage of protein prM, which probably has a regulatory function rather than a direct role in virus entry. Moreover, the results illustrate that mutation of the furin cleavage site is a convenient way to produce single-round infectious flavivirus particles, which may be useful in vaccine and vector development. INTRODUCTIONFusion of the viral membrane with a host cell membrane is an obligatory step of the entry processes of enveloped viruses (Hernandez et al., 1996;White, 1992). Viral surface proteins that mediate this event usually require the proteolytic cleavage of precursor proteins to achieve their fusogenic potential. In the case of so-called class I fusion proteins, which are present in orthomyxoviruses, paramyxoviruses, retroviruses and filoviruses, cleavage activation affects the fusion protein itself, i.e. a fusion-inactive precursor protein is cleaved to yield the mature fusogenic protein (Klenk & Garten, 1994). In contrast, class II fusion proteins (Lescar et al., 2001), which so far have been found in alphaviruses and flaviviruses, acquire their fusion competence through the endoproteolytic processing of an auxiliary protein, which, in its uncleaved precursor form, prevents the fusion protein from undergoing the structural changes necessary to induce fusion (reviewed by Heinz & Allison, 2000;Kielian et al., 2000).Activation cleavages are often carried out by the cellular protease furin, an enzyme that is concentrated in the transGolgi network (TGN) but also cycles between endosomes and the plasma membrane (Molloy et al., 1999; Plaimauer et al., 2001). In naturally occurring substrates, including not only viral surface proteins but also a large number of cellular proproteins, furin cleaves after the conserve...

Cleavage of the viral surface protein prM by the proprotein convertase furin is a key step in the maturation process of flavivirus particles. A mutant of tick-borne encephalitis virus (TBEV) carrying a deletion mutation within the furin recognition motif of protein prM (changing R-T-R-R to R-T-R) was previously shown to be noninfectious in BHK-21 cells. We now demonstrate how natural selection can overcome this lethal defect in two different growth systems by distinct resuscitating mutations. In BHK-21 cells, a spontaneous codon duplication created a minimal furin cleavage motif (R-R-T-R). This mutation restored infectivity by enabling intracellular prM cleavage. A completely different mutation pattern was observed when the mutant virus was passaged in mouse brains. The "pr" part of protein prM, which is removed by cleavage, contains six conserved Cys residues. The mutations selected in mice changed the number of Cys residues to five or seven by substitution mutations near the original cleavage site, probably causing a major perturbation of the structural integrity of protein prM. Although viable in mice, such Cys mutants could not be passaged in BHK-21 cells under normal growth conditions (37°C), but one of the mutants exhibited a low level of infectivity at a reduced incubation temperature (28°C). No evidence for the cleavage of protein prM in BHK-21 cells was obtained. This suggests that under certain growth conditions, the structural perturbation of protein prM can restore the infectivity of TBEV by circumventing the need for intracellular furin-mediated cleavage. This is the first example of a flavivirus using such a molecular mechanism.Flaviviruses are small enveloped viruses with a positivestranded RNA genome. Several of the members of the genus Flavivirus, in the family Flaviviridae, are important human pathogens, including Tick-borne encephalitis virus (TBEV), Yellow fever virus, Japanese encephalitis virus, West Nile virus, and the four serotypes of Dengue virus (2). All of the flaviviruses share very similar structural and functional properties (26). Their genomic RNA serves as the only viral messenger and encodes all viral proteins in a single long open reading frame. The translation product, a polyprotein, is cleaved by viral and cellular proteases to yield the three structural proteins, C (capsid protein), prM/M (membrane protein and its precursor protein), and E (envelope protein), as well as seven nonstructural proteins. Flavivirus virions consist of a nucleocapsid, which is formed by multiple copies of the basic and mostly alpha-helical protein C encapsulating the genomic RNA, and a surrounding host cell-derived lipid membrane, in which the two surface proteins, prM/M and E, are carboxy-terminally anchored (37).Protein E, which is much larger than protein M (the approximate sizes are 56 kDa and 8 kDa, respectively), forms almost the entire outer surface of the mature virion and mediates the viral entry functions. It is responsible for attachment of the virus to one or more host cell receptor mol...

The infectivity of flavivirus particles depends on a maturation process that is triggered by the proteolytic cleavage of the precursor of the M protein (prM). This activation cleavage is naturally performed by ubiquitous cellular proteases of the furin family, which typically recognize the multibasic sequence motif R-X-R/K-R. Previously, we demonstrated that a tick-borne encephalitis virus (TBEV) mutant with an altered cleavage motif, R-X-R, produced immature, noninfectious particles that could be activated by exogenous trypsin, which cleaves after single basic residues. Here, we report the adaptation of this mutant to chymotrypsin, a protease specific for large, hydrophobic amino acid residues. Using selection pressure in cell culture, two different mutations conferring a chymotrypsin-dependent phenotype were identified. Surprisingly, one of these mutations (Ser85Phe) occurred three positions upstream of the natural cleavage site. The other mutation (Arg89His) arose at the natural cleavage position but involved a His residue, which is not a typical chymotrypsin cleavage site. Efficient cleavage of protein prM and activation by the heterologous protease were confirmed using various recombinant TBEV mutants. Mutants with only the originally selected mutations exhibited unimpaired export kinetics and were genotypically stable during at least six cell culture passages. However, in contrast to the wild-type virus or trypsin-dependent mutants, chymotrypsin-dependent mutants were not neurovirulent in suckling mice. Our results demonstrate that flaviviruses with altered protease specificities can be generated and suggest that this approach can be used for the construction of viral mutants or vectors that can be activated on demand and have restricted tissue tropism and virulence.