A single amino acid mutation in the PA subunit of the influenza virus RNA polymerase inhibits endonucleolytic cleavage of capped RNAs

Abstract

The influenza A virus RNA-dependent RNA polymerase consists of three subunits-PB1, PB2, and PA. The PB1 subunit is the catalytically active polymerase, catalyzing the sequential addition of nucleotides to the growing RNA chain. The PB2 subunit is a cap-binding protein that plays a role in initiation of viral mRNA synthesis by recruiting capped RNA primers. The function of PA is unknown, but previous studies of temperature-sensitive viruses with mutations in PA have implied a role in viral RNA replication. In this report we demonstrate that the PA subunit is required not only for replication but also for transcription of viral RNA. We mutated evolutionarily conserved amino acids to alanines in the C-terminal region of the PA protein, since the C-terminal region shows the highest degree of conservation between PA proteins of influenza A, B, and C viruses. We tested the effects of these mutations on the ability of RNA polymerase to transcribe and replicate viral RNA. We also tested the compatibility of these mutations with viral viability by using reverse-genetics techniques. A mutant with a histidine-to-alanine change at position 510 (H510A) in the PA protein of influenza A/WSN/33 virus showed a differential effect on transcription and replication. This mutant was able to perform replication (vRNA-->cRNA-->vRNA), but its transcriptional activity (vRNA-->mRNA) was negligible. In vitro analyses of the H510A recombinant polymerase, by using transcription initiation, vRNA-binding, capped-RNA-binding, and endonuclease assays, suggest that the primary defect of this mutant polymerase is in its endonuclease activity.

FIG. 1.

Amino acid sequence similarity plot of influenza A, B, and C virus PA proteins. Influenza A/WSN/33, B/Panama/45/90, and C/JJ/50 virus PA sequences (GenBank accession numbers J02152, AF005738, and M28062, respectively) were aligned, and similarity was calculated and plotted for a window of 10 amino acids with programs in the Genetics Computer Group software package. Horizontal line, average level of overall similarity in the plot.

FIG. 2.

Alignment of amino acid sequences of PA proteins of influenza A, B, and C viruses and Thogoto virus. PA sequences of influenza A/WSN/33, B/Panama/45/90, and C/JJ/50 viruses and Thogoto virus (GenBank accession numbers J02152, AF005738, M28062, and 2253293, respectively) were aligned with CLUSTAL W from the Genetics Computer Group software package by using the default parameters (details available from us on request). Inclusion of the Thogoto virus PA sequence, the most divergent of the four PA sequences used in the alignment, could result in alternative alignments of some amino acid regions if different parameters of CLUSTAL were used. Amino acids selected for mutagenesis are boldfaced. Numbers refer to amino acid positions in the A/WSN/33 sequence. Only the C-terminal halves of the PA sequences are shown (amino acids 355 to 716 for A/WSN/33).

FIG. 3.

In vivo RNA synthesis mediated by mutant PA proteins. (A through D) Primer extension assays of vRNA, mRNA, and cRNA isolated from cells expressing influenza virus polymerase proteins and vRNA-like CAT-RNA. 293T cells were transfected, as indicated above the lanes, either with pcDNA3 (indicated by “C”), with pPOLI-CAT-RT, with pPOLI-CAT-RT, pcDNA-PB1, and pcDNA-PB2 (−PA), or with pPOLI-CAT-RT, pcDNA-PB1, pcDNA-PB2, and wild-type (WT) or mutant pcDNA-PA. (E) Primer extension assays of vRNA, mRNA, and cRNA isolated from cells expressing influenza virus polymerase proteins and cRNA-like CAT-RNA. 293T cells were transfected, as indicated above the lanes, either with pcDNA3 (indicated by “C”), with pPOLI-cCAT-RT, with pPOLI-cCAT-RT, pcDNA-PB1, and pcDNA-PB2 (−PA), or with pPOLI-cCAT-RT, pcDNA-PB1, pcDNA-PB2, and wild-type (WT) or mutant (H510A) pcDNA-PA. pcDNA-NP was included in all transfections except the negative controls C, pPOLI-CAT-RT, and pPOLI-cCAT-RT. Isolation of RNA and primer extension assays were performed as described in Materials and Methods. Positions of vRNA, mRNA, and cRNA signals are indicated on the right. Stars indicate nonspecific priming on cellular RNAs. M, ³²P-labeled 1-kb DNA ladder (Gibco BRL); sizes are indicated on the left.

FIG. 3.

In vivo RNA synthesis mediated by mutant PA proteins. (A through D) Primer extension assays of vRNA, mRNA, and cRNA isolated from cells expressing influenza virus polymerase proteins and vRNA-like CAT-RNA. 293T cells were transfected, as indicated above the lanes, either with pcDNA3 (indicated by “C”), with pPOLI-CAT-RT, with pPOLI-CAT-RT, pcDNA-PB1, and pcDNA-PB2 (−PA), or with pPOLI-CAT-RT, pcDNA-PB1, pcDNA-PB2, and wild-type (WT) or mutant pcDNA-PA. (E) Primer extension assays of vRNA, mRNA, and cRNA isolated from cells expressing influenza virus polymerase proteins and cRNA-like CAT-RNA. 293T cells were transfected, as indicated above the lanes, either with pcDNA3 (indicated by “C”), with pPOLI-cCAT-RT, with pPOLI-cCAT-RT, pcDNA-PB1, and pcDNA-PB2 (−PA), or with pPOLI-cCAT-RT, pcDNA-PB1, pcDNA-PB2, and wild-type (WT) or mutant (H510A) pcDNA-PA. pcDNA-NP was included in all transfections except the negative controls C, pPOLI-CAT-RT, and pPOLI-cCAT-RT. Isolation of RNA and primer extension assays were performed as described in Materials and Methods. Positions of vRNA, mRNA, and cRNA signals are indicated on the right. Stars indicate nonspecific priming on cellular RNAs. M, ³²P-labeled 1-kb DNA ladder (Gibco BRL); sizes are indicated on the left.

FIG. 3.

In vivo RNA synthesis mediated by mutant PA proteins. (A through D) Primer extension assays of vRNA, mRNA, and cRNA isolated from cells expressing influenza virus polymerase proteins and vRNA-like CAT-RNA. 293T cells were transfected, as indicated above the lanes, either with pcDNA3 (indicated by “C”), with pPOLI-CAT-RT, with pPOLI-CAT-RT, pcDNA-PB1, and pcDNA-PB2 (−PA), or with pPOLI-CAT-RT, pcDNA-PB1, pcDNA-PB2, and wild-type (WT) or mutant pcDNA-PA. (E) Primer extension assays of vRNA, mRNA, and cRNA isolated from cells expressing influenza virus polymerase proteins and cRNA-like CAT-RNA. 293T cells were transfected, as indicated above the lanes, either with pcDNA3 (indicated by “C”), with pPOLI-cCAT-RT, with pPOLI-cCAT-RT, pcDNA-PB1, and pcDNA-PB2 (−PA), or with pPOLI-cCAT-RT, pcDNA-PB1, pcDNA-PB2, and wild-type (WT) or mutant (H510A) pcDNA-PA. pcDNA-NP was included in all transfections except the negative controls C, pPOLI-CAT-RT, and pPOLI-cCAT-RT. Isolation of RNA and primer extension assays were performed as described in Materials and Methods. Positions of vRNA, mRNA, and cRNA signals are indicated on the right. Stars indicate nonspecific priming on cellular RNAs. M, ³²P-labeled 1-kb DNA ladder (Gibco BRL); sizes are indicated on the left.

FIG. 4.

In vitro RNA synthesis mediated by the H510A polymerase. (A) Transcription initiation primed with ApG and globin mRNA primers. Transcription reactions were carried out in vitro with nuclear extracts from cells transfected either with pcDNA3 (C) (lanes 1 and 4) or with pcDNA-PB1, pcDNA-PB2, and wild-type (WT) (lanes 2 and 5) or mutant (H510A) (lanes 3 and 6) pcDNA-PA in the presence of ApG dinucleotide (lanes 1 to 3) or globin mRNA (lanes 4 to 6) primers. The positions of transcription products (TP) are indicated. The star indicates the position of a nonspecific band. (B) Transcription initiation primed with a capped RNA primer. Transcription reactions were carried out in vitro with nuclear extracts from cells transfected either with pcDNA3 (C) (lane 1) or with pcDNA-PB1 and pcDNA-PB2 either without pcDNA-PA (−PA) (lane 2) or with wild-type (WT) (lane3) or mutant (H510A) (lane4) pcDNA-PA in the presence of an 11-nt ³²P-labeled capped RNA. The positions of transcription products (TP) and the primer are indicated.

FIG. 5.

RNA-binding and endonuclease activities of the H510A polymerase. (A) 5′-end vRNA-binding activity of the H510A polymerase. Nuclear extracts from cells transfected either with pcDNA3 (lane 1), with pcDNA-PB1 and pcDNA-PB2 (−PA) (lane 2), or with pcDNA-PB1, pcDNA-PB2, and either wild-type (WT) or H510A mutant pcDNA-PA (lanes 3 and 4, respectively) were used in gel shifts with ³²P-labeled 15-nt 5′-end vRNA as described in Materials and Methods. The position of the polymerase complex (3P) is indicated. The star indicates a nonspecific band. (B) Capped RNA-binding activity of the H510A polymerase. His-tagged polymerases, partially purified from cells transfected either with pcDNA-PB1 and pcDNA-PB2 (−PA) (lane1) or with pcDNA-PB1, pcDNA-PB2, and either pcDNA-PA-His₆ wild-type (WT) or H510A mutant pcDNA-PA-His₆ (lanes 2 and 3, respectively), were cross-linked to ³²P-labeled capped RNA as described in Materials and Methods. The positions of the PB1 and PB2 bands are indicated. (C) Endonuclease activity of the H510A polymerase. Nuclear extracts from cells transfected either with pcDNA3 (lane 1), with pcDNA-PB1 and pcDNA-PB2 (−PA) (lane 2), or with pcDNA-PB1, pcDNA-PB2, and either wild-type (WT) or H510A mutant pcDNA-PA (lanes 3 and 4, respectively) were incubated with poly(A)⁺-capped RNA, and cleavage products were analyzed as described in Materials and Methods. As a positive control, endonuclease cleavage was carried out with viral cores isolated from purified virus (RNP) (lane 5). The position of the specific cleavage product (CP) is indicated. The poly(A)⁺-capped RNA (indicated) runs as a smear due to the variety in the length of the poly(A) tail.