Envelope protein glycosylation status influences mouse neuroinvasion phenotype of genetic lineage 1 West Nile virus strains

Abstract

The introduction of West Nile virus (WNV) into North America has been associated with relatively high rates of neurological disease and death in humans, birds, horses, and some other animals. Previous studies identified strains in both genetic lineage 1 and genetic lineage 2, including North American isolates of lineage 1, that were highly virulent in a mouse neuroinvasion model, while other strains were avirulent or significantly attenuated (D. W. C. Beasley, L. Li, M. T. Suderman, and A. D. T. Barrett, Virology 296:17-23, 2002). To begin to elucidate the basis for these differences, we compared a highly virulent New York 1999 (NY99) isolate with a related Old World lineage 1 strain, An4766 (ETH76a), which is attenuated for mouse neuroinvasion. Genomic sequencing of ETH76a revealed a relatively small number of nucleotide (5.1%) and amino acid (0.6%) differences compared with NY99. These differences were located throughout the genome and included five amino acid differences in the envelope protein gene. Substitution of premembrane and envelope genes of ETH76a into a NY99 infectious clone backbone yielded a virus with altered in vitro growth characteristics and a mouse virulence phenotype comparable to ETH76a. Further site-specific mutagenesis studies revealed that the altered phenotype was primarily mediated via loss of envelope protein glycosylation and that this was associated with altered stability of the virion at mildly acidic pH. Therefore, the enhanced virulence of North American WNV strains compared with other Old World lineage 1 strains is at least partly mediated by envelope protein glycosylation.

FIG. 1.

Western blot showing the differing mobilities of E proteins of WNV strains consistent with the presence of Asn-Tyr-Ser glycosylation motif or mutant Ser-Tyr-Ser at residues 154 to 156. Proteins were separated on a 5%/10% discontinuous sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel, electrophoretically transferred to 0.2-μm nitrocellulose, and detected using WNV-specific monoclonal antibody 7H2. The suffix “mb” indicates strains isolated from the brain of a mouse that died following peripheral inoculation with the respective parent strain.

FIG. 2.

Comparisons of (a) growth kinetics in Vero cells at an MOI of approximately 0.02 and (b) sensitivity to inactivation at mildly acidic pH for WNV strains (approximately 10⁵ input PFU for each) with glycosylated or nonglycosylated E protein sequences. Error bars in panel a are 1 standard deviation.