Capsid protein C of tick-borne encephalitis virus tolerates large internal deletions and is a favorable target for attenuation of virulence

Abstract

Deletions ranging in size from 4 to 21 amino acid residues were introduced into the capsid protein of the flavivirus tick-borne encephalitis (TBE) virus. These deletions incrementally affected a hydrophobic domain which is present at the center of all flavivirus capsid protein sequences and part of which may form an amphipathic alpha-helix. In the context of the full-length TBE genome, the deletions did not measurably affect protein expression and up to a deletion length of 16 amino acid residues, corresponding to almost 17% of mature protein C, viable virus was recovered. This virus was strongly attenuated but highly immunogenic in adult mice, revealing capsid protein C as a new and attractive target for the directed attenuation of flaviviruses. Apparently, the larger deletions interfered with the correct assembly of infectious virus particles, and this disturbance of virion assembly is likely to be the molecular basis of attenuation. However, all of the mutants carrying large deletions produced substantial amounts of subviral particles, which as judged from density gradient analyses were identical to recombinant subviral particles as obtained by the expression of the surface proteins prM and E alone. The structural and functional flexibility of protein C revealed in this study and its predicted largely alpha-helical conformation are reminiscent of capsid proteins of other enveloped viruses, such as alphaviruses (N-terminal domain of the capsid protein), retroviruses, and hepadnaviruses and suggest that all of these may belong to a common structural class, which is fundamentally distinct from the classical beta-barrel structures of many icosahedral viral capsids. The possibility of attenuating flaviviruses by disturbing virus assembly and favoring the production of noninfectious but highly immunogenic subviral particles opens up a promising new avenue for the development of live flavivirus vaccines.

FIG. 1.

Characteristics of the TBE virus protein C wild-type and mutant amino acid sequences. (a) Sequence of the wild-type primary translation product, from which the amino-terminal methionine (shown in parentheses) and the carboxy-terminal 20 residues (presumed NS2B/3 cleavage site indicated by an arrow) are removed to yield mature protein C. Positively charged residues are underlined and bold. (b) Locations of the introduced deletion mutations (arrows) together with the designations of the corresponding mutants. The numbers in the names of the mutants indicate which residues were deleted. (c) Hydrophobicity plot calculated using the Kyte-Doolittle algorithm (window size 11) (20). The positions of two extended hydrophobic domains located in the center and at the carboxy terminus of the primary sequence are emphasized by grey shading. (d) Secondary-structure prediction revealing four predicted alpha-helical domains. The positions of the potential helices are indicated by shaded boxes and numbered consecutively by roman numerals. The first and last residue of each predicted helix are listed below the box. (e) Helical wheel representations of two sequence elements exhibiting features of leucine zipper motifs.

FIG. 2.

Expression of protein E and cell culture passage. (a to g) Wild-type (WT) and mutant RNAs (as indicated) were synthesized in vitro and transfected into BHK-21 cells. Expression of protein E was tested 48 h posttransfection by immunofluorescence, and protein E released into the supernatants was detected using a four-layer ELISA. Results of ELISA are given in the inserts as follows: −, optical density (OD) < 0.1; +, OD = 0.1 to 1.0; ++, OD > 1.0. (h to n) Supernatants were passaged onto new BHK-21 cell cultures as symbolized by the arrows, and the expression of protein E was determined 48 h postinoculation by immunofluorescence and ELISA as before.

FIG. 3.

Quantitative analysis of total protein E expression and export. BHK-21 cells were transfected with in vitro-synthesized wild-type (WT) and mutant RNAs as indicated. The amount of protein E expressed per cell (a) and the percentage of protein E released from the cell into the cell culture supernatant (b) were determined at 24 h (open bars) and 30 h (shaded bars) posttransfection. Results shown are mean values of three experiments (± standard errors of the means).

FIG. 4.

Particle analysis by rate zonal centrifugation. Control preparations of wild-type virus and RSPs as well as particles pelleted from BHK-21 cell supernatants after transfection with the indicated mutant RNA were fractionated on discontinuous sucrose gradients. The 10, 35, and 50% zones are indicated below the graphs. The protein E content of each fraction was determined by a quantitative SDS-ELISA.

FIG. 5.

Growth curve analysis of wild-type and mutant TBE viruses. CE cells were infected at a MOI of 1, and the amounts of infectious particles released into the supernatants during 1-h time periods were quantified at various time points postinfection.