Mutational analysis of conserved amino acids in the influenza A virus nucleoprotein

Abstract

The nucleoprotein (NP), which has multiple functions during the virus life cycle, possesses regions that are highly conserved among influenza A, B, and C viruses. To better understand the roles of highly conserved NP amino acids in viral replication, we conducted a comprehensive mutational analysis. Using reverse genetics, we attempted to generate 74 viruses possessing mutations at conserved amino acids of NP. Of these, 48 mutant viruses were successfully rescued; 26 mutants were not viable, suggesting a critical role of the respective NP amino acids in viral replication. To identify the step(s) in the viral life cycle that is impaired by these NP mutations, we examined viral-genome replication/transcription, NP localization, and incorporation of viral-RNA segments into progeny virions. We identified 15 amino acid substitutions in NP that inhibited viral-genome replication and/or transcription, resulting in significant growth defects of viruses possessing these substitutions. We also found several NP mutations that affected the efficient incorporation of multiple viral-RNA (vRNA) segments into progeny virions even though a single vRNA segment was incorporated efficiently. The respective conserved amino acids in NP may thus be critical for the assembly and/or incorporation of sets of eight vRNA segments.

FIG. 1.

Individual residues targeted by mutagenesis. (A) The NP regions responsible for binding to RNA (red), NP itself (orange), and PB2 (purple) are shown. (B) Amino acid sequence of WSN NP. Amino acids tested in this study are shaded. The unconventional NLS, the bipartite NLS, the cytoplasmic accumulation signal (CAS), and the NP tail loop, which mediates NP oligomerization, are shown in different colors. NP mutants flagged with an asterisk were further tested for intracellular localization and incorporation efficiency of a vRNA segment(s) into VLPs, in addition to replicative ability and polymerase activity.

FIG. 2.

Localization of NP mutants. MDCK cells transfected with plasmids expressing mutant NP or wild-type NP were subjected to indirect immunofluorescence assays. NP and DNA were stained with an anti-NP antibody and DAPI (4′,6′-diamidino-2-phenylindole), respectively. Samples were observed under a confocal laser microscope. Nine hours after transfection, wild-type and mutant NP exclusively localized to the nucleus. Shown are wild-type NP (A) and mutant R175A as a representative example of the mutants tested (B). Twenty-four hours after transfection, wild-type NP and all mutant NPs tested localized to both the nucleus and the cytoplasm, regardless of the addition of the translational inhibitor cycloheximide at 9 h posttransfection. The localization of the mutant NP proteins was indistinguishable from that of wild-type NP. Scale bars, 20 μm.

FIG. 3.

System to test vRNP virion incorporation efficiencies. VLPs were recovered from 293T cells transfected with eight PolI constructs for the eight RNA segments PB1, PB2, PA, NA, M, NS, HA-GFP, and NP(Met⁻), which lacks a start codon. These VLPs were used to infect MDCK cells in the presence of helper virus (left). In this experimental setting, GFP-positive MDCK cells are representative of VLPs that contain at least one vRNA segment (i.e., the HA-GFP vRNA). In a parallel experiment, VLPs derived from 293T cells were used to infect MDCK cells expressing NP (right). In this experimental setting, GFP-positive MDCK cells are indicative of VLPs that possess at least four vRNAs (i.e., the PB2, PB1, PA, and HA-GFP vRNAs).

FIG. 4.

Crystal structure of influenza A virus NP. The images were created with the program PYMOL (W. L. Delane; http://www.pymol.org), and the NP structure was obtained from the Protein Data Bank (41) (accession number 2IQH). The locations of the amino acid residues that affect the efficient incorporation of multiple vRNA segments into VLPs are shown in color.