Deep sequencing of the mouse lung transcriptome reveals distinct long non-coding RNAs expression associated with the high virulence of H5N1 avian influenza virus in mice

<p>Long non-coding RNAs (lncRNAs) play multiple key regulatory roles in various biological processes. However, their function in influenza A virus (IAV) pathogenicity remains largely unexplored. Here, using next generation sequencing, we systemically compared the whole-transcriptome response of the mouse lung infected with either the highly pathogenic (A/Chicken/Jiangsu/k0402/2010, CK10) or the nonpathogenic (A/Goose/Jiangsu/k0403/2010, GS10) H5N1 virus. A total of 126 significantly differentially expressed (SDE) lncRNAs from three replicates were identified to be associated with the high virulence of CK10, whereas 94 SDE lncRNAs were related with GS10. Functional category analysis suggested that the SDE lncRNAs-coexpressed mRNAs regulated by CK10 were highly related with aberrant and uncontrolled inflammatory responses. Further canonical pathway analysis also confirmed that these targets were highly enriched for inflammatory-related pathways. Moreover, 9 lncRNAs and 17 lncRNAs-coexpressed mRNAs associated with a large number of targeted genes were successfully verified by qRT-PCR. One targeted lncRNA (NONMMUT011061) that was markedly activated and correlated with a great number of mRNAs was selected for further in-depth analysis, including predication of transcription factors, potential interacting proteins, genomic location, coding ability and construction of the secondary structure. More importantly, NONMMUT011061 was also distinctively stimulated during the highly pathogenic H5N8 virus infection in mice, suggesting a potential universal role of NONMMUT011061 in the pathogenesis of different H5 IAV. Altogether, these results provide a subset of lncRNAs that might play important roles in the pathogenesis of influenza virus and add the lncRNAs to the vast repertoire of host factors utilized by IAV for infection and persistence.</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

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

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

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

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

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

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