Species-Specific Contribution of the Four C-Terminal Amino Acids of Influenza A Virus NS1 Protein to Virulence

Abstract

Large-scale sequence analyses of influenza viruses revealed that nonstructural 1 (NS1) proteins from avian influenza viruses have a conserved C-terminal ESEV amino acid motif, while NS1 proteins from typical human influenza viruses have a C-terminal RSKV motif. To test the influence of the C-terminal domains of NS1 on the virulence of an avian influenza virus, we generated a wild-type H7N1 virus with an ESEV motif and a mutant virus with an NS1 protein containing a C-terminal RSKV motif by reverse genetics. We compared the phenotypes of these viruses in vitro in human, mouse, and duck cells as well as in vivo in mice and ducks. In human cells, the human C-terminal RSKV domain increased virus replication. In contrast, the avian Cterminal ESEV motif of NS1 increased virulence in mice. We linked this increase in pathogenicity in mice to an increase in virus replication and to a more severe lung inflammation associated with a higher level of production of type I interferons. Interestingly, the human C-terminal RSKV motif of NS1 increased viral replication in ducks. H7N1 virus with a C-terminal RSKV motif replicated to higher levels in ducks and induced higher levels of Mx, a type I interferon-stimulated gene. Thus, we identify the C-terminal domain of NS1 as a species-specific virulence domain.

Length Variations in the NA Stalk of an H7N1 Influenza Virus Have Opposite Effects on Viral Excretion in Chickens and Ducks

“…The virus-inoculated ducks showed no clinical symptoms, which was in agreement with previous observations of an asymptomatic replication of influenza viruses in ducks (26). The lungs were overall histologically normal, except for a mild focal pneumonia that was observed in only 2 out of 20 ducks in each virusinoculated group.…”

“…3F, circles) corresponded to animals of the insNA virus group. The observed discrepancy between the substantial cloacal excretion and the infrequent detection of vRNA in the cecum samples could result from the rapid desquamation of differentiated intestinal epithelial cells, which were previously found to be the main site of replication of the H7N1 virus (26). Taken together, our observations suggest that the insNA virus replicated more efficiently than the wt virus in the intestinal tract of ducks.…”

“…A plasmid-based reverse genetics system for this virus has been previously described (26). The NA plasmid was modified by the insertion of a sequence encoding the 22 amino acids ITYENNTWVNQTYVNISNTNFL between residues I 53 and T 54 of the NA stalk, as found in the NA sequence of the A/chicken/Italy/1082/1999 (H7N1) LPAI virus.…”

Prevalence of the C-terminal truncations of NS1 in avian influenza A viruses and effect on virulence and replication of a highly pathogenic H7N1 virus in chickens

“…In addition to the wellknown role as antiviral antagonist, it can affect virus replication, host-range, virulence and tropism mainly shown in mammal models or in-vitro, whereas only few studies have been performed in poultry. 3,12,13,21 Although truncation of the CTE of NS1 has been previously reported using a limited number of sequences, 9,14,22 little is known about the prevalence and distribution of NS1 DCTE in avian influenza viruses of all subtypes. Moreover, the impact of NS1 truncation on virulence particularly the HPAIV H7 viruses in poultry has not been adequately studied.…”

“…10 In addition to its well-defined role as an antiviral antagonist, variations in the Postsynaptic density protein 95, Drosophila disc large tumor suppressor, and Zonula occludens 1 protein domain (PDZ) at the carboxyl terminal end (CTE) of NS1 resulted in expansion of the host range of some influenza viruses in different cell cultures and modulated virulence in mice and poultry. [11][12][13] Size variation of NS1 has been described, mainly due to a stop codon in the ED which results in truncation of the C-terminal end (DCTE). 9, 14 Nonetheless, prevalence and distribution of NS1 DCTE among AIV subtypes and their biological impact in poultry have not been well studied.…”