Identification of novel influenza A virus proteins translated from PA mRNA

Abstract

Many replication events are involved in the influenza A virus life cycle, and they are accomplished by different virus proteins with specific functions. However, because the size of the influenza virus genome is limited, the virus uses different mechanisms to express multiple viral proteins from a single gene segment. The M2 and NS2 proteins are produced by splicing, and several novel influenza A virus proteins, such as PB1-F2, PB1-N40, and PA-X, have recently been identified. Here, we identified novel PA-related proteins in influenza A virus-infected cells. These newly identified proteins are translated from the 11th and 13th in-frame AUG codons in the PA mRNA and are, therefore, N-terminally truncated forms of PA, which we named PA-N155 and PA-N182, respectively. The 11th and 13th AUG codons are highly conserved among influenza A viruses, and the PA-N155 and PA-N182 proteins were detected in cells infected with various influenza A viruses isolated from different host species, suggesting the expression of these N-truncated PAs is universal in nature among influenza A viruses. These N-truncated PAs did not show polymerase activity when expressed together with PB1 and PB2; however, mutant viruses lacking the N-truncated PAs replicated more slowly in cell culture and had lower pathogenicity in mice than did wild-type virus. These results suggest that these novel PA-related proteins likely possess important functions in the replication cycle of influenza A virus.

Fig 1

Expression of N-truncated PAs in plasmid-transfected cells. (A) 293T cells were transfected with a plasmid for the expression of PA vRNA or PA mutant vRNA (pPolI-PA or PA mutants) in combination with expression plasmids encoding WSN-PB1, -PB2, -NP (1 μg each), and -PA (0.2 μg); an expression plasmid, pCAGGS-PA; or N-truncated PAs (1 μg). Forty-eight hours later, total cell lysates were analyzed by Western blotting for the detection of the full-length PA and the N-truncated PAs by using a mixture of anti-PA monoclonal antibodies. (B) Schematic representation of N-truncated forms of PA. N-truncated PAs are translated from the indicated AUG codons at amino acid positions 155 and 182.

Fig 2

Detection of N-truncated PAs in virus-infected cells. MDCK cells were infected with WSN and PA mutant viruses (A) or several different influenza A virus strains (B) and at 3 h p.i. radiolabeled for 3 h in [³⁵S]methionine/cysteine-containing medium. After cell lysis, the supernatants were immunoprecipitated (IP) with a mixture of mouse anti-PA monoclonal antibodies (MAbs) or rabbit anti-PA antiserum and analyzed by SDS-PAGE, followed by visualization by autoradiography. The asterisk indicates a fourth band whose nature remains unknown.

Fig 3

Growth kinetics of PA mutant viruses lacking N-truncated PAs in cell culture. MDCK cells were infected with wild-type WSN or mutant viruses lacking N-truncated PAs at a multiplicity of infection of 0.0005. At different time points postinfection, virus titers in the supernatants of the infected cells were determined by means of plaque assays in MDCK cells. The mean titers from triplicate independent cultures ± standard deviations (SD) are shown.

Fig 4

Replication of PA mutant viruses lacking N-truncated PAs in the lungs of mice. Eight groups (n = 10 per group) of 6-week-old female BALB/c mice were infected intranasally with 10² PFU of virus, and 3 and 6 days later, lungs were collected for virus titration. Each bar represents the mean titer for each virus. The data are the mean titer and the SD for each virus. An asterisk indicates that the mean titer of the PA mutant virus was significantly lower than that of the wild-type WSN (P < 0.05). Detection limit, 2 log₁₀ PFU/g tissue.

Fig 5

Polymerase activities of mutant PAs lacking the expression of N-truncated PAs. 293 cells were transfected with expression plasmids encoding PB2, PB1, NP, and a vRNA replicon possessing a firefly luciferase gene between the noncoding regions of WSN-NP vRNA, along with the indicated PA- or PA mutant-expressing plasmid. Twenty-four or 40 h later, the luciferase activity was quantified. The data are the means ± SD of eight independent experiments plotted as the percent activity with wild-type PA for each experiment.

Fig 6

Functions of N-truncated PAs. (A) Polymerase activities of N-truncated PAs. 293 cells were transfected with expression plasmids encoding PB2, PB1, and NP and a vRNA replicon possessing a firefly luciferase gene, along with the indicated PA- or N-truncated PA-expressing plasmid. Twenty-four hours later, the luciferase activity was quantified. The data shown here are representative of three independent experiments. An asterisk indicates that the activity of the N-truncated PA was significantly lower than that of wild-type PA (P < 0.05). (B) Viral growth kinetics of PA mutant viruses that lack N-truncated PAs in Vero E6 cells. Vero E6 cells were infected with wild-type WSN or mutant viruses lacking N-truncated PAs at a multiplicity of infection of 0.005. At different time points postinfection, virus titers in the supernatants of the infected cells were determined by means of plaque assays. The mean titers from triplicate independent cultures ± standard deviations are shown.