The N-terminal extension of the influenza B virus nucleoprotein is not required for nuclear accumulation or the expression and replication of a model RNA

Abstract

The nucleoprotein (NP) of influenza B virus is 50 amino acids longer at the N-terminus than influenza A virus NP and lacks homology to the A virus protein over the first 69 residues. We have deleted the N-terminal 51 and 69 residues of the influenza B/Ann Arbor/1/66 virus NP and show that nuclear accumulation of the protein is unaffected. This indicates that the nuclear localization signal is not located at the extreme N terminus, as in influenza A virus NP. To determine if the N-terminal mutants could support the expression and replication of a model influenza B virus RNA, the genes encoding the subunits of the viral RNA-dependent RNA polymerase (PA, PB1, and PB2) were cloned. Coexpression of NP and the P proteins in 293 cells was found to permit the expression and replication of a transfected model RNA based on segment 4 of B/Maryland/59, in which the hemagglutinin-coding region was replaced by a chloramphenicol acetyltransferase gene. The expression and replication of the synthetic RNA were not affected by the replacement of NP with NP mutants lacking the N-terminal 51 or 69 residues, indicating that the N-terminal extension is not required for transcription or replication of the viral RNA. In addition, we report that the influenza B virus NP cannot be functionally replaced by type A virus NP in this system.

FIG. 1

Alignment of the amino acid sequences of the influenza A/PR/8/34 and B/AA/1/66 virus NPs at the N-termini. The alignment was generated with the FASTA program (Genetics Computer Group, University of Wisconsin) and predicts 37.7% identity and 76.2% similarity of the proteins in a 496-amino-acid overlap. The residues in influenza A/PR/8/34 virus NP reported by Wang et al. (34) to interact with NPI-1 and NPI-3 are shown in boldface below the influenza A virus sequence. Neumann et al. (25) have separately reported that residues Lys7 and Arg8 are crucial for nuclear import of the A/WSN/33 NP. Arrows indicate the extent of the N-terminal deletions in the type B virus NP made in this study, vertical lines denote amino acid identity, and colons refer to conservative changes.

FIG. 2

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the influenza B/AA/1/66 virus NP and NP deletion mutants (A) and of influenza B/AA/1/66 virus polymerase proteins (B) synthesized in vitro. Rabbit reticulocyte lysates (Promega) were primed with 50 ng of T7 transcripts from SmaI-linearized templates in the presence of [³⁵S]methionine according to the manufacturer’s instructions. Proteins were resolved on an SDS–10% PAGE gel and visualized by autoradiography. Numbers refer to the sizes in kilodaltons of protein standards.

FIG. 3

Localization of NP and NP deletion mutants in MDCK cells. MDCK cells (American Type Culture Collection; CCL 34) were grown on glass coverslips to 50 to 70% confluency and were transfected with 5 μg of pcDNA3-NP, pcDNA3-NPΔ51, pcDNA3-NPΔ69, or pHMG-NP (encoding A/PR/8/34 NP), by using 30 μg of Pfx-2 lipofection reagent (Invitrogen) in serum-free Eagle’s minimal essential medium. Twenty-four or forty-eight hours after transfection the cells were fixed and permeabilized with −20°C absolute ethanol for 5 min and then analyzed by indirect immunofluorescence with a mouse anti-B virus NP MAb (MAS774b; Harlan Sera-lab) and an anti-mouse immunoglobulin G-fluorescein isothiocyanate conjugate. Samples were mounted with Mowiol 40-88 and 1,4-diazabicyclo[2.2.2]octane (Aldrich) and analyzed with a Zeiss Axiovert 135 fluorescence microscope and a 100× oil immersion lens. The same localization of NP or the NP deletion mutants was observed if the cells were fixed with 3% (wt/vol) paraformaldehyde and permeabilized with 0.1% (vol/vol) Triton X-100 (data not shown).

FIG. 4

Expression of HABCAT RNA in cells supplying PA, PB1, PB2, and NP in trans. Approximately 10⁶ 293 cells in 35-mm-diameter dishes were transfected with 0.5 μg of pcDNA3-PA, 0.5 μg of pcDNA3-PB1, 0.5 μg of pcDNA3-PB2, and 1 μg of pcDNA3-NP by using 20 μg of Lipofectamine (GIBCO/BRL) in serum-free Eagle’s minimal essential medium (EMEM) according to the manufacturer’s instructions. At time zero and at 3, 6, 9, 12, and 24 h after transfection of the plasmids a separate mixture of 1 μg of HABCAT RNA and 20 μg of Lipofectamine was added. HABCAT RNA was synthesized in vitro in a 25-μl reaction mixture containing 1 μg of HgaI-linearized pT3HABCAT (4), 40 mM Tris-HCl (pH 8.0), 50 mM NaCl, 8 mM MgCl₂, 10 mM dithiothreitol, 2 mM spermidine, the four dNTPs (1 mM each), 30 U of human placental RNase inhibitor, and 50 U of T3 RNA polymerase. After incubation at 37°C for 1 h, 2 U of RQ1 RNase-free DNase (Promega) was added to remove the template and the RNA was extracted with phenol-chloroform and precipitated with ethanol. After 24 h at 37°C the cells were supplemented with 1 ml of EMEM containing 10% heat-inactivated fetal calf serum. Forty-eight hours posttransfection the cells were harvested into 100 μl of 250 mM Tris-HCl (pH 7.5) and lysed by freezing and thawing three times. Lysates (50 μl) were then processed for the detection of CAT as described elsewhere (21).

FIG. 5

Expression of HABCAT RNA is not affected by the removal of the N-terminal extension of influenza B virus NP. 293 cells were transfected with 0.5 μg of pcDNA3-PA, 0.5 μg of pcDNA3-PB1, 0.5 μg of pcDNA3-PB2, and 1 μg of either pcDNA3-NP, pcDNA3-NPΔ51, pcDNA3-NPΔ69, pHMG-NP, or pcDNA3 by using 20 μg of Lipofectamine. Immediately after, a separate mixture of 1 μg of HABCAT RNA and 20 μg of Lipofectamine was added.

FIG. 6

Replication of HABCAT RNA is not affected by removal of the N-terminal extension of influenza B virus NP. In order to synthesize the cRNA intermediate of HABCAT replication, the HABCAT cDNA was cloned under a T3 promoter in the reverse orientation to that in pT3HABCAT (4) by PCR with oligonucleotides 5′ gcgcgcaagcttgacgcatcgaAGTAGTAACAAGAGC 3′ and 5′ gcgcgcgaattcaattaaccctcactaaaAGCAGAAGCAGAGC 3′. 293 cells were transfected with 0.5 μg of pcDNA3-PA, 0.5 μg of pcDNA3-PB1, 0.5 μg of pcDNA3-PB2, and 1 μg of either pcDNA3-NP, pcDNA3-NPΔ51, pcDNA3-NPΔ69, pHMG-NP, or pcDNA3 by using 20 μg of Lipofectamine. Immediately after, a separate mixture of 1 μg of HABCAT cRNA and 20 μg of Lipofectamine was added.