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 pr eparations (MRPs) has previously been shown to select attenuated yellow fev er and Japanese encephalitis viruses. In this study, this methodology has b een extended to the tick-borne serocomplex of flaviviruses. Langat (LGT) vi rus strain TP21 was bound to mouse or human brain MRPs and viruses that esc aped binding were isolated and characterized. In addition, variant viruses escaping neutralization by the monoclonal antibody (MAb) 9F9 were also isol ated. All of the variant viruses were attenuated for mouse neurovirulence ( greater than or equal to 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 greater than or equal to 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 protei n immediately N-terminal to the prM/E cleavage site. MAb 9F9 neutralized no ne of the variant viruses and hemagglutination inhibition assays suggest th at the variant virus surface proteins have slightly different conformations compared to the parental virus. These data support previous work indicatin g that the stem-anchor region of the E protein is important to the surface architecture of the tick-borne flaviviruses. In addition, this study demons trates that the M protein is at least partially solvent accessible on the v irion surface and that the M protein plays a role in maintaining the confor mation of the M/E surface complex, (C) 2001 Academic Press.