Mapping of a Region of the PA-X Protein of Influenza A Virus That Is Important for Its Shutoff Activity

Abstract

Influenza A virus PA-X comprises an N-terminal PA endonuclease domain and a C-terminal PA-X-specific domain. PA-X reduces host and viral mRNA accumulation via its endonuclease function. Here, we found that the N-terminal 15 amino acids, particularly six basic amino acids, in the C-terminal PA-X-specific region are important for PA-X shutoff activity. These six basic amino acids enabled a PA deletion mutant to suppress protein expression at a level comparable to that of wild-type PA-X.

FIG 1

Comparisons of the shutoff activities of wild-type PA, wild-type PA-X, and C-terminal deletion mutant forms of PA. (A) Schematic representation of PA, PA-X, and C-terminal deletion mutant forms of PA. PA-X is composed of the N-terminal 191 amino acids of PA and a C-terminal PA-X-specific region of 61 amino acids. PA_191 and PA_252 contain the N-terminal 191 and 252 amino acids of PA, respectively. (B to D) Renilla luciferase activity (B), expression of Renilla luciferase (C), and expression of each viral protein (D) in cells transfected with pGL4.74[hRluc/TK] together with an empty plasmid or a plasmid encoding wild-type PA, wild-type PA-X, or deletion mutant forms of PA were evaluated by using a luciferase assay and Western blotting with an anti-Renilla luciferase and an anti-PA monoclonal antibody. The luminescence data in panel B are mean values ± standard deviations (n = 3). **, P < 0.01 (one-way analysis of variance, followed by Bonferroni correction). (C) The intensities of the Renilla luciferase and β-actin bands were quantified, and their ratios were calculated. The value of the mock lane was set to 100%. In panels C and D, β-actin served as a loading control. ORF, open reading frame; RLU, relative light units.

FIG 2

Importance of the N-terminal 15 amino acids in the PA-X-specific region for the shutoff activity of PA-X. (A) Schematic diagram of the C-terminal deletion mutant forms of PA-X. A series of C-terminal deletion mutant forms of PA-X was constructed to identify the region(s) important for the shutoff activity of PA-X. PA-X_X50, PA-X_X40, PA-X_X30, PA-X_X20, PA-X_X15, PA-X_X10, PA-X_X5, and PA_191 possessed 50, 40, 30, 20, 15, 10, 5, and no PA-X-specific amino acids, respectively. (B) Suppression of Renilla luciferase activity by each C-terminal deletion mutant form of PA-X in 293 cells. Renilla luciferase activity in cells cotransfected with wild-type PA-X, a series of C-terminal deletion mutant proteins, or PA_191 was measured with a luciferase assay. The luminescence in the each C-terminal deletion mutant sample was compared with that in the wild-type PA-X sample. Luminescence data are mean values ± standard deviations (n = 3). **, P < 0.01 (one-way analysis of variance, followed by Dunnett's test). RLU, relative light units.

FIG 3

Identification of amino acids in the PA-X-specific region that are important for the shutoff activity of PA-X. (A) Comparison of the N-terminal PA-X-specific 15 amino acids of PA-X with the identical region of PA. All six basic amino acids (K or R) in PA-X were the acidic amino acid E in PA. The six amino acids are underlined. (B) Substitution of the six basic amino acids in the PA-X-specific region. The six basic amino acids of PA-X were replaced with acidic amino acids (PA-X_6E or PA-X_6D), a neutral amino acid (PA-X_6A), or a basic amino acid (PA-X_6R, PA-X_6K, or PA-X_KR). (C to E) Shutoff activities of mutant PA-X proteins. In 293 cells, Renilla luciferase was coexpressed with wild-type PA-X or each mutant PA-X protein. Renilla luciferase activity (C) and Renilla luciferase (E) and wild-type and mutant PA-X (D) expression levels were evaluated by means of a luciferase assay or Western blotting. (F) Comparison of the N-terminal PA-X-specific 15 amino acids of PA-X with the identical region of PA_252. Six acidic amino acids in PA_252 (underlined) were replaced with those of PA-X (PA_252_6KR). (G to I) Shutoff activity of PA_252_6KR. In 293 cells, Renilla luciferase was coexpressed without (mock) or with wild-type PA-X, PA_252, or PA_252_6KR. Renilla luciferase activity (G) and levels of expression of Renilla luciferase (H) and each viral protein (I) were evaluated by means of a luciferase assay or Western blotting. The luminescence data in panels C and G are mean values ± standard deviations (n = 3). **, P < 0.01 (t test, followed by Bonferroni correction). The intensities of the Renilla luciferase and β-actin bands were quantified and their ratios were calculated (E and H). The value of the mock lane was set to 100%. In panels D, E, H, and I, β-actin served as a loading control. RLU, relative light units.

FIG 4

Effect of the N-terminal 20 amino acids in the PA-X-specific region on the shutoff activity of PA_252 and PA-X_6E. (A) Schematic diagram of PA_252_X20 and PA-X_6E_X20. These proteins contain the N-terminal 20 amino acids of the PA-X-specific region (gray) at the C terminus of PA_252 or PA-X_6E, respectively. (B to D) Shutoff activities of PA_252_X20 and PA-X_6E_X20. In 293 cells, Renilla luciferase was coexpressed without (mock) or with wild-type PA-X, PA_252, PA_252_X20, PA-X_6E, or PA-X_6E_X20. Renilla luciferase activity (B) and the expression of Renilla luciferase (C) and each viral protein (D) were evaluated by means of a luciferase assay or Western blotting. (C) The intensities of the Renilla luciferase and β-actin bands were quantified, and their ratios were calculated. The value of the mock lane was set to 100%. In panels C and D, β-actin served as a loading control. RLU, relative light units.