Paired charge-to-alanine mutagenesis of dengue virus type 4 NS5 generates mutants with temperature-sensitive, host range, and mouse attenuation phenotypes

Charge-to-alanine mutagenesis of dengue virus type 4 (DEN4) NS5 gene generated a collection of attenuating mutations for potential use in, a recombinant live, attenuated DEN vaccine. Codons for 80 contiguous pairs of charged amino acids in NS5 were individually mutagenized to create uncharged pairs of alanine residues, and 32 recombinant mutant viruses were recovered from the 80 full-length mutant DEN4 cDNA constructs. These mutant viruses were tested for temperature-sensitive (ts) replication in both Vero cells and HuH-7 human hepatoma cells. Of the 32 mutants, 13 were temperature sensitive (ts) in both cell lines, 11 were not ts in either cell line, and 8 exhibited a host range (tshr) phenotype. One tshr mutant was ts only in Vero cells, and seven were ts only in HuH-7 cells. Nineteen of the 32 mutants were 10-fold or more restricted in replication in the brains of suckling mice compared to that of wild-type DEN4, and three mutants were approximately 10,000-fold restricted in replication. The level, of temperature sensitivity of replication in vitro did not correlate with attenuation in vivo. A virus bearing two pairs of charge-to-alanine mutations was constructed and demonstrated increased temperature sensitivity and attenuation relative, to either parent virus. This large set of charge-to-alanine mutations specifying a wide range of attenuation for mouse brain should prove useful in fine-tuning recombinant live attenuated DEN vaccines.