The NS segment of an H5N1 highly pathogenic avian influenza virus (HPAIV) is sufficient to alter replication efficiency, cell tropism, and host range of an H7N1 HPAIV

Abstract

A reassortant avian influenza virus (designated FPV NS GD), carrying the NS-segment of the highly pathogenic avian influenza virus (HPAIV) strain A/Goose/Guangdong/1/96 (GD; H5N1) in the genetic background of the HPAIV strain A/FPV/Rostock/34 (FPV; H7N1), was rescued by reverse genetics. Remarkably, in contrast to the recombinant wild-type FPV (rFPV), the reassortant virus was able to replicate more efficiently in different human cell lines and primary mouse epithelia cells without prior adaptation. Moreover, FPV NS GD caused disease and death in experimentally infected mice and was detected in mouse lungs; in contrast, rFPV was not able to replicate in mice effectively. These results indicated an altered host range and increased virulence. Furthermore FPV NS GD showed pronounced pathogenicity in chicken embryos. In an attempt to define the molecular basis for the apparent differences, we determined that NS1 proteins of the H5N1 and H7N1 strains bound the antiviral kinase PKR and the F2F3 domain of cleavage and polyadenylation specificity factor 30 (CPSF30) with comparable efficiencies in vitro. However, FPV NS GD infection resulted in (i) increased expression of NS1, (ii) faster and stronger PKR inhibition, and (iii) stronger beta interferon promoter inhibition than rFPV. Taken together, the results shed further light on the importance of the NS segment of an H5N1 strain for viral replication, molecular pathogenicity, and host range of HPAIVs and the possible consequences of a reassortment between naturally occurring H7 and H5 type HPAIVs.

FIG. 1.

Growth curves of rFPV and reassortant FPV NS GD. MDCK cells were infected at an MOI of 0.001 with rFPV (⧫) or reassortant FPV NS GD (▴). Supernatants were monitored in multicycle replication for HA titers (A) and for infectious particles (PFU/ml) (B) at the indicated time points. The P value (n.s., not significant [>0.05]; *, <0.05; **, <0.01) is given in comparison to rFPV. The experiments were done in duplicate and analyzed in duplicate titrations. Embryonated chicken eggs (5 eggs per dose) were infected with the indicated amount of PFU of each virus (rFPV, black; FPV NS GD, gray) and analyzed 36 h p.i. for HA titers (C) and infectious particles (D). The production of infectious particles in single-cycle replication was also monitored (E). MDCK monolayers were infected with rFPV or reassortant FPV NS GD and incubated for 48 h under a viscous overlay, which was then removed. Cells were stained to visualize focus size (F).

FIG. 2.

Induction of the IFN-β promoter. (A) MDCK cells were transfected with the IFN-ß promoter luciferase reporter plasmid (pIFN-Luci). At 24 h p.t., cells were either mock infected or infected with the rFPV or the reassortant FPV NS GD (MOI of 1). Cell extracts were prepared at 4 h p.i. and assayed for luciferase activity. The results are representative of four independent transfections/infections and are given as the ratio of luciferase activity relative to activity of the mock-infected control set to 1. The P value (*, <0.05; **, <0.01; ***, <0.001) is given in comparison to either (−) virus or rFPV-infected cells. (B) MDCK cells were transfected with the IFN-ß promoter luciferase reporter plasmid (pIFN-Luci) and either an empty vector or a plasmid expressing the FPV or GD NS1 protein. Twenty-four hours p.t., cells were transfected with poly(I·C) and assayed 4 h later for reporter gene activity. Shown is the difference in reporter activity between poly(I·C)-stimulated cells and nonstimulated cells. Data represent the mean of three independent experiments and are given as relative luciferase activity.

FIG. 3.

Survival rate of C57BL/6 mice after infection with rFPV or FPV NS GD. Female C57BL/6 mice were intranasally infected with 10⁵ PFU of either rFPV (n = 9) or FPV NS GD (n = 10). (A) The average weight of the surviving mice infected with rFPV (⧫) and of surviving mice infected with FPV NS GD (▵) or of mice that succumbed to infection with FPV NS GD (▴) measured at the indicated time points. (B) Survival rate of mice infected with rFPV (⧫) and FPV NS GD (▴).

FIG. 4.

Transient expression of the GD NS1 enhances the rFPV titers. MDCK cells were transfected with either an empty vector or a plasmid expressing the FPV or GD NS1 protein. At 24 h p.t., cells were infected with rFPV (MOI of 1), and virus titers were determined at 8 h p.i. The results are representative of four (vector and FPV NS1) and two (GD NS1) independent experiments calculated in duplicate titrations. P value (n.s., not significant [>0.05]; **, <0.01) is given in comparison to FPV NS1- and GD NS1-transfected cells.

FIG. 5.

NS1 protein expression, inhibition of PKR activity, and PKR binding capacity. (A) Expression levels of NS1 and NP protein in cells infected with either rFPV (⧫) or FPV NS GD (▴) (MOI of 1). The relative NS1 and NP amounts from six independent experiments each determined by in-cell Western using NS1- and NP-specific MAbs at the indicated time points were normalized to the loading control (ERK2). (B) Inhibition of PKR activity. MDCK cells were infected with rFPV or FPV NS GD (MOI of 1), and eIF2α-phosphorylation was determined by Western blotting using phospho-specific anti-eIF2α MAb (P-eIF2α) at the indicated time points. The relative P-eIF2α amounts from four independent experiments were normalized to the loading control (eIF2α) and set in relation to the amount of NP (control for viral expression) normalized to the loading control (eIF2α). (C) PKR binding capacity of the NS1 protein of FPV and GD. In vitro translated NS1 proteins of FPV and GD were labeled with [³⁵S]methionine and incubated with the GST-PKR (K296R)-Sepharose beads. The precipitated proteins were separated by SDS-gel electrophoresis, and the amount of NS1 was visualized by autoradiography.

FIG. 6.

The NS1 proteins from FPV and GD have similar F2F3-binding capacities. In a GST pulldown assay, in vitro translated and ³⁵S-labeled NS1 proteins of FPV, GD, or PR8 were mixed with F2F3-GST or H₂O as a control and precipitated using glutathione-Sepharose (GS4B) after incubation. The precipitated proteins were separated by SDS-gel electrophoresis. The amounts of input and bound NS1 protein were visualized by autoradiography, and the amount of F2F3-GST was determined by Coomassie staining of the SDS-PAGE gel. Bound NS1 was normalized to the amount of F2F3-GST. Data presented represent the means of three independent experiments and are given as the relative amount of bound NS1.