Research ArticleVIROLOGY

Human TRA2A determines influenza A virus host adaptation by regulating viral mRNA splicing

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Science Advances  19 Jun 2020:
Vol. 6, no. 25, eaaz5764
DOI: 10.1126/sciadv.aaz5764
  • Fig. 1 TRA2A associates with vRNP.

    (A and B) A549 cells were infected with either the YS (A) or the PR8 (B) virus at a multiplicity of infection (MOI) of 0.01 for 24 hours; cell lysates were immunoprecipitated with an anti-TRA2A antibody or the control immunoglobulin G (IgG) and then analyzed by Western blotting. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (C) A549 cells were infected with the YS or PR8 virus at an MOI of 5. At 4 hours post-infection (hpi), cells were subjected to RNA FISH combined with immunofluorescence to detect M vRNA and TRA2A protein. Scale bars, 10 μm. DAPI, 4′,6′-diamidino-2-phenylindole. (D) A549 cells were infected with either YS or PR8 virus at an MOI of 0.01. At 24 hpi, cells were fixed and analyzed for the colocalization of TRA2A with NP. Scale bars, 20 μm. (E and F) Human embryonic kidney (HEK) 293T cells were transfected with the indicated plasmids for 24 hours. The cell lysates were treated with or without 100 U of ribonuclease A (RNase A) at 37°C for 1 hour. CoIP assay was performed using an anti-Flag antibody and analyzed by Western blotting. (G and H) HEK 293T cells were transfected with the indicated plasmids for 24 hours. Cell lysates were immunoprecipitated with an anti-hemagglutinin (HA) antibody and then analyzed by Western blotting.

  • Fig. 2 TRA2A inhibits YS replication but enhances PR8 replication.

    (A) A549 cells were seeded on the 96-well plates, and cell viability was detected by Cell Counting Kit-8 assay at 36, 48, and 60 hours after transfection. NT, non-treated. (B to H) A549 (B to E and H) or U251 (F and G) cells were transfected with either HA-TRA2A, HA-chTRA2A, or pCAGGS vector or either siNC (negative control) or siTRA2A for 24 hours and then infected with the YS (B, C, F, and H) or PR8 (D, E, and G) virus at an MOI of 0.01. Cell culture supernatants were collected at 12, 24, and 36 hpi. Virus titers were determined by TCID50 assay on MDCK (Madin-Darby canine kidney) cells. A549 cell lysates were analyzed by Western blotting, and the silence efficiency of TRA2A and changes of NP were quantified by ImageJ and normalized to GAPDH (C and E). (I) A549 cells were transfected with either siNC or siTRA2A for 24 hours and infected with an avian virus H9N2 or H7N9 or a human virus WSN/H1N1 strain at an MOI of 0.01. Cells lysates were subjected to Western blotting analysis. GAPDH was used as loading control. Each protein band was quantified by ImageJ and normalized to GAPDH levels. Means ± SD (error bars) of three independent experiments are indicated (*P < 0.05 and **P < 0.01).

  • Fig. 3 TRA2A inhibit YS-M and PR8-NS mRNA splicing.

    (A to G) A549 cells were transfected with either siNC or siTRA2A for 24 hours and infected with the YS (A, C, D, and G) or PR8 (B and E to G) virus at an MOI of 5. Cell lysates were collected at 3, 6, and 9 hpi and subjected to Western blotting analysis. Each protein band was quantified by ImageJ and normalized to GAPDH levels. The NEP/NS1 and M2/M1 protein ratios were also calculated (A and B). The splicing ratios of M (C and E) and NS (D and F) mRNAs were analyzed by the specific reverse transcription quantitative polymerase chain reaction (RT-qPCR). The M2/M1 ratios were analyzed by semi-qPCR (G). Means ± SD (error bars) of three independent experiments are indicated (*P < 0.05, **P < 0.01, and ***P < 0.001).

  • Fig. 4 TRA2A binds to the YS-M and PR8-NS mRNA through GAAARGARR motif.

    (A) A549 cells were infected with either the YS (left) or PR8 (right) virus at an MOI of 1 for 12 hours. RIPs and RT-qPCR were performed using specific primers detecting viral M, NS, and PB1 mRNA and cellular U87 small Cajal body-specific RNA (scaRNA). Fold enrichment of mRNA was calculated. Means ± SD (error bars) of three independent experiments are indicated (**P < 0.01). (B) Schematic illustration of the TRA2A binding sites in M and NS mRNA. (C and E) TRA2A bound to biotin-labeled YS or PR8 NS (C) or M (E) mRNAs or their truncated mRNAs. In vitro transcribed mRNAs were labeled with the biotin and immunoprecipitated with the NEs, and the bead eluate was then analyzed by Western blotting. (D and F) Pull-down TRA2A proteins using 20-bp NS (D) or M (F) biotin probes of PR8 and YS: Probes were immunoprecipitated with the NEs, and the bead eluate was analyzed by Western blotting. (G) TRA2A binding efficiency to PR8-NSWT and YS-NSWT was compared in RNA EMSA with increasing amounts of NEs. (H) TRA2A binding efficiency to YS-MWT and YS-M334C (left) and PR8-MWT and PR8-M334G (right) was compared in RNA EMSA with increasing amounts of NEs.

  • Fig. 5 M segment 334 site single mutation alters its mRNA splicing and virus replication.

    (A to D, F, and G) A549 cells were infected with either the PR8-WT, PR8-M334C, YS-WT, or YS-M334C virus at an MOI of 5; the total RNA was isolated and analyzed by RT-qPCR assay (A and B). Ratios of M2/M1 mRNA are presented (C and D). Cells lysates were subjected to Western blotting analysis using antibodies against respective influenza virus proteins. Each protein band was quantified by ImageJ and normalized to GAPDH levels (F and G). (E) A549 cells were infected with the WT or mutated YS (right) or PR8 (left) virus (MOI, 1) for 12 hours, and the mRNAs were purified with either the control IgG or the TRA2A antibody and analyzed by RT-qPCR assay. (H and I) Growth curves of the indicated virus. (J) A549 cells were transfected with siNC or siTRA2A for 24 hours and then infected with the indicated virus for 6 hours. Cells were stained with rabbit anti-HA antibody followed by fluorescein isothiocyanate (FITC)–goat anti-rabbit IgG and analyzed by flow cytometry. The data represent means ± SD (error bars) of three independent biological replicates (ns, not significant; *P < 0.05, **P < 0.01, and ***P < 0.001).

  • Fig. 6 Pathogenicity of WT YS or PR8 and their mutated viruses at 334 site in mice.

    Six-week-old female BALB/c mice were intranasally inoculated with the indicated doses of the PR8-WT, PR8-M334G, YS-WT, or YS-M334C virus or the phosphate-buffered saline (PBS) control. (A to D) Weight loss and mortality of mice infected with each indicted virus. Body weight of the WT and 334 mutant groups were compared and statistically analyzed. Error bars represent means ± SEM (n = 10). Statistical analysis was used by two-tailed analysis of variance (ANOVA) with Bonferroni post test. PFU, plaque-forming units. (E) Pathological lesions in the lungs of mice infected with the indicated virus at 3 dpi with hematoxylin and eosin (H&E) staining. Scale bars, 100 μm. (F) Immunofluorescent staining of lung sections of mice infected with the indicated virus at 3 dpi. The viral NP antigen was stained green, and the nucleus was stained blue. Scale bars, 50 μm. (G) Virus titers in the lungs of infected mice (n = 3) at 3 dpi (left) and 5 dpi (right). Error bars represent means ± SD. Statistical analysis was performed by using one-tailed method. EID50, 50% egg infectious dose. (H) Model for avian IAV overcomes the huTRA2A host barrier. *P < 0.05, **P < 0.01, and ***P < 0.001.

Supplementary Materials

  • Supplementary Materials

    Human TRA2A determines influenza A virus host adaptation by regulating viral mRNA splicing

    Yinxing Zhu, Ruifang Wang, Luyao Yu, Huimin Sun, Shan Tian, Peng Li, Meilin Jin, Huanchun Chen, Wenjun Ma, Hongbo Zhou

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