Attenuation of tick-borne encephalitis virus by structure-based site-specific mutagenesis of a putative flavivirus receptor binding site

The impact of a specific region of the envelope protein E of tick-borne enc ephalitis (TBE) virus on the biology of this virus was investigated by a si te-directed mutagenesis approach. The four amino acid residues that were an alyzed in detail (E308 to E311) are located on the upper-lateral surface of domain III according to the X-ray structure of the TEE 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 substitut ions, as well as combinations of mutations, were constructed and analyzed f or their virulence in mice, growth properties in cultured cells, and geneti c stability. The most significant attenuation in mice was achieved by mutag enesis of threonine 310. Combining this mutation with deletion mutations in the 3'-noncoding region yielded mutants that were highly attenuated, The b iological 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) th at arose spontaneously during infection of a mouse, Mutagenesis of the othe r positions provided evidence for the functional importance of residue 308 (Asp) and its charge interaction with residue 311 (Lys), whereas residue 30 9 could be altered or even deleted without any notable consequences. Deleti on of residue 309 was accompanied by a spontaneous second-site mutation (Ph e to Tyr) at position 332, which in the three-dimensional structure of prot ein E is spatially close to residue 309. The information obtained in this s tudy is relevant for the development of specific attenuated flavivirus stra ins that may serve as future live vaccines.