Specific mutations in H5N1 mainly impact the magnitude and velocity of the host response in mice

Tchitchek, Nicolas; Eisfeld, Amie J; Tisoncik-Go, Jennifer; Josset, Laurence; Gralinski, Lisa E; Bécavin, Christophe; Tilton, Susan C; Webb-Robertson, Bobbie-Jo; Ferris, Martin T; Totura, Allison L; Li, Chengjun; Neumann, Gabriele; Metz, Thomas O; Smith, Richard D; Waters, Katrina M; Baric, Ralph; Kawaoka, Yoshihiro; Katze, Michael G

All recombinant influenza virusesincluding H1N1 CA04 wild-type, the H5N1 VN1203 wild-type and the panel of four H5N1 VN1203 mutant viruses encoding changes in specific pathogenicity markerswere generated by using reverse genetics coupled with site-directed mutagenesis (for mutant viruses), essentially as described previously [18, 44, 45] .The H5N1 VN1203 mutant viruses included the following: a virus in which the hemagglutinin (HA) surface protein poly-basic cleavage site (RERRRKKR↓G) was mutated to (RETR↓G) (referred to as H5N1 VN1203-HAavir mutant), which has been previously described [2] ; a virus possessing a K→E amino acid substitution at residue 627 of the PB2 protein (referred to as H5N1 VN1203-PB2627E), which was previously described [4] ; a newly generated virus in which a stop codon mutation was introduced into the NS1 protein open reading frame at amino acid 124, which was achieved by a T→A nucleotide substitution at position 400 of the H5N1 VN1203 NS gene segment (referred to as H5N1 VN1203-NS1trunc); and a newly generated virus in which the PB1-F2 open reading frame was eliminated by introduction of three stop codon mutations (affected nucleotide positions in the H5N1 VN1203 PB1 gene segment: T120C, C153G, G291A; referred to as H5N1 VN1203-PB1F2del). Mutations in the PB1-F2 and NS1 open reading frames did not affect the coding sequences of collinear PB1 and NS2 proteins, respectively. Primer sequences are available upon request.