Amino acid substitution(s) in the stem-anchor region of langat virus envelope protein attenuates mouse neurovirulence

The identification of variants that are unable to bind membrane receptor preparations (MRPs) has previously been shown to select attenuated yellow fever and Japanese encephalitis viruses. In this study, this methodology has been extended to the tick-borne serocomplex of flaviviruses. Langat (LGT) virus strain TP21 was bound to mouse or human brain MRPs and viruses that escaped binding were isolated and characterized. In addition, variant viruses escaping neutralization by the monoclonal antibody (MAb) 9F9 were also isolated. All of the variant viruses were attenuated for mouse neurovirulence (≥13-fold). Sequence analysis of the prM/E region of the variant viruses identified mutations within the stem-anchor region of the E protein in variants isolated following incubation with mouse or human brain MRPs at a pH ≥ 7.0. The MAb 9F9 variants and MRP variants isolated at pH 5.0, which should induce a conformational shift in the viral E protein, had nearly identical mutations in the prM/M protein immediately N-terminal to the prM/E cleavage site. MAb 9F9 neutralized none of the variant viruses and hemagglutination inhibition assays suggest that the variant virus surface proteins have slightly different conformations compared to the parental virus. These data support previous work indicating that the stem-anchor region of the E protein is important to the surface architecture of the tick-borne flaviviruses. In addition, this study demonstrates that the M protein is at least partially solvent accessible on the virion surface and that the M protein plays a role in maintaining the conformation of the M/E surface complex.