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Chapter 2b: The molecular and antigenic structure of TBEV

for rapid signal sequence cleavage at its N- engineered into an infectious TBEV clone
terminus during viral polyprotein processing. (resulting in the deletion of one of the arginines
After their formation in the ER, immature virus at P1,P2; Table 1) did not impair the assembly
particles are transported through the exocytic of immature particles but completely abolished
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pathway of the cell. As a crucial step of virus infectivity. Infectivity could be restored by in
maturation, the prM protein is cleaved in the vitro trypsin cleavage, which is likely to cleave at
trans-Golgi network (TGN) by the cellular one of the R residues that was retained at the
protease furin, generating membrane anchored furin cleavage site (Table 1).
protein M and the proteolytic fragment pr.
So far, the structure of immature virions has
Increasing the pH in the TGN of TBEV-infected only been determined for mosquito-borne
cells by acidotropic agents (such as ammonium- flaviviruses, which were shown to carry 60
chloride) or by bafilomycin A1 (a specific spikes of trimers of prM-E heterodimers.
inhibitor of the vacuolar type H+ ATPase) led to 16,19,21,31 Considering the high degree of
the release of immature particles with a 20 – 50 structural conservation of viral proteins and
fold lower specific infectivity and hemaggluti- mature particles, it is justified to assume that
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nation (HA) activity than mature viruses. This immature TBE virions are similar to those of
suggested that a conformational change in the mosquito-borne flaviviruses. In the course of
prM-E complex is induced by the slightly acidic exocytosis of immature viral particles, the acidic
pH in the TGN, which is required for furin cleav- pH in the TGN causes a major re-arrangement
age. Evidence that the maturation cleavage is of the viral glycoprotein interactions, resulting
conferred by the TGN-resident protease furin in the conversion of the trimeric prM-E spikes
was obtained in experiments with a furin- into a herringbone-like shell of 90 E protein
deficient human cell line (LoVo), which dimers (Figure 1G). Data obtained with dengue
produced only immature viruses, as well as a virus show that the pr fragment remains
specific furin inhibitor that blocked furin associated with the particles at acidic pH after
cleavage, and by in vitro cleavage experiments furin cleavage but dissociates at neutral pH
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with recombinant furin. Treatment of im- when the particles are released from the cells
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mature TBEV particles with furin resulted in a (Figure 2).
100-fold increase in specific infectivity and the
acquisition of hemagglutination as well as Mature virions display the herringbone-like
membrane fusion activities. arrangement of E that was induced in immature
particles when encountering the low pH in the
Importantly, furin cleavage itself did not require TGN. The release of the pr fragment leaves E in
an acidic pH, but the conformational change a metastable conformation, poised to undergo
exposing the cleavage site in the prM-E complex dramatic low pH-induced structural changes
was acid pH-dependent. The low-pH-induced that mediate viral fusion in endosomes upon
reorganization of the protein complex was virus entry (see below). The function of prM and
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shown to be irreversible in the case of TBEV , the pr fragment is thus to protect E in the acidic
but appears to be reversible in the case of TGN and to avoid membrane fusion already at
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dengue viruses. this stage of the viral life cycle.
The furin cleavage site of TBEV corresponds to a The static pictures of fully immature and fully
consensus sequence also found in other mature particle structures determined by cryo
flavivirus prM proteins (Table 1). The depen- EM cannot be reconciled with all experimental
dence of virus maturation on this conserved data obtained in studies of flavivirus entry and
sequence element in prM was demonstrated virus interactions with antibodies. 34, 35 First,
directly by a genetic approach. A specific some antibodies binding to seemingly inacces-
mutation in the furin recognition sequence sible (cryptic) epitopes in E neutralized viral
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