Intracellular Distribution of NS1 Correlates with the Infectivity and Interferon Antagonism of an Avian Influenza Virus (H7N1)

Highly pathogenic avian influenza viruses of subtype H7N1 that emerged during an outbreak in 1999 and 2000 in Italy differ from their low-pathogenicity precursor viruses by changes in several genes, including three mutations in the NS1 protein. Two of them involve amino acid exchanges located within or closely adjacent to the nuclear export signal of NS1. The third mutation resulted in a new stop codon and thereby a C-terminal truncation of the NS1 protein of the highly pathogenic viruses. To find out whether these mutations contribute to the phenotypic differences between the highly pathogenic and low pathogenic viruses, we generated recombinants of the highly pathogenic A/ostrich/Italy/984/00 strain that contained the nuclear export signal and/or the extended C terminus of NS1 of a low pathogenic virus (A/chicken/Italy/1082/99). Using these recombinants we could demonstrate that replication rate and spread of infection in chicken fibroblast cultures, as well as infectivity for chicken embryos is reduced, whereas the mean death time for chicken embryos is increased, when the highly pathogenic virus acquires the NS1 motifs of the low pathogenic virus. Analysis of beta interferon transcription in chicken fibroblasts infected with the recombinants revealed that the mutations observed in the nuclear export signal of the highly pathogenic viruses were responsible for the enhanced interferon antagonism of these viruses. Cell fractionation and immunofluorescence studies in chicken fibroblasts showed that the nuclear export signal of the highly pathogenic viruses is responsible for cytoplasmic accumulation of NS1, whereas the C-terminal truncation promotes transport into the nucleoli. Comparative analysis in human A549 cells indicated that intracellular distribution of NS1 is host specific. Taken together, these observations support the concept that compartmentalization of NS1 within the cell contributes to the pathogenicity of avian influenza viruses.