Differential use of importin-α isoforms governs cell tropism and host adaptation of influenza virus

Abstract

Influenza A viruses are a threat to humans due to their ability to cross species barriers, as illustrated by the 2009 H1N1v pandemic and sporadic H5N1 transmissions. Interspecies transmission requires adaptation of the viral polymerase to importin-α, a cellular protein that mediates transport into the nucleus where transcription and replication of the viral genome takes place. In this study, we analysed replication, host specificity and pathogenicity of avian and mammalian influenza viruses, in importin-α-silenced cells and importin-α-knockout mice, to understand the role of individual importin-α isoforms in adaptation. For efficient virus replication, the polymerase subunit PB2 and the nucleoprotein (NP) of avian viruses required importin-α3, whereas PB2 and NP of mammalian viruses showed importin-α7 specificity. H1N1v replication depended on both, importin-α3 and -α7, suggesting ongoing adaptation of this virus. Thus, differences in importin-α specificity are determinants of host range underlining the importance of the nuclear envelope in interspecies transmission.

Figure 1. Growth curves of avian and mammalian H7N7 mutant viruses in importin-silenced human cells.

(a) Endogenous importins (α1–α7) were silenced using siRNA in human lung cells (A549). Importin-α7 antibody crossreacts with importin-α5. The doublet represents importin-α5 (upper band) and importin-α7 (lower band). (b–h) Virus growth in importin-α-silenced A549 cells infected with (b) SC35, (c) SC35M, (d) SC35-PB1_SC35M, (e) SC35-PA_SC35M, (f) SC35-PB2_SC35M, (g) SC35-NP_SC35M and (h) the mutant SC35-PB2_701N-NP_319K. Growth curves show controls (black, filled diamonds), α1 (blue, squares), α3 (green, filled triangles), α4 (black, filled squares), α5 (black diamonds) and α7 (red triangles) silenced cells.

Figure 2. Pathogenicity of SC35M in WT and importin-α-knockout animals.

WT (black square; n=18), α4^−/− (black triangle; n=16), α5^−/− (black cross; n=16), α7^−/− (red triangle; n=16) and α7^ΔIBB/ΔIBB (orange circle; n=18) animals were infected with 100-fold LD₅₀ of SC35M. Control groups received PBS (black diamond). (a) Survival and (b) weight loss were monitored for 14 days. (c) Virus titres in the lung, brain and liver homogenates of controls (black columns) and infected WT (dark grey columns), α7^−/− (light grey columns) and α7^ΔIBB/ΔIBB (white columns) animals were determined by plaque assays. The error bars indicate the standard deviation of viral titres detected in three animals per time point. The detection limit was ≥30 p.f.u. (d) In situ hybridization (upper panels) shows severe infection of epithelial cells with classical signs of primary viral pneumonia with extensive infiltration and destruction of the alveolae in WT mice (upper left panel) in contrast to mostly intact lung tissues of α7^−/− or α7^ΔIBB/ΔIBB animals (upper middle and right panel). Double-labelling experiments (lower panels) demonstrate virus RNA-positive Mac-3+ macrophages in the lungs of WT mice (lower left panel, arrows and inset) but not in α7^−/− or α7^ΔIBB/ΔIBB animals infected for 3 days (lower middle and right panel), respectively. The statistical significance of differences in lung titres of WT and importin-knockout animals were assessed by Student's t-test (*P<0.05; **P<0.01).

Figure 3. Growth curves of various avian and human influenza viruses in importin-silenced human cells.

Endogenous human importins (α1–α7) were silenced using siRNA in human lung cells (A549) and infected with (a) A/FPV/Rostock/1/34 (H7N7), (b) A/Thai/KAN-1/04 (H5N1), (c) A/Victoria/3/75 (H3N2), (d) A/Sachsen-Anhalt/101/09 (H1N1v) or (e) A/Hamburg/NY1580/09 (H1N1v). Growth curves show controls (black, filled diamonds), α1 (blue, squares), α3 (green, filled triangles), α4 (black, filled squares), α5 (black diamonds) and α7 (red triangles)-silenced cells.

Figure 4. Pathogenicity of H5N1 and H1N1v viruses in WT and importin-α7-knockout animals.

WT (black square; n=16), α7^−/− (red triangle; n=16) or α7^ΔIBB/ΔIBB (orange triangle; n=16) animals were infected with 50-fold LD₅₀ of (a, b) A/Thai/KAN-1/04 (H5N1), (c, d) 10-fold LD₅₀ of A/Sachsen-Anhalt/101/09 (H1N1v) or (e, f) 100-fold LD₅₀ of A/Hamburg/NY1580/09 (H1N1v). Control groups received PBS (black diamond). (a, c, e) Survival was monitored for 14 days. (b, d, f) Virus titres in the lung, brain and liver homogenates and in the blood of infected WT, α7^−/− and α7^ΔIBB/ΔIBB animals were determined by plaque assays. The statistical significance of differences in lung titres of WT and importin-knockout animals were assessed by Student's t-test (*P<0.05; **P<0.01). The error bars indicate the standard deviation of viral titres detected in three animals per time point. The detection limit was ≥30 p.f.u.