The Functional Role of the 3′ Untranslated Region and Poly(A) Tail of Duck Hepatitis A Virus Type 1 in Viral Replication and Regulation of IRES-Mediated Translation

Figures

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  FIGURE 1. Plasmids construction. (A) RNA-launched infectious clones absent/present poly(A) tail or 3′ UTR were established. Besides, the mutated RNA-launched infectious clones possessed various length of poly(A) tail were also constructed and named pR-DHAV-An (n = 0, 5, 10, 15, 20, 25, 30, and 40). (B) Construction of the DHAV-1 monocistronic reporter system. The plasmid pDHAV-3′UTR-A25 contains the following elements 5′ to 3′ in a pcDNA3.1/V5-His A vector: the CMV immediate early promoter, a T7 promoter, the entire DHAV-1 5′ UTR (nucleotides 1–626, strain LY0801), and the FLuc gene and the entire DHAV-1 3′ UTR followed by a poly(A) tail containing 25 adenines. pDHAV-Δ3′UTR-ΔA25, or pDHAV-3′UTR-ΔA25, or pDHAV-Δ3′UTR-A25 were obtained from pDHAV-3′UTR-A25 to remove 3′ UTR plus poly(A) tail, or poly(A) tail, or 3′ UTR, respectively. The mutated monocistronic reporter plasmids possessed various length of poly(A) tail was constructed and named as pDHAV-3′UTR-An (n = 0, 5, 10, 15, 20, 25, 30, and 40).

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  FIGURE 2. Effects of poly(A) tail length and 3′ UTR on RNA stability. DEFs transfected with 0.8 μg of the in vitro transcribed RNA were collected at 4 hpt and were immediately used for total RNA extraction. The samples were analyzed by 1.5% formaldehyde-agarose gel electrophoresis and autoradiography. Digoxigenin (DIG)-labeled RNA probes (based on 81–351 of LY0801) was used to in this assay. The gels show the amount of input RNA remaining at 4 hpt. (A) The remaining amount of pR-DHAV-1-An or pDHAV-1-An was measured at 4 hpt. (B) The grayscale value of represent the mean ± standard deviation of three replicate experiments for poly(A) n group. The grayscale value of parental group was set as 100%. (C) The remaining amount of complete/mutated RNA-launched infectious clone or reporter RNA was measured at 4 hpt. The upper panel shows the ethidium bromide staining of the agarose gel loaded with aliquots of 800 ng RNAs. (D) The average grayscale values of three replicate experiments for each group. The grayscale value of pR-DHAV-1 or pDHAV-3′UTR-A25 was set as 100%.

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  FIGURE 3. Growth characteristics of DHAV-1 with various length of poly(A) tail. 0.8 μg of in vitro transcribed product of pR-DHAV-3′UTR-An were transfected into DEFs, cell lysates were collected from 12 to 60 hpt and were immediately used for total RNA extraction. Viral growth characteristics were measured by RT-qPCR as previously described. Bars represent the mean ± S.D of three replicate experiments for each group. 0.1 μg of pGMLR-TK plasmid was co-transfected with RNA samples, and the RLuc activity was measured at each detection point. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ns represent P > 0.05.

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  FIGURE 4. The 3′ UTR is essential for viral genome replication. (A) 0.8 μg of the in vitro transcribed products of pR-DHAV-1 or pR-DHAV-Δ3′UTR-A25 were transfected into DEFs, cell lysates were collected from 12 to 60 hpt and viral copy number was measured by RT-qPCR. (B) Equal amount of in vitro transcribed product of pR-DHAV-1 or pR-DHAV-R3′UTR-A25 were transfected into DEFs, cell lysates were collected at 4 hpt to measure the remaining amount of viral RNAs. (C) Equal amount of 0.8 μg of the in vitro transcribed products of pR-DHAV-1 or pR-DHAV-R3′UTR-A25 were transfected into DEFs to measure viral growth characteristics as previously described. Bars represent the means ± standard deviations of three replicate experiments.

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  FIGURE 5. The effect of the DHAV-1 3′ UTR plus the poly(A)25 tail on IRES-mediated translation efficiency. 0.8 μg of of the in vitro transcribed products of the linearized plasmids pDHAV-3′UTR-A25 and pDHAV-Δ3′UTR-ΔA25 were co-transfected into DEFs with 0.1 μg of pGMLR-TK plasmid, and the FLuc/RLuc activities were measured from 4 to 12 hpt. Columns and bars represent the means and standard deviations, respectively, of three independent transfections. ∗P < 0.05; ∗∗∗P < 0.001.

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  FIGURE 6. The 3′ UTR and poly(A) tail function as individual elements to stimulate IRES-mediated translation. (A) The in vitro transcribed products of plasmid pDHAV-3′UTR-ΔA25 or pDHAV-Δ3′UTR-A25 were transfected into DEFs, and reporter RNAs of pDHAV-3′UTR-A25 or pDHAV-Δ3′UTR-ΔA25 were used as positive or negative controls, respectively. The stimulation values between 3′ UTR-containing RNAs and poly(A)-containing RNAs were compared. 0.1 μg of pGMLR-TK plasmid was co-transfected with reporter RNAs, and the RLuc activity was measured at each detection point. Bars represent the means ± standard deviations of three replicate experiments. ∗∗∗P < 0.001, ∗∗P < 0.01. (B) After transfection of pR-DHAV-1-3′UTR-ΔA25 or pR-DHAV-1-Δ3′UTR-A25 into DEFs, cell lysates were collected at 4 hpt and used for western blot analysis, with pR-DHAV-1 and pR-DHAV-1-Δ3′UTR-ΔA25 used as controls. The anti-DHAV-1 monoclonal antibody 4F8 (dilution: 1:500) and HRP-labeled goat anti-mouse antibody (dilution: 1:3000) were used for western blot analysis.

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  FIGURE 7. The effects of poly(A) tail length on IRES-mediated translation efficiency. (A) In vitro transcribed RNAs (0.8 μg) derived from linearized monocistronic reporter plasmids pDHAV-1-An containing various lengths of the poly(A) tail (n: 0, 5, 10, 15, 20, 25, 30, and 40) were co-transfected into DEFs with 0.1 μg of pGMLR-TK plasmid. Cell lysates were collected at 4, 6, 12, and 24 hpt to measure FLuc/RLuc activity. Columns and bars represent means and standard deviations, respectively, of three independent transfections. ∗∗∗P < 0.001, ∗∗P < 0.01. (B) The RNA-launched infectious clone pR-DHAV-1-An was transfected into DEFs, and the cell lysates were collected at 4 hpt for western blot analysis with anti-DHAV-1 monoclonal antibody 4F8 and HRP-labeled goat anti-mouse antibody. The grayscale value in poly(A)25 group was set as 100%.

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  FIGURE 8. Effects of 3′ UTR plus poly(A) tail on IRES-mediated translation efficiency in various cell lines. Equal amount (∼2 × 105) of DEFs, or BHK-21, or HEK 293T were seeded into 24-well plate 12 h before transfection. 0.8 μg of the Fluc activity of the pDHAV-Δ3′UTR-ΔA25 or pDHAV-3′UTR-A25 were co-transfected into DEFs, BHK-21 cells, and HEK 293T cells with 0.1 μg of pGMLR-TK plasmid to measure the FLuc/RLuc activity at 4 hpt. Data represent three replicate experiments for each group. ∗∗∗P < 0.001.