Attenuation of Tick-Borne Encephalitis Virus by Structure-Based Site-Specific Mutagenesis of a Putative Flavivirus Receptor Binding Site

Abstract

The impact of a specific region of the envelope protein E of tick-borne encephalitis (TBE) virus on the biology of this virus was investigated by a site-directed mutagenesis approach. The four amino acid residues that were analyzed in detail (E308 to E311) are located on the upper-lateral surface of domain III according to the X-ray structure of the TBE virus protein E and are part of an area that is considered to be a potential receptor binding determinant of flaviviruses. Mutants containing single amino acid substitutions, as well as combinations of mutations, were constructed and analyzed for their virulence in mice, growth properties in cultured cells, and genetic stability. The most significant attenuation in mice was achieved by mutagenesis of threonine 310. Combining this mutation with deletion mutations in the 3-noncoding region yielded mutants that were highly attenuated. The biological effects of mutation Thr 310 to Lys, however, could be reversed to a large degree by a mutation at a neighboring position (Lys 311 to Glu) that arose spontaneously during infection of a mouse. Mutagenesis of the other positions provided evidence for the functional importance of residue 308 (Asp) and its charge interaction with residue 311 (Lys), whereas residue 309 could be altered or even deleted without any notable consequences. Deletion of residue 309 was accompanied by a spontaneous second-site mutation (Phe to Tyr) at position 332, which in the three-dimensional structure of protein E is spatially close to residue 309. The information obtained in this study is relevant for the development of specific attenuated flavivirus strains that may serve as future live vaccines.Tick-borne encephalitis (TBE) virus is a human pathogenic member of the genus Flavivirus (family Flaviviridae) (31). Many members of this genus can cause severe human diseases, the most important representatives besides TBE virus being the mosquito-borne viruses yellow fever (YF) virus, Japanese encephalitis (JE) virus, and the four serotypes of dengue virus (18). In spite of the availability of attenuated live vaccines (in the case of YF virus) and formalin-inactivated killed vaccines (TBE virus, JE virus) which have proven to be effective for the prevention of flavivirus infections, there is a strong demand for the development of novel and improved vaccines against these and other flavivirus infections. For the rational design of live vaccines, a detailed understanding of the molecular basis of virulence and pathogenesis is a major goal. With the availability of modern molecular techniques and high-resolution structural information, it is now possible to alter viral structures in a specific and rational way in order to understand structurefunction relationships. This knowledge can then be applied to achieve the desired biological property, such as attenuation of the virus.Flavivirus virions are relatively simple particles consisting of a nucleocapsid composed of a single capsid protein (C) surrounded by a lipid membrane that contains two viral protein...

Capsid Protein C of Tick-Borne Encephalitis Virus Tolerates Large Internal Deletions and Is a Favorable Target for Attenuation of Virulence

“…Moreover, all of the known major antigenic targets required for the development of a protective immune response, in particular glycoprotein E, which contains important neutralizing epitopes, are fully intact in these constructs. This should mean a major advantage over approaches that achieve attenuation through various protein E mutations (29,34,37). Our data suggest that the attenuation achieved by deletions within the central hydrophobic domain (helix I) of the capsid protein is mainly based on the disturbance of the assembly of infectious virus particles, while still allowing subviral particles to be formed.…”

“…The characterization of mutant viruses in the mouse model was performed as in previous studies (29,33,34). Briefly, groups of 10 5-week-old (body weight, approximately 20 g) outbred Swiss albino mice were inoculated subcutaneously, and survival was recorded for 28 days.…”

On a mouse monoclonal antibody that neutralizes all four dengue virus serotypes

“…Despite this variety of host-cell surface receptors, one viral component consistently shown to bind directly to cellular receptors is the Ig-like domain III of the E protein (Beasley & Barrett, 2002;Hung et al, 2004;Lin & Wu, 2003;Mandl et al, 2000;Thullier et al, 2001). Subtle structural variations at the surface of domain III are thus likely to influence virus interactions with attachment molecules (cell tropism) and also with antibodies from the host that define serotypes.…”

Incorporation of Tick-Borne Encephalitis Virus Replicons into Virus-Like Particles by a Packaging Cell Line

“…Specific mutagenesis of the TBE virus genome by means of an infectious cDNA clone has been performed in several studies during past years (7,23,31), but no replicons of this virus have so far been generated. RSPs of TBE virus derived by transient expression have been extensively characterized with respect to their structural, functional, and immunochemical properties (1,8,11,39), but as yet there is no stable cell line for the production of such particles.…”