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

The H5N1 VN1203-WT virus is extremely virulent in mice [5] , and elicits a host response that contributes in part to its pathogenicity [6] . The H5N1 VN1203-HAavir mutant virus harbors an altered multi-basic cleavage sitea virulence factor important for expanded tissue range [7] [8] [9] and exhibits restricted systemic viral spread due to limited HA susceptibility to furin protease activity. The H5N1 VN1203-PB2627E mutant possesses an amino acid substitution (Lys-to-Glu) at position 627 in the PB2 polymerase subunit. This mutation is known to confer increased polymerase activity in mammalian cells [10] , and also modulates anti-viral activity, apoptosis, and viral clearance [11] . Our newly generated H5N1 VN1203-NS1trunc mutant virus produces a 91 amino acid Cterminal truncation in the effector domain of the NS1 host response antagonist protein. The NS1 protein inhibits RIG-I activation [12] and cellular mRNA processing [13] , and also promotes PI3K activation [13] . The truncation results in the loss of the NS1 nuclear localization signal, a PI3K-binding motif, and binding domains that support interactions with the cellular nuclear proteins CPSF and poly (A)-binding protein II (PABII)two factors that function in the 3'-end-processing of cellular pre-mRNAs [14, 15] . The newly generated H5N1 VN1203-PB1F2del mutant lacks expression of the PB1-F2 protein, potentially impacting an array of functions. PB1-F2 is a viral pathogenicity factor in mammals and birds [16] , and has been shown to modulate viral polymerase activity [17, 18] , enhance lung inflammation [19] , modulate innate immune responses [20, 21] , and demonstrate pro-apoptotic activity [14] . Finally, the CA04-WT virus, which is an H1N1 isolate from the 2009 pandemic season, induces lower pathogenicity in mice relative to VN1203 [5, 22] .