Targeting Importin-α7 as a Therapeutic Approach against Pandemic Influenza Viruses

Abstract

Viral drug resistance is believed to be less likely to occur if compounds are directed against cellular rather than viral proteins. In this study, we analyzed the feasibility of a crucial viral replication factor, namely, importin-α7, as a cellular drug target to combat pandemic influenza viruses. Surprisingly, only five viral lung-to-lung passages were required to achieve 100% lethality in importin-α7⁻/⁻ mice that otherwise are resistant. Viral escape from importin-α7 requirement was mediated by five mutations in the viral ribonucleoprotein complex and the surface glycoproteins. Moreover, the importin-α7⁻/⁻ mouse-adapted strain became even more virulent for wild-type mice than the parental strain. These studies show that targeting host proteins may still result in viral escape by alternative pathways, eventually giving rise to even more virulent virus strains. Thus, therapeutic intervention strategies should consider a multitarget approach to reduce viral drug resistance. IMPORTANCE Here, we investigated the long-standing hypothesis based on in vitro studies that viral drug resistance occurrence is less likely if compounds are directed against cellular rather than viral proteins. Here, we challenged this hypothesis by analyzing, in an in vivo animal model, the feasibility of targeting the cellular factor importin-α7, which is crucial for human influenza virus replication and pathogenesis, as an efficient antiviral strategy against pandemic influenza viruses. In summary, our studies suggest that resistance against cellular factors is possible in vivo, and the emergence of even more virulent viral escape variants calls for particular caution. Thus, therapeutic intervention strategies should consider a multitarget approach using compounds against viral as well as cellular factors to reduce the risk of viral drug resistance and potentially increased virulence.

FIG 1

Adaptation of 2009 pH1N1 influenza virus to α7^−/− mice. (A) Pathogenicity of pH1N1-MA7 influenza virus. WT (black squares) or α7^−/− (open squares) mice were intranasally inoculated with 10⁵ PFU of pH1N1-MA7 virus. As controls, a group of α7^−/− mice were infected with the parental strain 2009 pH1N1 (open circles) or received PBS only (gray circle). Weight loss was monitored for 14 days. Data shown represent means ± SEM (n = 5 to 13). (B) MLD₅₀ of pH1N1 and pH1N1-MA7 strains in WT and α7^−/− mice. (C) Amino acid sequence differences are shown for parental pH1N1 and adapted pH1N1-MA7 viruses.

FIG 2

Pathogenicity of pH1N1-MA7 recombinant viruses in WT and α7^−/− mice. (A) WT (black square) or α7^−/− (open square) mice were intranasally inoculated with recombinant viruses, i.e., 10⁴ PFU of adapted pH1N1-MA7_rec, 10⁵ PFU of pH1N1-PA_MA7, 10⁵ PFU of pH1N1-NP_MA7, 10⁵ PFU of pH1N1-PA,NP_MA7, 10⁵ PFU of pH1N1-HA_MA7, and 10⁵ PFU of pH1N1-NA_MA7. Weight loss and survival was monitored for 14 days. Control mice received PBS only (gray circle). Data shown represent means ± SEM (n = 5 to 15). (B) Weight loss has been summarized as representing the mean area under the curve (AUC). WT (black bars) or α7^−/− (white bars) mice were infected with 10⁵ PFU of wild-type viruses (pH1N1 and pH1N1-MA7) and recombinant viruses (pH1N1-PA_MA7, pH1N1-NP_MA7, pH1N1-PA,NP_MA7, pH1N1-HA_MA7, and pH1N1-NA_MA7). Control mice received PBS (gray bar). Data shown represent means ± SEM (n = 5 to 15; P < 0.05 [*], P < 0.01 [**], and P < 0.001 [***], all by Student's t test compared to the control group).

FIG 3

Localization of the adaptive mutations in pH1N1 protein structure and their effect on structural stability. (A) Structural models of the regions surrounding the adaptive mutations of pH1N1-MA7 with homologous structures. Adaptive mutations are indicated in blue. Relevant sites in the structures are indicated in orange (PA endonuclease active site, RNA binding site and oligomerization domain of NP, HA receptor binding site, and NA active site). (B) Global z score (as boxplots) and local energy comparison between the mutants of pH1N1-MA7 and their respective wild types (pH1N1). The x axis shows the corresponding amino acid position, and the y axis shows the energy change. Each set is composed of 30 structural models. Pair (red), surface (blue), and combined (black) energy are shown for each analysis. Dots placed above the z score distributions of the pH1N1 models represent the score of the structural template.

FIG 4

Biological activity of recombinant RNPs with adaptive mutations in epithelial HEK and HEK-shα7 cell lines. (A) Human HEK-WT (black bars) and HEK-shα7 (white bars) cells were cotransfected with plasmids expressing PB1, PA, NP, and PB2, as well as a plasmid encoding Renilla luciferase and a plasmid encoding firefly luciferase in negative polarity, flanked by the nontranslated regions of influenza NP segment. Plasmid expressing PB1 was omitted as a negative control. Plasmids encoding PA_MA7 and NP_MA7 were used when indicated, and both were used in pH1N1-MA7. At 20 h posttransfection, luciferase accumulation was determined. Values were normalized to Renilla expression, and the activity of the pH1N1 RNP in HEK-WT cells was set to 100% (means ± SEM; n = 9 to 18; ***, P < 0.001 by Student's t test compared to pH1N1 RNP activity in HEK-WT cells). (B) Confirmation of importin-α7 knockdown in human HEK cells by Western blotting. Since importin-α7 antibody cross-reacts with importin-α5, the doublet represents importin-α5 (upper band) and importin-α7 (lower band). GAPDH was used as a loading control.

FIG 5

Importin-α binding affinities to NP monomers with adaptive mutation. (A) Human HEK-WT cells were cotransfected with plasmids encoding FLAG-tagged importins (α1, α3, or α7) and NP or NP_MA7. NP-only transfected cells served as a control (cont). At 48 h posttransfection, importins were immunoprecipitated using the FLAG tag, and coimmunoprecipitated NP was determined by Western blotting. β-Actin was used as a loading control in the input samples. (B) Quantification of NP (black bars) or NP_MA7 (dashed bars) binding to overexpressed importins. Values of NP were normalized against precipitated importin-α levels, and the relative amount of NP bound to importin-α1 was set to 100%. Data shown represent means ± SEM (n = 3).

FIG 6

Replication kinetics in epithelial A549 and A549-shα7 cell lines infected with pH1N1-MA7 virus. (A) Human A549-WT and A549-shα7 cells were infected at an MOI of 0.1 with parental (pH1N1) or adapted (pH1N1-MA7) viruses. Virus titers of the supernatants were measured by plaque assay at the indicated hours p.i. Data are expressed as means ± SEM of viral titers (n = 8 to 12). (B) Confirmation of importin-α7 knockdown in human A549 cells by Western blotting. Since importin-α7 antibody cross-reacts with importin-α5, the doublet represents importin-α5 (upper band) and importin-α7 (lower band). GAPDH was used as a loading control.

FIG 7

Viral RNA accumulation in WT and α7^−/− MEFs infected with pH1N1-MA7 virus. (A) WT and α7^−/− MEFs were infected at an MOI of 2 with parental (pH1N1), adapted (pH1N1-MA7), or SGR (pH1N1-PA_MA7, pH1N1-NP_MA7, pH1N1-PA,NP_MA7, pH1N1-HA_MA7, and pH1N1-NA_MA7) viruses. After 24 h, total RNA was isolated and analyzed by NP gene-specific primer extension. A primer specific for 5S rRNA was used for normalization. (B) vRNA accumulation in MEF-WT (black bars) and MEF-α7^−/− (white bars) infected with the parental and adapted viruses. Values were expressed relative to vRNA levels in MEF-WT infected with pH1N1 virus, which was set to 100%. (C) vRNA accumulation was expressed relative to vRNA levels in MEF-WT, which was set to 100% for every virus tested. Data shown represent means ± SEM (n = 9 to 12). (D) Importin-α7 expression in MEF cells was checked by Western blotting. Since importin-α7 antibody cross-reacts with importin-α5, the doublet represents importin-α5 (upper band) and importin-α7 (lower band). GAPDH was used as a loading control.