Spontaneous Mutations Restore the Viability of Tick-Borne Encephalitis Virus Mutants with Large Deletions in Protein C

Abstract

The capsid protein, C, of tick-borne encephalitis virus has recently been found to tolerate deletions up to a length of 16 amino acid residues that partially removed the central hydrophobic domain, a sequence element conserved among flaviviruses which may be crucial for virion assembly. In this study, mutants with deletion lengths of 19, 21, 27, or 30 residues, removing more or all of this hydrophobic domain, were found to yield viable virus progeny, but this was without exception accompanied by the emergence of additional mutations within protein C. These point mutations or sequence duplications were located downstream of the engineered deletion and generally increased the hydrophobicity, suggesting that they may compensate for the loss of the central hydrophobic domain. Two of the second-site mutations, together with the corresponding deletion, were introduced into a wild-type genetic backbone, and the analysis of these "double mutants" provided direct evidence that the viability of the deletion mutant indeed depended on the presence of the second-site mutation. Our results corroborate the notion that hydrophobic interactions of protein C are essential for the assembly of infectious flavivirus particles but rule out the possibility that individual residues of the central hydrophobic domain are absolutely required for infectivity. Furthermore, the double mutants were found to be highly attenuated and capable of inducing a protective immune response in mice at even lower inoculation doses than the previously characterized 16-amino-acid-residue deletion mutant, suggesting that the combination of large deletions and second-site mutations may be a superior way to generate safe, attenuated flavivirus vaccine strains.Tick-borne encephalitis (TBE) virus is a representative of the genus Flavivirus (family Flaviviridae), which also includes several other important human pathogens, such as yellow fever virus, Japanese encephalitis virus, West Nile virus, and the dengue viruses (33). Flaviviruses are small, round, enveloped particles that contain only three structural virus proteins, i.e., the membrane-anchored surface proteins M and E and the capsid protein, C (18). The last is a relatively small protein (approximately 11 kDa) with a large content of positively charged amino acid residues that shows significantly less sequence homology among members of the genus than do the other two structural proteins (21). Nevertheless, a number of characteristics of its amino acid sequence, including the clustering of basic residues in certain sections of the sequence, the presence of two distinctive hydrophobic segments located approximately in the center of the sequence and at the carboxy-terminal end, and a predicted high propensity to adopt a predominantly alpha-helical conformation, are generally maintained among different flaviviruses, suggesting an overall conserved structural and functional organization of protein C (14,18,25).In the absence of protein C, the surface proteins M (which is first synthesized as a precursor prote...

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“…Previous investigations have revealed a remarkable functional flexibility of protein C, allowing the generation of infectious viral mutants carrying deletions that removed up to almost one-third of this protein (11,12). We now demonstrate that a very large deletion (approximately two-thirds of the protein) results in an entirely noninfectious but RNA-replication-competent phenotype.…”

“…1). Previous studies had indicated that deletion of the hydrophobic helix I in conjunction with spontaneously occurring compensatory mutations in helix II or III was tolerated by the virus, yielding infectious progeny (11,12). In mutant C(⌬28-89), all three of these helices were removed by a deletion extending from residues 28-89.…”

“…Previously analyzed smaller capsid deletion mutants of TBEV were found to be infectious or were able to revert to an infectious phenotype after inoculation into suck- ling mouse brain (11,12), the most sensitive growth system for this virus (25). Corresponding experiments performed with mutants C(⌬28 -89) and C(⌬28 -89)-S in BHK-21 cells and suckling mice, however, yielded no evidence of infectivity or genetic reversion events.…”

“…The flavivirus protein C is a small protein with a large proportion of positively charged amino acid residues and a high percentage of ␣-helical secondary structures (11,12,24). A large deletion was engineered into the protein C-coding region of the full-length TBEV genome (Fig.…”

Solution structure of dengue virus capsid protein reveals another fold

“…1), spanning the C-terminal half of ␣1 and the N-terminal half of ␣2 in DEN2C. Larger deletions that extended to the N terminus of ␣3, residues 32-64, failed to produce viable virus, but second-site revertants to these lethal deletions restored virus viability (24). Many of these revertants involved single amino acid substitutions to an apolar residue.…”