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Neuroblastoma cell-adapted yellow fever virus: mutagenesis of the E protein locus involved in persistent infection and its effects on virus penetration and spread

Thomas J. Chambers

Department of Molecular Microbiology and Immunology, St Louis University Health Sciences Center, 1402 South Grand Avenue, St Louis, MO 63104, USA

Correspondence
Thomas J. Chambers
chambetj{at}slu.edu

Persistent infection of mouse neuroblastoma NB41A3 cells with yellow fever 17D virus generates viral variants which exhibit defective cell penetration, poor cell-to-cell spread, small plaque size and reduced growth efficiency, caused by substitution of glycine for aspartic acid or glutamic acid at positions 360 and 362 in the envelope protein. These positions occur within a charge cluster, Asp₃₆₀-Asp₃₆₁-Glu₃₆₂, located in domain III, near its interface with domain I. To characterize further the molecular basis for the variant phenotype, a series of mutant viruses containing substitutions at position 360, 361 and 362, were studied for effects on the cell culture properties typical of the neuroblastoma-adapted variant. Most substitutions at position 360 gave rise to viruses that were very defective in cell penetration, growth efficiency and cell-to-cell spread, whereas substitution with glutamic acid yielded a virus indistinguishable from parental yellow fever 17D. Substitution with lysine was not tolerated and substitution with asparagine resulted in frequent wild-type revertants. A glycine residue was not tolerated at position 361, but substitution at 362 yielded a small plaque virus, similar to the effect of substitution at position 360. These data indicate that the yellow fever virus E protein contains a locus within domain III where a negative-charge cluster is important for optimal function of this domain in virus-cell interactions beyond the stage of virus attachment. Modelling predictions suggest that the mutations alter the local properties of the loop within domain III, and may compromise interactions of this domain with an adjacent region of domain I during conformational changes that occur in the E protein in association with virus entry.

Present address: Merck & Co. Inc., PO Box 4, BLB-22, West Point, PA 19486, USA.

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