ABSTRACT
The authors studied a possible role of the caspase cleavage motif located in the nucleoprotein (NP) of pandemic influenza virus H1N1 in the regulation of viral virulence properties. A reverse genetics method was used to obtain chimeric seasonal-like mouse-adapted influenza virus hvA/PE/8/34 (H1N10) carrying either the NP gene of wild type pandemic virus with incomplete caspase motif ETGC or mutated pandemic NP with natural caspase cleavage site of human type ETDG. The wild-type NP gene of the pandemic virus was found to poorly fit to the gene pattern of closely related seasonal-like hvA/PR/8/34 virus (H1N1) and did not rescue mature virus production whereas a mutated NP with human-type caspase cleavage site maintained gene fitness, giving rise to a chimeric virus. The generated chimeric virus hvA/PR/8/34 carrying the mutated pandemic NP successfully replicated in the murine lung, but was attenuated and did not reach the virulence level of seasonal-like mouse-adapted virus hvA/PR/8/34. The findings indicate that the NP caspase cleavage site plays a role in viral adaptation and viral virulence in mammals.
Subject(s)
Caspases/metabolism , Genes, Viral , Influenza A Virus, H1N1 Subtype , RNA-Binding Proteins , Viral Core Proteins , Adaptation, Biological , Animals , Birds , Cell Line , Chick Embryo , Humans , Influenza A Virus, H1N1 Subtype/pathogenicity , Influenza A Virus, H1N1 Subtype/physiology , Influenza in Birds/virology , Influenza, Human/virology , Lung/virology , Mice , Molecular Sequence Data , Mutant Proteins/chemistry , Mutant Proteins/genetics , Mutant Proteins/metabolism , Nucleocapsid Proteins , Point Mutation , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Swine , Viral Core Proteins/chemistry , Viral Core Proteins/genetics , Viral Core Proteins/metabolism , Virulence/genetics , Virus Replication/geneticsABSTRACT
A reverse genetics approach was applied to generate variants of avian influenza virus A/FPV/Ro/34 (H7N1) containing mutations in the caspase cleavage sites of NP and M2 proteins. Mutation Gly16 --> Asp in avian virus NP made this protein (NPgd) sensitive to caspases, like human virus NP, and permitted its cleavage in infected cells. Mutant recombinant virus NPgd was able to replicate and stably carried Gly --> Asp mutation during passages in cultured cells, chicken eggs, and chickens. This variant was found to have significantly decreased virulence for chickens comparatively to wild type recombinant virus (wtr). Virus variants characterized by deletion Gly16 in NP (NPdel) and mutated caspase cleavage site VDVDD87 --> VNVND87 in M2 (M2nn) protein were shown to lack intracellular caspase-dependent cleavage of NP and M2, respectively, and to retain their ability to replicate in different hosts. Variant NPdel, like wide type virus, displayed a high chicken virulence whereas M2nn, like NPgd one, was found to possess a low virulent phenotype. The findings suggest that the mutations altering natural caspase cleavage motifs in NP and M2 do not restrict virus replication ability but can significantly reduce the virulent potential of the mutant viruses. Recombinant virus variants with altered caspase cleavage motifs could be proposed as a matrix for the design of live recombinant vaccines.
