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. 2008 Mar 18;105(11):4381-6.
doi: 10.1073/pnas.0800482105. Epub 2008 Mar 11.

A new influenza virus virulence determinant: the NS1 protein four C-terminal residues modulate pathogenicity

David Jackson  1 , Md Jaber Hossain, Danielle Hickman, Daniel R Perez, Robert A Lamb
Affiliations

A new influenza virus virulence determinant: the NS1 protein four C-terminal residues modulate pathogenicity

David Jackson et al. Proc Natl Acad Sci U S A. .

Abstract

The virulence of influenza virus is a multigenic trait. One determinant of virulence is the multifunctional NS1 protein that functions in several ways to defeat the cellular innate immune response. Recent large-scale genome sequence analysis of avian influenza virus isolates indicated that four C-terminal residues of the NS1 protein is a PDZ ligand domain of the X-S/T-X-V type and it was speculated that it may represent a virulence determinant. To test this hypothesis, by using mice as a model system, the four C-terminal amino acid residues of a number of influenza virus strains were engineered into the A/WSN/33 virus NS1 protein by reverse genetics and the pathogenicity of the viruses determined. Viruses containing NS1 sequences from the 1918 H1N1 and H5N1 highly pathogenic avian influenza (HPAI) viruses demonstrated increased virulence in infected mice compared with wt A/WSN/33 virus, as characterized by rapid loss of body weight, decreased survival time, and decreased mean lethal dose. Histopathological analysis of infected mouse lung tissues demonstrated severe alveolitis, hemorrhaging, and spread of the virus throughout the entire lung. The increase in pathogenicity was not caused by the overproduction of IFN, suggesting the NS1 protein C terminus may interact with PDZ-binding protein(s) and modulate pathogenicity through alternative mechanisms.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
The generation of mutant viruses. (A) Mutant (rNS1) influenza A viruses were generated by reverse genetics in which mutations were introduced into the C terminus of the A/WSN/33 virus NS1 protein. The C-terminal four amino acids (RSEV) were mutated to introduce sequences from a number of influenza virus strains. Mutations are underlined. Early H1N1, influenza viruses isolated before the 1950s; Late H3N2, H3N2 influenza viruses isolated after 1990; Fiji/83 H1N1-like, the only naturally occurring H1N1 strain isolated containing glutamic acid at residue 227 of a 237-aa NS1 protein (A/Fiji/83). rNS1-trunc contains a four amino acid truncation. Because NS1 and NS2/NEP use overlapping reading frames, the mutant viruses also contained the following amino acid changes in NS2/NEP: rNS1-RSKV, no mutations; rNS1-KSEV, G70S mutation; rNS1-ESEV, G70S mutation; rNS1-ESEV + 7, G70S and F73L mutations; rNS1-EPEV, G70S mutation; rNS1-trunc, L69F and G70S mutations. As argued in Discussion, it seems extraordinarily unlikely that these changes in NS2/NEP are responsible for the observed phenotypes. (B) The expression of viral proteins from infected MDCK cells.
Fig. 2.
Fig. 2.
Viral growth kinetics in MDCK cells. (A) MDCK cells were infected with wt and mutant rNS1 viruses at an MOI of 0.001, supernatants were harvested every 12 h, and samples were titrated by plaque assay in MDCK cells. Results represent the average of three independent experiments. (B) Plaque phenotype of wt and mutant rNS1 influenza viruses in MDCK cells. After titration of viruses cells were fixed in 1% glutaraldehyde and stained with napthylene blue.
Fig. 3.
Fig. 3.
Mouse experiments. Five-week-old BALB/c mice were inoculated intranasally with 104 pfu of wt or mutant rNS1 influenza viruses, with four mice per group. (A) The body weight of mice infected was measured up to 14 days p.i. Mice were euthanized after the loss of 30% of their initial body weight. (B) The data expressed as the percentage survival of mice infected with 104 pfu. (C) The titers of infectious wt or mutant rNS1 influenza virus isolated from homogenized lung tissues 2, 4, or 6 days p.i. Results represent the average infectious titers from the lungs of four infected mice per group.
Fig. 4.
Fig. 4.
Histopathological analysis of infected mouse lung tissue. Wt or mutant rNS1 influenza virus-infected mouse lung samples were preserved in formalin, embedded in paraffin, and sectioned into serial 4-μm sections. Tissues were stained with H&E or antigen-stained by using a goat anti-Udorn polyclonal antiserum. Images were captured at ×20 magnification.
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