Interaction of polymerase subunit PB2 and NP with importin alpha1 is a determinant of host range of influenza A virus

Abstract

We have previously reported that mutations in the polymerase proteins PB1, PB2, PA, and the nucleocapsid protein NP resulting in enhanced transcription and replication activities in mammalian cells are responsible for the conversion of the avian influenza virus SC35 (H7N7) into the mouse-adapted variant SC35M. We show now that adaptive mutations D701N in PB2 and N319K in NP enhance binding of these proteins to importin alpha1 in mammalian cells. Enhanced binding was paralleled by transient nuclear accumulation and cytoplasmic depletion of importin alpha1 as well as increased transport of PB2 and NP into the nucleus of mammalian cells. In avian cells, enhancement of importin alpha1 binding and increased nuclear transport were not observed. These findings demonstrate that adaptation of the viral polymerase to the nuclear import machinery plays an important role in interspecies transmission of influenza virus.

Figure 1. Interaction of PB2 and NP with Importin α1 in Mammalian Cells

293T cells were transfected with plasmids encoding SC35-PB2 (30 μg), SC35M-PB2 (30 μg), and SC35-PB2 D701N (30 μg) (A) and SC35-NP (10 μg) and SC35M-NP (10 μg) (B) proteins. As a control, non-transfected cells were used (Mock). 48 h after transfection cells were harvested and immunoprecipitated using the respective PB2 or NP antibody. Precipitated eluates (1–3) and non-precipitated whole cell lysates of transfected cells were subjected to SDS gel electrophoresis, and PB2, NP, and importin α1 were detected by Western blot analysis. In (B) two NP bands can be seen of which the minor one most likely represents a cleavage product [39]. The total amount of importin α1 bound in all three eluates to PB2 (C) and NP (D) was quantitated using Image J software (http://rsb.info.nih.gov/ij/). An arbitrary unit is the amount of importin α1 bound to SC35 PB2 and SC35 NP, respectively.

Figure 2. Interaction of PB2 and NP with Importin α1 in Avian Cells

CEC-32 cells were transfected with plasmids encoding SC35-PB2 (30 μg), SC35M-PB2 (30 μg), and SC35-PB2 D701N (30 μg) (A) and SC35-NP (10 μg) and SC35M-NP (10 μg) (B) proteins. As a control, non-transfected cells were used (Mock). 48 h after transfection cells were harvested and immunoprecipitated using the respective PB2 or NP antibody. Precipitated eluates (1–3) and non-precipitated whole cell lysates of transfected cells were subjected to SDS gel electrophoresis, and PB2, NP, and importin α1 were detected by Western blot analysis. Importin α1 bound to PB2 (C) and NP (D) was quantitated as described in the legend of Figure 1.

Figure 3. Importin α1 Distribution in Mammalian and Avian Cells

293T or CEC-32 cells were infected at MOI 2 with SC35 or SC35M. 293T cells were harvested 6 h p.i. (A) and 12 h p.i. (B), and CEC-32 cells were harvested 9 h p.i. (C) and 18 h p.i. (D) and separated into nuclear and cytoplasmic fractions. NP, importin α1, importin ß1, and ß-actin were determined by Western blot analysis.

Figure 4. Kinetics of Nuclear Transport of Importin α1

293T cells were infected at MOI 2 with SC35 and SC35M, and the localization of human importin α1 was determined by immunofluorescence assays at different time points after infection (2 h p.i., 4 h p.i., 6 h p.i., and 8 h p.i.). For detection of importin α1, FITC-coupled secondary antibody was used. Cell nuclei were stained with DAPI.

Figure 5. Distribution of Importin α1 in Mammalian Cells

293T cells were infected at MOI 2 with SC35, SC35M, SC35-PB1_SC35M, SC35-PB2_SC35M, SC35-PA_SC35M, SC35-NP_SC35M, SC35-PB2_701N, and SC35-PB2_714R. Cells were harvested 6 h p.i. and separated into nuclear and cytoplasmic fractions. NP, importin α1, importin ß1, and ß-actin were determined by Western blot analysis.

Figure 6. Localization of PB2, NP, and Importin α1 in Mammalian Cells by Immunofluorescence Analysis

A549 cells were infected with SC35, SC35M, SC35-PB2_SC35M, and SC35-PB2_701N (MOI 2). 6 h p.i. cells were stained with antisera specific for importin α1 (secondary antibody FITC-coupled) and PB2 (secondary antibody Rhodamine-coupled) (A) or importin α1 (secondary antibody Rhodamine-coupled) and NP (secondary antibody FITC-coupled) (B). The amounts of PB2, NP, and importin α1 in the nucleus and the cytoplasm are indicated by bars. Quantification was done as described in Materials and Methods.

Figure 7. Localization of PB2, NP, and Importin α1 in Avian Cells

CEC-32 cells were infected with SC35 and SC35M at MOI 2. 12 h p.i. cells were stained with antisera specific for importin α1 (secondary antibody FITC-coupled) and viral PB2 (secondary antibody Rhodamine-coupled) (A) or importin α1 (secondary antibody Rhodamine-coupled) and NP (secondary antibody FITC-coupled) (B). The amounts of PB2, NP, and importin α1 in the nucleus and the cytoplasm are indicated by bars. Quantification was done as described in Materials and Methods.