Mutational analysis of the West Nile virus NS4B protein.
West Nile virus NS4B is a small hydrophobic nonstructural protein approximately 27 kDa in size whose function is poorly understood. Amino acid substitutions were introduced into the NS4B protein primarily targeting two distinct regions; the N-terminal domain (residues 35 through 60) and the central hydrophobic domain (residues 95 through 120). Only the NS4B P38G substitution was associated with both temperature-sensitive and small-plaque phenotypes. Importantly, this mutation was found to attenuate neuroinvasiveness greater than 10,000,000-fold and lower viremia titers compared to the wild-type NY99 virus in a mouse model. Full genome sequencing of the NS4B P38G mutant virus revealed two unexpected mutations at NS4B T116I and NS3 N480H (P38G/T116I/N480H), however, neither mutation alone was temperature sensitive or attenuated in mice. Following incubation of P38G/T116I/N480H at 41°C, five mutants encoding compensatory substitutions in the NS4B protein exhibited a reduction in the temperature-sensitive phenotype and reversion to a virulent phenotype in the mouse model.
Department of Microbiology and Immunology, Sealy Center for Vaccine Development, Institute for Human Infections and Immunity, University of Texas Medical Branch at Galveston, TX 77555-0609, USA.
Jason A
JA
Wicker
Melissa C
MC
Whiteman
David W C
DW
Beasley
C Todd
CT
Davis
Charles E
CE
McGee
J Ching
JC
Lee
Stephen
S
Higgs
Richard M
RM
Kinney
Claire Y H
CY
Huang
Alan D T
AD
Barrett
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D004252:DNA Mutational Analysis; D005260:Female; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D020125:Mutation, Missense; D016415:Sequence Alignment; D017361:Viral Nonstructural Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D014774:Virulence; D014902:West Nile virus / Q000737:chemistry / Q000235:genetics* / Q000254:growth & development / Q000472:pathogenicity
D017361:Viral Nonstructural Proteins