Amino acid residues in the N-terminal region of the PA subunit of influenza A virus RNA polymerase play a critical role in protein stability, endonuclease activity, cap binding, and virion RNA promoter binding

Abstract

The RNA-dependent RNA polymerase of influenza virus is a heterotrimer formed by the PB1, PB2, and PA subunits. Although PA is known to be required for polymerase activity, its precise role is still unclear. Here, we investigated the function of the N-terminal region of PA. Protease digestion of purified recombinant influenza virus A/PR/8/34 PA initially suggested that its N-terminal region is folded into a 25-kDa domain. We then systematically introduced point mutations into evolutionarily conserved amino acids in the N-terminal region of influenza virus A/WSN/33. Most alanine-scanning mutations between residues L109 and F117 caused PA degradation, mediated by a proteasome-ubiquitin pathway, and as a consequence interfered with polymerase activity. Three further PA mutations, K102A, D108A, and K134A, were investigated in detail. Mutation K102A caused a general decrease both in transcription and replication in vivo, whereas mutations D108A and K134A selectively inhibited transcription. Both the D108A and K134A mutations completely inhibited endonuclease activity in vitro, explaining their selective defect in transcription. K102A, on the other hand, resulted in a significant decrease in both cap binding and viral RNA promoter-binding activity and consequently inhibited both transcription and replication. These results suggest that the N-terminal region of PA is involved in multiple functions of the polymerase, including protein stability, endonuclease activity, cap binding, and promoter binding.

FIG. 1.

Digestion of the PA subunit by trypsin. (A) Purified PA (8 μg) was incubated with increasing amounts of trypsin (10, 40, and 100 ng; lanes 2 to 5) or without trypsin (lane 1) at 37°C for 30 min. The reaction products were resolved by 12% SDS-PAGE and stained with Coomassie blue. The positions of marker proteins are indicated, in kilodaltons, on the left. (B) N-terminal amino acid sequences of the two ∼56-, the ∼51-, and the ∼25-kDa fragments determined by Edman degradation (underlined). The identity of the neighboring amino acids is also shown. The arrows represent the predicted trypsin cleavage sites.

FIG. 2.

Alignment of residues 1 to 402 of the PA subunits of the RNA polymerases of influenza A, B, and C viruses and Thogoto virus. Sequence accession numbers are X17336 for influenza virus A/WSN/33, AF102022 for influenza virus B/Victoria/2/87, M28062 for influenza virus C/JJ/50, and AF006073 for Thogoto virus. Sequences were aligned with Omiga software, version 2.0 (Oxford Molecular Ltd., Oxford, United Kingdom). Numbers refer to amino acid positions in the A/WSN/33 sequences, and amino acids selected for mutagenesis are highlighted. Identical amino acids are boldfaced.

FIG. 3.

Effects of mutations in the PA subunit on RNA synthesis in vivo. 293T cells were transfected with plasmids expressing vRNA-like CAT RNA reporter, NP, PB1, PB2, and either a wild-type (WT) PA or mutant PAs. Cell extracts were tested for polymerase activity 24 h posttransfection by measuring the levels of vRNA, mRNA, and cRNA by primer extension. Sizes are indicated, in nucleotides, on the left. Positions of vRNA, mRNA, and cRNA signals are indicated on the right. The extension product of the 5S rRNA, with an expected size of 62 nt, is indicated on the right as an internal control.

FIG. 4.

Effects of mutations in the PA subunit on the assembly of the influenza virus RNA polymerase complex. (A) Expression and assembly of the polymerase subunit PB1, TAP-tagged PB2, and PA mutants in 293T cells. (B) Expression of TAP-tagged PA mutants alone in 293T cells. (C) Expression of PA mutants alone in total cell lysates of 293T cells. TAP-tagged proteins were partially purified from lysates of transfected 293T cells 24 h posttransfection with immunoglobulin G (IgG)-Sepharose, and bound proteins were cleaved by tobacco etch virus protease. The samples were analyzed by silver-stained 7.5% SDS-PAGE. (D₁ and D₂) Effects of the proteasome inhibitor on PA mutants. TAP-tagged PA was purified from cell lysates with IgG Sepharose and analyzed by 7.5% SDS-PAGE and silver staining. The positions of PB1, PB2-TAP, PA, and PA-TAP are shown on the right. The positions of marker proteins are indicated, in kilodaltons. WT, wild type.

FIG. 5.

Transcription initiation activity of PA mutants in vitro. 293T cells were transfected with plasmids expressing PB1 and PB2-TAP, either without PA (−PA), with a wild-type PA (WT), or with mutant PAs (K102A, D108A, and K134A). TAP-tagged polymerases were partially purified from cell extracts by immunoglobulin G-Sepharose and used in vitro. As a negative control (C), cell extracts from untransfected cells were prepared. (A) Silver staining of purified TAP-tagged polymerases separated by 7.5% SDS-PAGE. The positions of PB1, PB2-TAP, and PA are shown on the right. (B) Transcription activity on a model vRNA promoter of mutant polymerases primed with a globin mRNA primer. The positions of the 27- or 28-nt-long transcription products (TP) are indicated. (C) Transcription activity of mutant polymerases primed with an 11-nt ³²P-labeled capped RNA primer (see Materials and Methods). The positions of the 27- or 28-nt-long transcription products (TP) of capped RNA primer are indicated. (D) Endonuclease activity (see Materials and Methods) of mutant polymerases. TAP-purified polymerases were incubated with [³²P]poly(A)⁺-capped RNA. The positions of the poly(A)⁺-capped RNA and specific 11-nt cleavage product are indicated on the right. (D, E, and F) Quantification of results obtained in panels B, C, and D, respectively, by phosphorimaging analysis. Data are expressed as percentages relative to the wild type (mean ± standard deviation; n = 3).

FIG. 6.

RNA binding activity and replication initiation activity of PA mutants. (A) UV cross-linking analysis of the binding of RNA with polymerases containing wild-type PA (WT) or mutant PAs (K102A, D108A, and K134A). Purified TAP-tagged polymerases were incubated with ³²P-labeled capped RNA in the presence of both 5′ vRNA and 3′ vRNA (upper panel) or with ³²P-labeled 3′ vRNA in the presence of 5′ vRNA (lower panel). The reaction mixtures were subsequently exposed with UV light and analyzed by 7.5% SDS-PAGE. The positions of the cross-linked products are indicated on the right. (B) Replication initiation activity of the polymerases containing WT PA or mutant PAs (K102A, D108A, and K134A). The position of the specific ApG product is indicated on the right. (C and D) Quantification of results obtained in panels A and B, respectively, by phosphorimaging. Data are expressed as percentages relative to the wild type (mean ± standard deviation; n = 3). (C) Black bars, cap binding; white bars, model vRNA promoter binding. (D) Black bars, model vRNA template; white bars, model cRNA template.

FIG. 7.

Model of the endonuclease active site of the influenza virus RNA polymerase. Amino acids 508, 519, and 522 derive from the PB1 subunit, while amino acids 108, 134, and 510 are from PA. Acidic amino acids in PB1 (E508, E519, and D522) and PA (D108) interact with a divalent metal ion (Mg²⁺) in the endonuclease active site. Two other amino acids in PA (K134 and H510) are also involved (see the text for details).