Identification of novel viral proteins translated from PA mRNA.
To seek as-yet-unidentified viral proteins expressed from the PA gene of influenza A virus, we transfected pPolI-PA, the plasmid for the expression of PA vRNA, from WSN (H1N1) into 293T cells, along with pCAGGS-WSN-PB2, -PB1, -PA, and -NP, to provide polymerases and the NP protein. Forty-eight hours later, the cell lysates were analyzed by Western blotting using a mixture of five anti-PA monoclonal antibodies. Two bands, migrating to approximately 62 and 60 kDa, that were reactive with the anti-PA antibodies were detected, in addition to a strong signal for the full-length PA (83 kDa) (, third lane from the left).
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 (more ...)
We speculated that translation initiations at downstream AUG codons are involved in the expression of the additional smaller PA-related proteins, as is the case with PB1-F2 and PB1-N40 from the PB1 gene (6
). Based on their molecular weights and the presence of Kozak initiation consensus sequences, we hypothesized that the 11th AUG codon at amino acid position 155 and the 13th AUG codon at amino acid position 182 on the PA gene were used as the initiation codons for these additional PA gene products.
First, we mutated these two AUG codons to CUA, which encodes leucine. The smaller PA-related proteins expressed from the PA mutant plasmids were analyzed by Western blotting using a mixture of anti-PA monoclonal antibodies (). When the CUA mutation was introduced on the 11th AUG codon (PA-M155L), a full-length PA and a smaller protein were detected, but the upper, smaller PA-related protein was undetectable. For the PA-M182L construct, which contains the AUG-to-CUA mutation on the 13th codon, the lower, smaller PA-related protein was undetectable. For the PA-DM construct, which possesses the AUG-to-CUA mutation at both the 11th and 13th codons, neither smaller PA-related protein was detected. To further confirm that the 11th and 13th AUG codons are the initiation codons for these smaller PA-related proteins, we made a plasmid encoding a PA-N155 protein, which is an N-terminal 154-amino-acid-truncated form of PA, and a plasmid encoding a PA-N182 protein, which is an N-terminal 181-amino-acid-truncated form of PA (pCAGGS-PA-N155 and pCAGGS-PA-N182) (), and analyzed them by Western blotting. These N-truncated PAs comigrated with the smaller proteins expressed from the PA gene. In addition, transfection of an expression plasmid encoding PA (pCAGGS-PA) also produced these smaller PA-related proteins, although the band for PA-N182 is weak due to the low expression level. These results indicate that, in addition to authentic PA protein, N-terminally truncated forms of PA gene products, PA-N155 and PA-N182, are produced from the 11th and 13th AUG codons, respectively, in cells transfected with plasmids carrying PA genes ().
Detection of N-truncated PAs in influenza A virus-infected cells.
Next, we investigated whether PA-N155 and PA-N182 are detectable in virus-infected cells. The WSN PA mutant viruses, M155L, M182L, and DM, were generated by use of reverse genetics. These viruses possess replacements of methionine (AUG) with leucine (CUA) at amino acid position 155 (M155L), 182 (M182L), or both (DM). MDCK cells were infected with wild-type WSN or with each of the PA mutant viruses and then radiolabeled with [35S]methionine/cysteine. The cell lysates were treated with 1% SDS to disrupt noncovalent interactions among the polymerase subunits and then immunoprecipitated with a mixture of five anti-PA monoclonal antibodies, followed by SDS-PAGE and autoradiography. Two smaller PA-related proteins, in addition to the full-length PA, were detected in WSN-infected cells (). One smaller protein was not detected in each of the M155L- and M182L-infected cells. The DM-infected cells expressed neither of these two proteins. In addition to the two smaller proteins and the wild type, a fourth band was detected in WSN- and PA mutant-infected cells (indicated by the asterisk in ). Although the nature of this protein remains unknown, the signal intensity of this fourth band was weaker in a non-SDS-treated sample (right lane), indicating that the fourth protein may be a cleavage product of PA. These results indicate that both N-truncated PAs, PA-N155 and PA-N182, are synthesized in WSN virus-infected cells.
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 (more ...)
To evaluate the importance of PA-N155 and PA-N182 in influenza viruses in general, we analyzed the conservation of the 11th and 13th AUG codons at amino acid positions 155 and 182, respectively, among influenza viruses by using publically accessible PA genes in GenBank. While these AUG codons were not conserved among influenza B and C viruses (17
), the AUG codon at position 155 was highly conserved among influenza A viruses, being absent in only 10 strains of the 11,023 PA sequences of influenza A virus examined (). The AUG codon at position 182 was also well conserved, being absent in 79 strains of the 11,023 PA sequences examined (). The viruses possessing non-AUG codons at position 182 were mainly avian and swine H9N2 viruses (), indicating that these viruses could spread at least among these hosts and that the AUG at position 182 may be dispensable for the viruses. In contrast, viruses possessing non-AUG codons at position 155 were isolated only sporadically, suggesting that the viruses could not spread efficiently. In addition, we found no strain that lacked both AUG codons at positions 155 and 182 in the PA gene. These results suggest that these AUG codons, especially that at position 155, might be important in the evolution of influenza A virus.
Influenza A virus strains lacking the AUG codon at amino acid position 155 of PA in GenBank
Influenza A virus strains lacking the AUG codon at amino acid position 182 of PA in GenBank
Accordingly, we investigated whether the expression of PA-N155 and PA-N182 is common among influenza A viruses. MDCK cells were infected with a human-pandemic H1N1 2009 virus, a seasonal H3N2 virus, a swine virus, and a duck virus, in addition to the WSN strain. The radiolabeled cell lysates were immunoprecipitated with anti-PA monoclonal antibodies or anti-PA antiserum as described above. The PA and N-truncated PAs expressed in WSN-infected cells were immunoprecipitated by anti-PA antiserum, as well as by anti-PA monoclonal antibodies (). In all viruses tested, we detected smaller PA-related proteins whose mobilities were slightly different among viruses but similar to those of PA-N155 and PA-N182 expressed in WSN-infected cells (); similarly, the mobilities of the full-length PA proteins differed among the viral strains. An unidentified fourth short band was also detected in all samples (indicated by the asterisk), which was likely identical to the fourth band detected in (asterisk). In addition to these proteins, several other bands were detected, although the nature of these bands is unknown. These results suggest that the expression of N-truncated PAs is not limited to the WSN strain but is a universal feature of influenza A viruses.
Growth properties of N-truncated PA-deficient viruses.
To assess the importance of these N-truncated forms of PA in virus replication, we compared the growth properties of mutant viruses that were unable to express the N-truncated PAs with those of wild-type WSN in MDCK cells (). The M182L mutant replicated as well as wild-type WSN. In contrast, the titers of the M155L mutant, which lacks PA-N155, and the DM mutant, which lacks both PA-N155 and PA-N182, were significantly lower than that of wild-type WSN at every time point (P < 0.05). The differences between these mutants and the wild type were particularly remarkable at the early time points (24, 28, and 32 h p.i.); for example, the titers of the M155L and DM mutants were about 1.1 and 1.5 log10 units lower than that of the wild type at 24 h p.i., respectively, indicating that the M155L and DM mutant viruses, which lack the expression of PA-N155, showed slower replication kinetics in cells. These results suggest that even though the N-truncated PAs are not essential for replication, PA-N155 possesses some functions that are important for efficient virus replication.
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 (more ...) Pathogenicity of N-truncated PA-deficient viruses in mice.
To assess whether the N-truncated forms of PA are important for viral pathogenicity in vivo, we compared the MLD50s of the PA mutant viruses with that of wild-type WSN. Although the pathogenicity of the M182L mutant was similar to that of the wild-type virus, the M155L and DM mutants were attenuated compared with the wild-type virus (the MLD50 value was approximately 1.5 log10 units higher than that of the wild-type virus) (). In addition, the virus titers in the lungs of mice infected with the M155L and DM mutants were more than 1.0 log10 unit lower than that of wild-type WSN on day 3, although the differences in virus titers between the PA mutants and WSN on day 6 were smaller than on day 3 (). These results suggest that PA-N155 likely plays important roles in virus pathogenicity in animals.
Comparison of pathogenicities of PA mutant viruses lacking N-truncated PAs in mice
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. (more ...) Polymerase functions of mutant PA proteins.
To understand the mechanisms behind the reduced growth of the mutant viruses M155L and DM, we tested the effects of the PA mutations on polymerase activity. 293 cells were cotransfected with a plasmid encoding wild-type PA or each mutant PA protein, along with plasmids for the expression of PB2, PB1, NP, and a vRNA replicon possessing the luciferase gene. At 24 and 40 h p.t., luciferase activity was measured (). All PA mutants, PA-M155L, PA-M182L, and PA-DM, supported viral RNA replication and transcription at levels similar to that of wild-type PA at both 24 and 40 h p.t. These results indicate that the expression levels of the N-truncated PAs have little effect on viral polymerase activity. They also suggest that the reduced replication and lower pathogenicity of the PA mutant viruses, M155L and DM, were not the result of reduced polymerase activity of the mutant PAs, at least as detected by the above-described mini-genome assay.
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 (more ...) Functions of N-truncated PA proteins.
Since N-truncated PAs lack the regions required for endonuclease activity (24
), they should not be functionally equivalent to full-length PA. We therefore tested whether N-truncated PAs support viral polymerase activity in the mini-genome assay, as described above. Only background levels of luciferase activity were detected in the absence of the PA protein (empty vector), and both PA-N155 and PA-N182 also produced only background levels of luciferase (), indicating that the N-truncated PAs lack polymerase activity. These results also suggest that PA-N155, which was shown to be important for viral replication in vitro
and in vivo
, is likely involved in viral replication steps other than the transcription and replication of the viral genome.
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 (more ...)
We then investigated whether PA-N155 was an antagonist of the antiviral response induced by type I interferon by comparing the growth properties of mutant viruses that lack the N-truncated PAs with those of wild-type WSN in Vero E6 cells, which do not produce type I interferon. As is true for virus growth in MDCK cells, the M182L mutant replicated as well as wild-type WSN (). The titers of the M155L and DM mutants were significantly lower than that of wild-type WSN at 12, 24, and 72 h p.i. in M155L and at 12, 24, and 48 h p.i. in DM (P < 0.05). These results indicate that mutant viruses that lack PA-N155 exhibit slower replication kinetics in Vero E6 cells, suggesting that PA-N155 is not an antagonist of the antiviral response induced by type I interferon.
We then examined the intracellular distribution of the N-truncated PAs. The PA protein has two nuclear localization signals (NLSs), which are located at amino acid positions 124 to 139 and 186 to 247 (26
). Since both of the N-truncated PAs lack the former NLS, we hypothesized that the N-truncated PAs would show a localization pattern different from that of full-length PA. By using an immunofluorescence assay, we examined the intracellular localization of PA-N155 and PA-N182 and compared it with that of full-length PA. To distinguish full-length PA from the N-truncated PAs expressed from the plasmid encoding PA (pCAGGS-PA) (), we used both the anti-PA monoclonal antibody 58/1, which recognizes the N-terminal region of the PA protein (amino acid positions 101 to 107), and anti-PA antiserum, which recognizes both full-length PA and N-truncated PA ().
When cells transfected with pCAGGS-PA were treated with the anti-PA monoclonal antibody 58/1, signals were detected in both the cytoplasm and the nucleus (data not shown). Cells transfected with pCAGGS-PA-N155 or pCAGGS-PA-N182 did not react with the antibody. These results indicate that full-length PA localized to both the cytoplasm and the nucleus. When we used the anti-PA antiserum, positive signals were detected in both the cytoplasm and the nuclei of cells transfected with pCAGGS-PA, and there was no apparent difference in PA detection between the monoclonal antibody and the antiserum (data not shown). The N-truncated PAs were also located in both the cytoplasm and the nuclei of the plasmid-transfected cells, and there was no significant difference in localization between full-length PA and N-truncated PAs (data not shown).
We then investigated the intracellular localization of the N-truncated PAs in the presence of PB1, since PA and PB1 interact with each other in the cytoplasm and are transported into the nucleus as a heterodimer (27
) and the N-truncated PAs possess the regions responsible for binding to PB1 (located at positions 601 to 692 of PA) (30
). In the absence of PA, PB1 was detected predominantly in the cytoplasm, whereas when PA was coexpressed, PB1 was largely found in the nucleus (data not shown). PA was predominantly detected in the nucleus in the presence of PB1, although it localized to both the cytoplasm and the nucleus in the absence of PB1 (data not shown), confirming that PA interacts with PB1 and together they are transported into the nucleus. The N-truncated PAs showed localization patterns similar to that of PA; the N-truncated PAs were predominantly found in the nucleus, as was PB1 in cells expressing both proteins. These results indicate that PA-N155 and PA-N182 interact with PB1 and are transported into the nucleus.