Strain-Specific Contribution of Eukaryotic Elongation Factor 1 Gamma to the Translation of Influenza A Virus Proteins

Abstract

Influenza A virus exploits multiple host proteins during infection. To define the virus-host interactome, our group conducted a proteomics-based screen and identified 299 genes that contributed to virus replication and 24 genes that were antiviral. Of these genes, we focused on the role during virus replication of eukaryotic elongation factor 1 gamma (eEF1G), which is a subunit of the eukaryotic elongation factor-1 complex and known to be a pro-viral host protein. Using the CRISPR/Cas9 system, we obtained two clones that were defective in eEF1G expression. In both of these clones, A/WSN/33 (H1N1) virus growth and protein expression were significantly suppressed, but viral mRNA, vRNA, and cRNA expression were not reduced. However, the replication and protein expression of A/California/04/2009 (H1N1pdm) virus in both clones were similar to those in parental cells. We found that the PB2 and PA proteins of WSN virus were responsible for the eEF1G-dependent replication. Our data show that eEF1G plays a role in the translation of virus proteins in a strain-specific manner. Additional analyses may be needed to further understand the role of strain-specific host proteins during virus replication.

Keywords: PA; PB2; eEF1G; host protein; influenza virus; protein translation.

FIGURE 1

Acquisition of clones 1–24 and 3–9. (A) A model of the eEF1 complex. The eEF1 complex consists of two eEF1A molecules and an eEF1B complex. eEF1B2, eEF1D, and eEF1G comprise the eEF1B complex. (B) Expression of eEF1G, eEF1B2, and eEF1D in clones 1–24 and 3–9. Total cell lysate of each cell line was analyzed by western blotting; β-actin served as a loading control. (C) Nucleotide sequences of the eEF1G mRNA expressed in clones 1–24 and 3–9. The eEF1G mRNA sequence was obtained by RT-PCR, followed by Sanger sequencing. Corresponding amino acids are presented above the nucleotide sequences. Dashed lines indicate deletion of the corresponding nucleotides. (D) Schematic diagram of eEF1G expressed in clones 1–24 and 3–9. eEF1G in clone 1–24 had a single amino acid substitution at position 182, followed by a 4-amino acid deletion. eEF1G in clone 3–9 was composed of the N-terminal authentic 280 amino acids and C-terminal frameshifted 62 amino acids (gray). (E) Intracellular localization of eEF1G in clones 1–24 and 3–9. eEF1G (green) was detected by using a rabbit anti-eEF1G mAb, followed by the Alexa Fluor 488 anti-rabbit IgG. Nuclei (blue) were stained with Hoechst 33342. Scale bar: 20 μm.

FIGURE 2

The eEF1G defect in clones 1–24 and 3–9 does not inhibit cell proliferation or endogenous protein expression. (A) Cell proliferation of clones 1–24 and 3–9. The data are shown as means ± SD (n = 3). ^∗∗P < 0.01 according to a two-way ANOVA followed by Dunnett’s test. (B) Proteins expressed in clones 1–24 and 3–9. Total cell lysates were subjected to SDS–PAGE and visualized with CBB staining. (C) Expression of endogenous proteins in clones 1–24 and 3–9. Total cell lysates were analyzed by western blotting using antibodies against eIF3B, SNRPA, α-tubulin, and β-actin.

FIGURE 3

Growth of WSN virus in clones 1–24 and 3–9. Wild-type A549 cells, clone 1–24, and clone 3–9 (A) or wild-type A549 cells, clone 1–24, clone 1-24#1, and clone 1-24#4 (B) were infected with WSN virus at an MOI of 0.001. Virus titers were determined by use of plaque assays in MDCK cells. The data are shown as means ± SD (n = 3). ^∗∗P < 0.01 according to a two-way (A) or one-way (B) ANOVA followed by Dunnett’s test. Expression of eEF1G, eEF1B2, and eEF1D was evaluated by western blotting; β-actin served as a loading control (B).

FIGURE 4

Role of eEF1G during the replication cycle of WSN virus. (A–C) Relative vRNA, cRNA, and mRNA expression in wild-type A549 cells, clone 1–24, and clone 3–9. The indicated cells were infected with WSN virus at an MOI of 10. Relative vRNA, cRNA, and mRNA expression was measured by strand-specific real-time RT-qPCR. The expression of vRNA, cRNA, and mRNA in wild-type A549 cells at 2 hpi was set to 1. The data are shown as means ± SD (n = 3). ^∗P < 0.05, ^∗∗P < 0.01 according to a one-way ANOVA with Bonferroni correction. (D) Expression of the viral proteins M1 and NP in infected clones 1–24 and 3–9. The indicated cells were infected with WSN at an MOI of 10. At 3, 6, 9, and 12 hpi, total cell lysates were analyzed by western blotting with anti-M1 and anti-NP antibodies; β-actin served as a loading control (left panel). The intensity of the M1 signals was measured and that in the wild-type A549 cells at each timepoint was set to 100% (right panel). The quantified data are shown as means ± SD (n = 3). ^∗∗P < 0.01 according to a one-way ANOVA with Bonferroni correction.

FIGURE 5

eEF1G is important for the propagation of Perth16 virus but not for CA04 virus. (A) Growth of CA04 virus in clones 1–24 and 3–9. The indicated cells were infected with CA04 virus at an MOI of 0.01. (B) Growth of Perth16 virus in clones 1–24 and 3–9. The indicated cells were infected with A/Perth/16/2009 (H3N2) virus at an MOI of 0.001. (A,B) Virus titers were determined by use of plaque assays in MDCK cells. The data are shown as means ± SD (n = 3). ^∗∗P < 0.01 according to a two-way ANOVA followed by Dunnett’s test. (C,D) Expression of the viral protein M1 in CA04- or Perth16-infected cells. The indicated cells were infected with CA04 (C) or Perth16 (D) virus at an MOI of 10. At 3, 6, 9, and 12 hpi, total cell lysates were analyzed by western blotting with an anti-M1 antibody; β-actin served as a loading control.

FIGURE 6

Growth of reassortant viruses in clone 1–24. (A,B) Schematic diagram of reassortant viruses. Black and gray indicate a viral segment derived from WSN and CA04 virus, respectively. (C,D) Growth of reassortant viruses in clone 1–24. Wild-type A549 cells and clone 1–24 were infected with the indicated viruses at an MOI of 0.001 for WSN and its reassortant viruses or an MOI of 0.01 for CA04 and its reassortant viruses. Virus titers at 48 hpi were determined by using plaque assays in MDCK cells. The data are shown as means ± SD (n = 3). ^∗P < 0.05, ^∗∗P < 0.01 according to a Student’s t-test with Bonferroni correction.