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

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

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

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

Infectivity of different genotypic strains on non-tumor mammalian cells.

Viral titers at 72hpi in (A) Vero, (B) sp2/0, (C) MDCK, (D) HcMEC/D3, (E) CHO, (F) 293T, (G) 16HBE, (H) TCID50 value of ten additional genotype VII NDV strains in HeLa cells. Representative data, shown as the mean ± SD (n = 3), were analyzed with one-way ANOVA. ****, P (TIF)</p

Prediction of disordered domains and infectivity assays of recombinant viruses.

(A and B) PONDR was used to predict the IDR of HNP (A) and INP (B) (http://www.pondr.com/). VL3 Predictor (Developed by P. Radivojac and A.K. Dunker) was used. Regions with a score greater than 0.5 are considered disordered regions. (C and D) PSIPRED was used to predict the IDR of HNP (A) and INP (B) (http://bioinf.cs.ucl.ac.uk/psipred/?disopred=1). Regions with a score greater than 0.5 are considered disordered regions. (E) Schematic diagram of the cloning strategy for exchanging the whole N-core and the first IDR of the N-tail domain between rHerts/33 and rI4. The virulence of the different recombinant viruses was determined by measuring the ICPI in day-old chickens. (F, G, H, and I) TCID50 value of recombinant strains after replacement of the whole N-core and the first IDR of N-tail domain. (J, K, L, M) TCID50 values of mutant NDVs at 72hpi on several non-tumor cell lines. Representative data, shown as the means ± SDs (n = 3), were analyzed with two-way ANOVA. ****, P (TIF)</p

Proposed model of the differential oncolytic capability mediated by NP Protein in NDV strains.

(A) Translation of oncolytic NDV (Herts/33) mRNA: i HNP(450th-L-NP) efficiently recruits ribosomes and interacts with eIF4A1 through its 366-489aa, ii The efficiency of viral mRNA lacking 2′-O-methylation modification loading onto ribosomes is higher. The translation of cellular mRNA with complex structures in the 5’UTR, dependent on eIF4A1, is inhibited. These two processes together resulting in low efficiency of cellular mRNA loading onto ribosomes and distribution in areas unrelated to ribosomes, iii Efficient translation of viral mRNA, iv Production of a large number of viral particles. (B) Translation of non-oncolytic NDV (I4) mRNA: i INP(450th-F-NP) has weak binding ability with ribosomes and cannot interact with eIF4A1, ii Cellular mRNA containing 2’-O-methylation modification is efficiently loaded onto ribosomes. The translation of cellular mRNA with a complex structure in the 5’UTR, dependent on eIF4A1, is unaffected. Few viral mRNA is loaded onto ribosomes and more viral mRNA is distributed in regions unrelated to ribosomes, iii Extremely low efficiency of viral mRNA translation, iv Production of a small number of viral particles.</p

Primers sequences used for the construction of plasmids in the BiFC experiment.

Primers sequences used for the construction of plasmids in the BiFC experiment.</p

Primer sequences for the construction of NP protein prokaryotic expression plasmid.

Primer sequences for the construction of NP protein prokaryotic expression plasmid.</p

Differences occur during the viral protein translation.

(A) Expression of GFP was detected at 24 h in CEF cells after transfecting 0.5 μg or 1.5 μg minigenome with anti-GFP, anti-NP, and anti-β-actin. (B) HeLa cells were treated with 100μg/ml CHX for 1h and then infected with NDV (10MOI) at 37°C for 0.5h. After that, cells were collected at 0h, 1h, 2h, 4h, and 8h. (TIF)</p

Natural immunity and stress response are not the primary causes of differential infectivity.

HeLa cells were transfected with control siRNA or specific siRNA targeting RIG-I and PKR. After 36 h, cells were infected with NDV at 1 MOI or 10 MOI and collected at 24 hpi for analysis by immunoblotting with anti-RIG-I, anti-HN, anti-p-PKR, anti-PKR, or anti-β-actin antibodies. (TIF)</p