Single amino acid substitutions in Puumala virus envelope glycoproteins G1 and G2 eliminate important neutralization epitopes.
Two monoclonal antibody escape virus mutants (MARs), rescued from a human MAb to glycoprotein 2 (G2) and a bank vole monoclonal antibody (MAb) directed to glycoprotein 1 (G1) of Puumala virus, strain Sotkamo, were produced by using a combination of neutralization tests and antigen detection. The MARs and the original virus were analyzed by nucleotide sequencing and the responsible mutations were defined and characterized. The G1 mutation was found to constitute an A to T nucleotide substitution, giving raise to an aspartic acid to valine mutation at residue 272, potentially increasing the hydrophobicity of this region. The G2 mutation was found to constitute a C to T substitution, altering the residue 944 from serine into the more hydrophobic phenylalanine and resulting in secondary structure alterations. The mutation was found to be in close vicinity to a glycosylation site. Synthetic peptides covering the regions of the native virus, defined by the MARs, were produced and evaluated for reactivity with the corresponding MAb. The peptides were not recognized by the MAbs, and did not inhibit the binding of the MAbs in competition assays. Sera from mice immunized with the peptides were not able to recognize the native protein. This indicates that the epitopes are non-linear and/or glycosylated in the native state, or alternatively, that the G1 and G2 MAbs binds to regions away from the mutations.
Hörling
J
J
Swedish Institute of Infectious Disease Control, Stockholm, Sweden. Jan.Horling@eu.pnu.com
Lundkvist
A
A
D000595:Amino Acid Sequence; D000818:Animals; D000956:Antigens, Viral / Q000235:genetics / Q000276:immunology*; D001483:Base Sequence; D002522:Chlorocebus aethiops; D004279:DNA, Viral; D001330:Electronic Data Processing; D000939:Epitopes / Q000235:genetics / Q000276:immunology*; D006023:Glycoproteins / Q000235:genetics / Q000276:immunology*; D006476:Orthohantavirus / Q000235:genetics / Q000276:immunology*; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009500:Neutralization Tests; D010455:Peptides; D017354:Point Mutation; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D014709:Vero Cells; D014759:Viral Envelope Proteins / Q000235:genetics / Q000276:immunology*
D000956:Antigens, Viral; D004279:DNA, Viral; D000939:Epitopes; D006023:Glycoproteins; D010455:Peptides; D014759:Viral Envelope Proteins