The predicted stem-loop structure in the 3′-end of the human norovirus antigenomic sequence is required for its genomic RNA synthesis by its RdRp

Figures

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  Figure 1. Schematic illustration of a nonradioactive labeling method to detect RNA synthesis by RdRp in vitro and characterization of the purified RdRp.A, as shown by red or blue arrows, respectively, the region of the RNA (positive sense or antisense (complementary) sequences of the 5′- or 3′-end 100 nt of the human norovirus genomic RNA) used as templates in this study is shown. Template single-stranded RNA (ssRNA) and norovirus RNA-dependent RNA polymerase (RdRp) were mixed and incubated to synthesize complementary RNA. The newly synthesized ssRNAs formed double-stranded RNA (dsRNA) with an excess of ssRNA templates. The ssRNAs in the reaction mixture were digested with S1 ribonuclease to visualize dsRNA only. RNA molecules before and after S1 ribonuclease treatment were separated by nondenaturing polyacrylamide gel electrophoresis (native PAGE) and stained with SYBR Green II. B, 31.7 nmol (lane 1) or 2.3 pmol (lanes 2 and 3) of the purified RdRp was performed using SDS-PAGE. After SDS-PAGE, the gel was visualized using Coomassie dye staining (CDS; lane 1) or detected with Western blotting using anti-RdRp antiserum (lane 2) or using anti-His-tag antibody (lane 3). M, molecular weight…

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  Figure 2. The RdRp synthesizes the complementary RNA from 100AS RNA template.A, in vitro synthesized RNAs separated in native PAGE. The template ssRNAs [100S RNA (lane 1), 100AS RNA (lane 2)], and ds100 RNA (lane 3) were used as size markers. Five pmol of each template RNA, i.e., 100S (100 nM, lanes 4–7) or 100AS (100 nM, lanes 8–11), and the purified HuNV RdRp (0–32 μM) were mixed. Half of the total volume of samples was mixed with 6× gel-loading buffer and loaded on native PAGE (the upper column). S1 ribonuclease was added to the remaining half of the samples, and the samples were incubated and loaded on native PAGE (the lower column). The dsRNA products separated in native PAGE before and after S1 ribonuclease treatment are shown in the upper and lower columns, respectively. Red arrow indicates newly synthesized RNA products, which had formed dsRNAs with an excess of template RNA and identically migrated to dsRNA markers (lanes 4–11). B, the relative amounts of the newly synthesized dsRNA products (the transcriptional activity) were analyzed using ImageJ v.2.1.0/1.53c computer software and shown as the graph. The amounts shown were the averages of three experiments. The standard…

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  Figure 3. The predicted secondary structure of 100AS HuNV RNA.A, the 100AS RNA secondary structure was predicted using the computer software (23). The region of 31AS RNA (nt 70–100 of the U201 strain) is surrounded by a dotted line, and red characters indicate nt 74 to 87, which form the predicted stem-loop structure. B, alignments of 21 kinds of norovirus genotypes at the 3′-end regions of the antisense genome sequences (nt 100–45 of 100AS RNA of the GII. P3 U201 strain and its corresponding regions of other genotypes) are shown. The nucleotides with more than 71% identity (the same nucleotide in more than 15 of the total 21 genotypes) are indicated in bold. RdRp-coding genes are used to classify the genotypes (24). Horizontal blue lines separate genogroups. Red line on the top indicates highly conserved regions (nt 100–74 regions of the GII.P3 U201 strain). The 31AS RNA region is indicated by separating with a vertical black line between nt 69 and 70. Four vertical blue lines show the separation in the stem and loop regions in the stem-loop regions. The GII. P3 U201 strain that was used in this paper is shown in red characters.

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  Figure 4. The RNA synthesis with mutated template RNAs by the HuNV RdRp. Five pmol of each template RNA (final concentration, 100 nM) and 0.8 pmol HuNV RdRp (final concentration, 16 μM) were mixed. After incubation, half of the samples were loaded on native PAGE (the upper column). The other half of the samples were treated with S1 ribonucleases and loaded on native PAGE (the lower column). The results using templates, Δ31S and Δ31AS RNAs (A); add31AS-S and add31S-AS RNAs (B); Δ6AS, Δ9AS, and Δ12AS RNAs (C); ΔslS and ΔslAS RNAs (D); swS and swAS RNAs (E); and lpS and lpAS RNAs (F), were shown, respectively. The template ssRNA (lanes 1, 2, 8, and 9) and dsRNA (lanes 3 and 10) were used as size markers in A, B, and D–F. The template ssRNA (lanes 1, 5, 9, and 13) and dsRNA (lanes 2, 6, 10, and 14) were used as size markers in C. The positions of each dsRNA are indicated with red arrows in the lower columns. All results are representative of at least triplicate experiments. The schematic structures of the 100S, 100AS, and its mutated template RNAs are shown on the right side of each figure.

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  Figure 5. Interaction of the template 100AS RNA and its mutated RNAs with RdRp by EMSA.A, the 32P-labeled 5′-end-labeled RNAs (100AS; lanes 1–4, Δ31AS; lanes 5–8, or 31AS; lanes 9–12, 1 pmol each) and the RdRp (0–600 pmol) were incubated. B, the 32P-labeled 5′-end-labeled 100AS RNA (0.2 pmol), the indicated number of non-labeled competitor RNAs; 100AS (lanes 1–5), Δ31AS (lanes 6–10), or 31AS RNA (lanes 11–15) and the RdRp (600 pmol) were incubated. C, the 32P-labeled 5′-end-labeled RNAs (1 pmol); 100AS (lanes 1–4), add31AS-S (lanes 5–8), or Δ31AS (lanes 9–12) RNAs and the RdRp (0–600 pmol) were incubated. After incubation, EMSA was used to examine all the samples. The relative amounts of RNAs that were complexed with the RdRp (the shifted RNAs) were analyzed using ImageJ v. 2.1.0/1.53c computer software and shown as the graph. The amounts were the averages of three times experiments. The standard deviations are shown in the graph. Each result of the three independent experiments is also shown by the three kinds of shapes (circles, rectangles, and hexagons). Black arrows indicate the position of the bottom of wells. Red arrows indicate the RNA-RdRp complex. The schematic structures of…