Characterization of nucleolar localization signals in the avian infectious bronchitis virus nucleocapsid protein and their critical role in viral replication

Figures

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  Fig 1. Nucleolar localization of IBV N protein during viral infection and transfection. (A) Subcellular localization of the IBV N protein during viral infection. CEK cells were infected with IBV at an MOI of 0.1, fixed at 12, 24, 36, and 48 hpi, and processed for IFA. Cells were immunolabeled with anti-IBV N and anti-fibrillarin antibodies, followed by incubation with secondary antibodies conjugated to Alexa Fluor 488 and Alexa Fluor 555. Nuclei were counterstained with DAPI. Red arrows indicate sites of colocalization between the IBV N protein and the nucleolar marker fibrillarin. Scale bars: 10 μm. (B) Subcellular localization of the N protein following plasmid transfection. Vero E6 cells were transfected with pCMV-Flag-N and fixed 24 h post-transfection. Samples were stained using the same IFA procedure as in panel A. Fluorescence intensity line-profiles and PCC were used to quantify the degree of colocalization between the N protein and fibrillarin. Scale bars: 10 μm.

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  Fig 2. Mapping of four NoLSs and one NLS in the IBV N protein. (A–D) Nucleolar localization analysis of full-length IBV N protein and truncated mutants. Left panels present schematic representations of the full-length N protein and various truncated mutants fused to EGFP. Constructs were designed based on the structural domains of the N protein and regions rich in basic amino acids (aa 72–85, 162–167, 177–188, 199–218, 330–349, and 356–368). Right panels present representative confocal micrographs of the corresponding subcellular localization patterns. Vero E6 cells were individually transfected with the indicated expression plasmids. At 24 h post-transfection, the cells were fixed with 4% paraformaldehyde and processed for IFA. EGFP-fused proteins are shown in green, fibrillarin in red, and nuclei in blue. Scale bars: 10 μm. (E) Sequence conservation of the four identified NoLSs (NoLS1–NoLS4). Multiple sequence alignment of N proteins from diverse IBV strains was used to generate WebLogo representations. The height of each individual symbol represents the relative frequency of that specific amino acid at the corresponding position.

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  Fig 3. NoLS3 and NoLS4 are the primary functional NoLSs of the IBV N protein. (A) Subcellular localization of the wild-type and NoLS alanine substitution mutant N proteins. Vero E6 cells were individually transfected with plasmids expressing the wild-type N protein (N-WT), single-NoLS alanine-substitution mutants (all basic residues in the targeted NoLS replaced with alanine), or combinatorial NoLS mutants. At 24 h post-transfection, the cells were fixed and subjected to an IFA. N protein is shown in green, fibrillarin in red, and nuclei in blue. Scale bars: 10 μm. (B) Quantitative analysis of the N protein nucleolar localization. For each construct, the percentage of transfection-positive cells exhibiting distinct nucleolar accumulation of N protein was calculated. Data are shown as mean ± SEM from three independent experiments (n = 3); >200 transfected cells were scored in each experiment; ***P < 0.001; ns, not significant by one-way ANOVA, followed by Dunnett post hoc test.

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  Fig 4. Cluster and single-point mutational analyses reveal critical basic residues for NoLS3 function. (A) Schematic representations of the amino acid sequences for the wild-type NoLS3 and its clustered alanine substitution mutants. The original basic amino acid residues are underlined, and the introduced alanine substitutions are indicated by red "A." (B) Vero E6 cells were individually transfected with plasmids expressing the indicated clustered mutants. At 24 h post-transfection, the cells were fixed and subjected to an IFA. N protein is shown in green, fibrillarin in red, and nuclei in blue. Scale bars: 10 μm. (C) Quantitative analysis of the N protein nucleolar localization. The percentage of N-expressing cells exhibiting distinct nucleolar localization of the N protein was calculated for each group. (D) Schematic representations of the NoLS3 mutants with single alanine substitutions of basic amino acids. (E) Confocal microscopy images showing the subcellular localization of the corresponding single-point mutants in Vero E6 cells at 24 h post-transfection. The staining strategy is identical to that described in panel B. Scale bars: 10 μm. (F) Quantitative analysis of the nucleolar…

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  Fig 5. Cluster and single-point mutational analyses reveal critical basic residues for NoLS4 function. (A) Schematic representations of the amino acid sequences for the wild-type NoLS4 and its clustered alanine substitution mutants. The original basic amino acid residues are underlined, and the introduced alanine substitutions are indicated by red "A"s. (B) Vero E6 cells were individually transfected with plasmids expressing the indicated clustered mutants. At 24 h post-transfection, the cells were fixed and subjected to an IFA. N protein is shown in green, fibrillarin in red, and nuclei in blue. Scale bars: 10 μm. (C) Quantitative analysis of the N protein nucleolar localization. The percentage of N-expressing cells exhibiting distinct nucleolar localization of the N protein was calculated for each group. (D) Schematic representations of the NoLS4 mutants with single alanine substitutions of basic amino acids. (E) Confocal microscopy images showing the subcellular localization of the corresponding single-point mutants in Vero E6 cells at 24 h post-transfection. The staining strategy is identical to that described in panel B. Scale bars: 10 μm. (F) Quantitative analysis of the nucleolar…

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  Fig 6. Disruption of NoLS4 abolishes nucleolar targeting of the N protein and impairs viral replication. (A) Schematic representation of the construction strategy for recombinant IBV mutants. Key alanine substitutions were introduced into the NoLS3 and NoLS4 regions of the N protein within the IBV genome using a reverse genetics platform (the introduced alanine mutations are indicated by red "A"s). (B) Sequence verification of the recombinant viruses. The N gene of the P5 recombinant viruses was verified by Sanger sequencing. The red dashed box highlights the nucleotide sequence of the NoLS4 region. (C) Assessment of viral infectivity in ECEs. Representative phenotypes of chicken embryos inoculated with PBS (MOCK), the wild-type recombinant virus (rYN-WT), or the mutant virus (rYN-N-K356A/R359A) are shown. (D) Assessment of viral replication in CEK cells by IFA. Left: CEK cells were infected with rYN-WT or rYN-N-K356A/R359A at an MOI of 0.01. Cells were fixed at 18, 24, 36, and 48 hpi and processed for IFA. The N protein is shown in green, and nuclei were counterstained with DAPI (blue). Scale bars: 400 μm. Right: Quantification of the percentage of IBV-positive cells at the indicated…