Primer and probe sequences used in the qRT-PCR experiments.

Primer and probe sequences used in the qRT-PCR experiments.</p

Construction strategy and primers for generating recombinant viruses.

Construction strategy and primers for generating recombinant viruses.</p

450th-L-NP mediates a higher translation efficiency of GFP mRNA from the minigenome.

(A) Cells were collected at 24h after transfection of minigenome or the control plasmid, and half of them were lysed to isolate ribosomes. Top: Total cytoplasmic ribosomes were separated by sucrose density gradient centrifugation, and the absorbance of each fraction was measured at 254nm. Cycloheximide was present in each sample. Lower panel: Protein in half of each fraction’s volume was subjected to TCA precipitation and subsequently utilized for immunoblotting with anti-His and anti-rpS6 antibodies. (B) The remaining half of the cells were extracted for total RNA to detect the mRNA of GFP by quantitative RT-PCR. The results represent the mean ± SD of a representative quantitative RT-PCR experiment conducted in triplicate. (C) Samples from the remaining half of each fraction after ribosome isolation were extracted for RNA and assayed for the distribution of mRNA of GFP in complex with ribosomes by quantitative RT-PCR. Results are the mean ± SD of a representative quantitative RT-PCR experiment performed in duplicate three times. Significance was analyzed by two-way ANOVA. (** means pp<0.001). (D) Schematic representation of NP protein deletion mutants. Boxes indicate the protein product of each truncated NP gene, with amino acid positions indicated above the boxes. Straight lines indicate the region of deletion. (E) Residues 122–366 and 366–489 of NP are sufficient for its localization to the ribosome. Multiple c- and n-terminal truncated NPs were expressed in HeLa cells. Cell extracts from transfected cells were subjected to 10–50% sucrose density gradient ultracentrifugation. RNase (100U/mL) was added to the cell lysate to eliminate the impact of varying RNA levels on polyribosome enrichment. Protein in each fraction was subjected to TCA precipitation and subsequently utilized for immunoblotting with anti-His and anti-rpS6 antibodies.</p

NP is the main protein responsible for the phenotype.

(A) Schematic diagram of the cloning strategy for replacement of NP, P, and L genes between rHerts/33 and rI4. The construction strategy is described in the S1 Table. The virulence of the different recombinant viruses was determined by measuring the ICPI in 1-day-old chickens. (B and C) TCID50 value of the virulent strains after simultaneous replacement of NP, P, and L at 72hpi on tumor cell lines. (D) Expression of viral proteins on HeLa cells by recombinant viruses after simultaneous replacement of NP, P, and L. Western blot analysis was performed by anti-NP and anti-HN at 24h after infection with NDVs at 1MOI and 10MOI, respectively. (E and F) TCID50 value of the virulent strains after individual gene replacement of NP, P, or L at 72hpi on tumor cell lines. (G-H) Viral proteins expression on HeLa cells by recombinant viruses after individual gene replacement of NP, P, or L. Western blot analysis was performed by anti-NP and anti-HN at 24h after infection with NDVs at 1MOI and 10MOI, respectively. Representative data, shown as the means ± SDs (n = 3), were analyzed with two-way ANOVA. ****, P<0.0001.</p

Viruses containing phenylalanine residues at 450 of NP are primarily found in genotype VII.

(A) ML phylogenetic tree of 890 NDV strains based on full-length F-gene sequences. The evolutionary tree was constructed using PhyloSuite in a SYM model. Pie chart representing the number of various residues at position 450 of amino acids for 890 NDV strains. (B) The pie chart illustrates all possible amino acids at position 450 of the NP protein and their proportions. (C) The pie chart shows the distribution of 450aa-phe-NP strains in each genotype. (D) The pie chart displays all the possibilities and their frequencies of the 450th amino acid position of the genotype VII strains’ NP protein.</p

No synergistic effect was found among the homologous NP, P, and L proteins.

(A) The cloning strategy schematic for simultaneous replacement of NP and P, NP, and L, or P and L genes between rHerts/33 and rI4. The virulence of the different recombinant viruses was determined by measuring the Intracerebral Pathogenicity Index (ICPI) in 1-day-old chickens. (B and C) TCID50 value of the virulent strains after simultaneous replacement of NP and P, NP and L, or P and L genes. Representative data, shown as the means ± SDs (n = 3), were analyzed with two-way ANOVA. ****, P (TIF)</p

Variations in viral replicative capability occur during the translation of viral mRNA.

(A) HeLa cells and DF1 cells were infected with NDVs at 10MOI at 37°C for 1h and then incubated with anti-HN mouse monoclonal antibody and goat anti-mouse IgG/FITC at 4°C. After that, cells were washed and assessed by flow cytometry. (B, C, D) HeLa cells were infected with NDV (10MOI) at 37°C for 0.5h and then were collected at 0h, 1h, 2h, 4h, and 8h. Total RNA was extracted and reverse-transcribed using specific primers for gRNA (B), mRNA (C), and cRNA (D) of NDVs. Copy numbers were determined using quantitative RT-PCR. (E and F) Total cellular RNA was extracted at 12h and 24h after transfection of 1.5 μg minigenome into HeLa cells. Reverse transcription was performed using specific primers to detect genomic RNA (E) and mRNA (F) of GFP by quantitative RT-PCR. (G) Expression of GFP was detected at 24h in HeLa cells after transfecting 0.5μg or 1.5μg minigenome with anti-GFP, anti-NP, and anti-β-actin. (H) After transfection with different minigenome systems for 24h, cells were treated with 100μg/ml CHX, and then cells were harvested at 4, 8, and 12 hours. Expression of GFP and NP was detected with anti-GFP, anti-NP, and anti-β-actin. (I, J, K) HeLa cells were treated with 100μg/ml CHX for 30mins and then infected with NDV (10MOI) at 37°C for 0.5h. After that, cells were collected at 0h, 1h, 2h, 4h, and 8h. Total RNA was extracted and reverse-transcribed using specific primers for gRNA (I), mRNA (J), and cRNA (K) of NDVs. Copy numbers were determined using quantitative RT-PCR. Data are presented as means from three independent experiments. Significance is analyzed by two-way ANOVA (****, p<0.0001).</p

HNP inhibits the eIF4A1-dependent translation.

(A) A lentiviral packaging system was used to express NDV-NP protein in HeLa cells, and NP expression was detected by Western blotting. (B) The statistical plot of the number of differentially expressed genes (DEGs) in each group (fc>1, p1, pp<0.0001.</p

HNP protein physically interacts with eIF4A1 with 366-489aa.

(A) Co-immunoprecipitation (Co-IP) of NP protein with endogenous eIF4A1 during NDV infection. HeLa cells were infected with rHerts/33, rI4, or mock-infected and subjected to IP using anti-NP protein or anti-eIF4A1 antibodies. Immunoblotting was performed using the indicated antibodies. (B) HeLa cells were transfected with His-tagged HNP or INP and HA-tagged eIF4A1. After 36 hours, HNP’s interaction with eIF4A1 was confirmed through Co-IP using both anti-NP and anti-HA antibodies. (C) GST pull-down assay. Glutathione beads were conjugated with GST or GST-NP fusion protein and incubated with lysate from cells overexpressing eIF4A1. Eluted proteins were subjected to Western Blot, and eIF4A1 presence was detected using anti-HA antibody. GST, GST-HNP, and GST-INP protein expression was confirmed with anti-GST antibody. (D) Redistribution and colocalization of eIF4A1 with HNP protein. HeLa cells were transfected with His-tagged HNP or INP and HA-tagged eIF4A1, fixed at 24 hpi, stained with anti-eIF4A1 and anti-NP antibodies, and visualized using confocal microscopy. (E) Direct interaction between eIF4A1 and HNP confirmed by Bifc assay. Venus luminescence was observed after separate or co-transfection of VC155-eIF4A1, VN173-HNP, and VN173-INP. (F) The C-terminal region 366-489aa of NP is sufficient for heterologous protein association with eIF4A1. Multiple C-terminal and N-terminal truncations of His-tagged NP were expressed in HeLa cells and subjected to IP experiments using anti-eIF4A1 antibody, followed by analysis with specific antibody immunoblotting.</p

Primers for generating HeLa cell lines with stable expression of the NP protein.

Primers for generating HeLa cell lines with stable expression of the NP protein.</p