Attenuated Viral Replication of Avian Infectious Bronchitis Virus with a Novel 82-Nucleotide Deletion in the 5a Gene Indicates a Critical Role for 5a in Virus-Host Interactions

FIG 1. Flow cytometry analysis of IBV N-positive cells. CEK cells were infected with rYN or rYN-Δ5a, then stained at various time points with mouse antibodies specific for the IBV structural protein N, and subjected to flow cytometry to measure the percentage of EGFP-positive (infected) cells. The background fluorescence of the cells was subtracted based on data from uninfected cells. Artificially constructed IBV-EGFP viruses were used as a positive control. At each time point, three samples from each group were tested, and the mean infected cell percentages were statistically evaluated using a two-way ANOVA test adjusted for post hoc analysis, followed by Bonferroni’s multiple-comparison tests. Statistically significant differences between groups are highlighted; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG 2. Genomic RNA and sgmRNA copy numbers in CEK cells infected with rYN or rYN-Δ5a. CEK cells were inoculated with rYN or rYN-Δ5a and then harvested at various time points for use in qRT-PCR to measure the genomic RNA and sgmRNA copy numbers. Leader-F was used as the forward primer, and different reverse primers were used to measure the genome replication (1ab) and sgmRNA synthesis (S, 3ab&E, M, 5ab, and N) levels (see Table 2). All assays were run in triplicate, and sample copy numbers were calculated based on a standard curve. Data were statistically evaluated by a two-way ANOVA test adjusted for post hoc analysis, followed by Bonferroni’s multiple-comparison tests. Statistically significant differences between groups are highlighted; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG 3. N synthesis and offspring virus release into the supernatant in CEK cells infected with rYN or rYN-Δ5a. CEK cells were inoculated with rYN or rYN-Δ5a. (A) Representative Western blot analysis of whole-cell lysates using antibodies recognizing IBV N and β-actin. The expression level of IBV N was quantified by densitometry and normalized to the β-actin expression level. Results shown are the means of three independent determinations (± standard error of the mean [SEM]). *, P < 0.05. (B) qRT-PCR with primers Leader-F and 1ab-R was conducted on the supernatant of cells harvested at various time points postinfection. All assays were run in triplicate, and values were calculated based on a standard curve. Data were statistically evaluated by a two-way ANOVA test adjusted for post hoc analysis, followed by Bonferroni’s multiple-comparison tests. Statistically significant differences between groups are highlighted; *, P < 0.05; ***, P < 0.001. (C) Representative Western blot analysis of the supernatant proteins at various time points using antibodies recognizing IBV N. The N expression levels were taken as a proxy measurement of released offspring virus. The expression level of IBV N was…

FIG 4. Expression, cellular sublocalization pattern, and interacting host proteins of EGFP-tagged and Flag-tagged 5a fusion protein. Vero cells were transfected with eukaryotic expression vectors for EGFP- or Flag-tagged 5a protein. (A) Western blot analysis of whole-cell lysates using antibodies recognizing EGFP or Flag-tag. (B) The sublocalization pattern of 5a protein in the cells was observed under a confocal microscope through EGFP fluorescence or ani-Flag antibodies in Vero cells transfected with pRK5-5a-EGFP or pRK5-5a-Flag, respectively. The results were observed at 24 h posttransfection. Nuclei were counterstained with DAPI (blue). Scale bar = 20 μm. (C) The constructed protein-protein interaction (PPI) network for 5a protein-correlated host proteins. The functional annotation of the genes included in the right-side nodules are listed separately in Table 2.

FIG 5. Expression and co-IP of HA-tagged XPOT, XPO1, TNPO1, TNPO3, RanBP1, and EIF2B4 with Flag-tagged 5a fusion protein. (A) Western blot analysis of whole-cell lysates of 293T cells transfected with eukaryotic expression vectors for HA-tagged XPOT, XPO1, TNPO1, TNPO3, RanBP1, or EIF2B4, probed with antibodies recognizing HA-tag. (B) Co-IP blots of anti-Flag antibody immunoprecipitations from 293T cells cotransfected with eukaryotic expression vectors for HA-tagged XPOT, XPO1, TNPO1, TNPO3, RanBP1, or EIF2B4 proteins and for Flag-tagged 5a, probed with anti-Flag and anti-HA antibodies. An unlabeled, antibody-coupled protein A/G-coupled agarose affinity gel was used as the negative control for nonspecific binding.

FIG 6. Cellular sublocalization pattern of HA-tagged XPOT, XPO1, TNPO1, TNPO3, RanBP1, and EIF2B4 in the presence or absence of 5a protein. (A and B) Vero cells were transfected with eukaryotic expression vectors for HA-tagged XPOT, XPO1, TNPO1, TNPO3, RanBP1, or EIF2B4 proteins alone (A) or were cotransfected with those expression vectors and an expression vector for Flag-tagged 5a (B). At 48 h posttransfection, paraformaldehyde-fixed, cyro-embedded cells were stained with antibodies against Flag (green) or HA (red) tag. Nuclei were counterstained with DAPI (blue). Images were captured using a Nikon A1 confocal microscope. Scale bar = 10 and 20 μm in panels A and B, respectively.

FIG 7. Cellular sublocalization pattern of IBV N in rYN or rYN-△5a infected CEK cells. CEK cells were inoculated with rYN or rYN-Δ5a. Expression plasmid for Flag-tagged 5a protein was electroporated into CEK cells after infection to supplement additional 5a protein (+5a). Flag-tag plasmid was set as the negative control (+k). (A) Sublocalization of IBV-N protein. Infected CEK cells were stained for IBV-N with anti IBV-N antibody (green) at 24 hpi. Nuclei were stained with DAPI (blue). Cells were analyzed by Nikon fluorescence microscopy using a 20× objective, and representative images are shown. Below the image is a profile showing the intensity of EGFP (green line) and DAPI (blue line) fluorescence in the image. (B) Expression of IBV-N protein in the cytoplasmic and nuclear extracts of CEK cells. Cytoplasmic and nuclear extracts of infected CEK cell lysates were prepared using a nuclear and cytoplasmic protein extraction kit and analyzed by Western blotting with anti-IBV-N antibody. Cytoplasmic extraction was analyzed using α-tubulin antibody, and nuclear extraction was analyzed using histone H3 antibody.

FIG 8. Cellular sublocalization pattern of IBV N in the presence or absence of 5a, XPOT, XPO1, TNPO1, TNPO3, RanBP1, and EIF2B4. Vero cells were transfected with PCI-N alone or were cotransfected with PCI-N and expression vectors for Flag-tagged 5a or HA-tagged XPOT, XPO1, TNPO1, TNPO3, RanBP1, or EIF2B4. At 48 h posttransfection, paraformaldehyde-fixed, cyro-embedded cells were stained with antibodies against Flag tag (red) and IBV N (green) or HA tag (red). Nuclei were counterstained with DAPI (blue). Images were captured using a Nikon A1 confocal microscope. Scale bar = 20 μm.