41 research outputs found

    Diagram of the hybrid BCoV DI RNA and the head-to-tail strategy used to detect the synthesis of (−)-strand DI RNA.

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    <p>(A) Upper panel: the composition of naturally occurring BCoV DI RNA relative to the full-length BCoV genome. Lower panel: the modified BCoV DI RNA construct BM 25A used in this study. The 65-nt leader sequence is illustrated with a filled rectangle. (B) Strategy for determining the synthesis of (–)-strand DI RNA. RNA extracted at 8 hpt of DI RNA transcript was treated with TAP and head-to-tail ligated. MHV-A59 3′ UTR-negative-strand-specific primer 1 MHV3UTR3(–) was used for RT with the ligated RNA as template. MHV-A59 3′ UTR-negative-strand-specific primer 2 MHV3UTR6(–) and BCoV 5′ UTR-positive-strand specific primer 3 BCoV23-40(+) were used for PCR.</p

    Effect of leaderless DI RNA on translation.

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    <p>(A) DI RNA constructs with a His-tag used for replication and translation assay. Each DI RNA construct has an open reading frame (open box), followed by an in-frame 18-nt His-tag coding region (stippled box) and MHV 3′ UTR. (B) Replication of DI RNA by Northern blot assay. RNA samples collected at 48 hpi of VP1 were used to determine the replication of the DI RNA. (C) and (F) Protein expression from the DI RNA constructs. BCoV-infected HRT-18 cells were transfected with the indicated DI RNA construct at 2 hpi, and total intracellular proteins or RNA was extracted at 4, 8, and 21 hpt for analysis. Western blotting was used to measure the abundance of His-tagged protein and β-actin. The levels of DI RNA, N sgmRNA, and 18S rRNA were measured by Northern blotting. (D) and (G) Quantitation analysis of the His-tagged protein from individual DI RNA constructs at different time points. (E) and (H) RT-PCR to detect a potential recombinant between the BCoV genome and DI RNA. The primers MHV3′UTR2(+), which anneal to the MHV 3′ UTR and was used for RT, and M3(−), which anneal to the BCoV M protein gene, were used for PCR to detect potential recombination between the BCoV genome and BM65Ahis (Fig. 2E, lane 2), Δ69-BM65Ahis (Fig. 2E, lane 3), BM65AhisΔ5 (Fig. 2H, lane 2), or Δ69-BM65AhisΔ5 (Fig. 2H, lane 3). The recombinant DNA of 1,639 nt shown in lane 4 of Figs. 2E and 2H was generated by overlap RT-PCR and was used as a size marker for the product generated using the primers MHV 3′ UTR2(+) and M3(−). The values (D) and (G) represent the mean±SEM of three individual experiments. *p<0.05, **p<0.01.</p

    Effect of coronaviral leaderless DI RNA on transcription.

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    <p>(A) Constructs of DI RNA with the insertion of the intergenic sequence (IS) followed by the EGFP gene to test the effect of leaderless DI RNA on coronavirus transcription. (B) RT-PCR products with a length of 120 nt were observed from DIEGFP- (lanes 5–9, arrowhead) or Δ69EGFP- (lanes 12–17, arrowhead) transfected BCoV-infected cells. 18S rRNA, DI RNA, and helper virus N sgmRNA were used as internal controls. RNA extracted at 0 hpt was from mock-transfected BCoV-infected HRT-18 cells. To detect a potential recombinant between the BCoV genome and input DI RNA, the primers EGFP1(+), which anneals to the EGFP sequence and was used for RT, and M3(−), which anneals to the BCoV M protein gene, were used for PCR to detect potential recombination between the BCoV genome and DIEGFP (lane19) or Δ69EGFP (lane 20). A recombinant DNA of 1,309 nt (lane 21) was produced by overlap RT-PCR and was used as a size marker for the product generated using the primers EGFP1(+) and M3(−). (C) Sequence of the cDNA-cloned 120-nt RT-PCR product from Fig. 4B, lane 7 (left panel) and lane 15 (right panel) showing the leader-body junction (indicated with vertical bar), the IS UCUAAAC (indicated with thin line), and the AUG translation start codon (indicated with thick line) for EGFP. (D) Quantitation analysis of the 120-nt RT-PCR products from the individual DI RNA constructs shown in Fig. 4B. The efficiency of sgmRNA synthesis was normalized to the levels of the internal controls including 18S rRNA, DI RNA, and helper virus N sgmRNA. (E) Fluorescence of EGFP expressed from DIEGFP- or Δ69EGFP-derived sgmRNA. Panels 1 and 3 are mock-infected cells transfected with DIEGFP and Δ69EGFP, respectively; panels 2 and 4 are BCoV-infected cells transfected with DIEGFP and Δ69EGFP, respectively. In all cases, the cells were examined for fluorescence at 24 hpt. **p<0.01, ***p<0.001.</p

    Alignment of the 3′-terminal 55-nt sequence within the individual subgroups of betacoronaviruses.

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    <p>The conserved sequence within individual subgroups is identified by shading. Position numbers −1 and −55 below the sequence indicate the first and 55th nt counted from the poly(A) tail, respectively. Abbreviations: CoV, coronavirus; BCoV, bovine coronavirus-Mebus; OC43, human coronavirus-OC43; HECoV-4408, human enteric coronavirus-4408; HEV, porcine hemagglutinating encephalomyelitis virus-TN11; ECoV, equine coronavirus-NC99; MHVA59, mouse hepatitis virus-A59; MHVJ, mouse hepatitis virus-JHM; PV, puffinosis virus; SARS-CoV, SARS coronavirus; B-S-HKU3, bat SARS coronavirus HKU3; B-S-HKU4, bat SARS coronavirus HKU4; B-S-HKU5, bat SARS coronavirus HKU5; BCHKU9-1, bat coronavirus HKU9-1; BCHKU9-2, bat coronavirus HKU9-2; BCHKU9-3, bat coronavirus HKU9-3; BCHKU9-4, bat coronavirus HKU9-4. GenBank Accession Nos. for the sequences studied here are as follows: U00735 for BCoV-Mebus, AF523847 for HCoV-OC43, AF523848 for HECoV-4408, AF523849 for HEV-TN11, AF523850 for ECoV-NC99, NC001846 for MHV-A59, X00990 for MHV-JHM, AJ544718 for PV, NC004718 for SARS-CoV, NC009694 for B-S-HKU3, NC009019 for B-S-HKU4, NC009020 for B-S-HKU5, EF065513.1 for BCHKU9-1, EF065514.1 for BCHKU9-2, EF065515.1 for BCHKU9-3, and EF065516.1 for BCHKU9-4.</p

    Effect of nucleotide species at the -1 position of 3′ terminal sequence in BCoV DI RNA on (−)- and (+)-strand RNA synthesis.

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    <p>(A) DI RNA constructs with nucleotide substitution (underlined) at the −1 position of 3′ terminal sequence. (B) The relative levels of (−)-strand DI RNA synthesis. BCoV-infected HRT-18 cells were transfected with the indicated DI RNA construct at 2 hpi, and the total cellular RNA was extracted at 8 hpt. The synthesis of the (−)-strand DI RNA from the substitution mutant was quantitated by qRT-PCR and compared with that from wt BM25A. Control A: total cellular RNA from mock-infected cells. Control B: total cellular RNA from BCoV-infected cells. Control C: total cellular RNA from DI RNA-transfected mock-infected cells. Control D: a mixture of BCoV-infected cellular RNA extracted at 10 hpi and 200 ng of BM25A transcript. (C) Upper panel: the synthesis of (+)-strand DI RNA as detected by Northern blot assay with N sgmRNA and 18S rRNA as internal controls. Middle panel: the relative levels of (+)-strand DI RNA synthesis. Lower panel: the sequence of the BCoV DI RNA at 48 hpi of VP1 as determined by RT-PCR and sequencing analysis. The values (B) and (C) represent the mean ±SD of three individual experiments. SD: standard deviation, wt: wild type, mut: mutant. *p<0.05, ***p<0.001.</p

    Identification of leaderless genomic RNA during BCoV persistent infection.

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    <p>(A) Strategy to identify positive-strand leaderless genomic RNA. Poly(A)-containing RNA was selected from total cellular RNA extracted from BCoV-persistently infected cells, treated with alkaline phosphatase, decapped with tobacco acid pyrophosphatase, head-to-tail ligated with T4 RNA ligase I, and used as the template for RT-PCR with the BCoV 5′ UTR-(+)-strand-specific primer 2: BCV107(+) (for RT) and BCoV 3′ UTR-(−)-strand-specific primer 1: BCV3′UTR1(−). (B) RT-PCR product synthesized by the method described in Fig. 1A. RT-PCR products with a size of more than 200 bp (lanes 2–7, marked with black arrowhead) and with a size of less than 200 bp (lanes 6–7, marked with white arrowhead) were observed. (C) The upper panel shows part of the first 88-nt sequence of the 5′ UTR in the positive-strand BCoV genomic RNA. The positions (1 and 70) are given on the top of the sequence, and the intergenic sequence (IS) UCUAAAC is underlined. The lower panel shows the sequence (shown in the negative strand) of the cDNA-cloned RT-PCR product with a size of more than 200 bp from lane 7, as indicated with a black arrowhead in Fig. 1B. (D) The upper panel shows the sequence of the 5′UTR on the positive-strand BCoV genomic RNA, which lacks the first 69 nts; position 70 is given on the top of the sequence. The lower panel shows the sequence (shown in the negative strand) of the cDNA-cloned RT-PCR product with a size of less than 200 bp from lane 7, as indicated with a white arrowhead in Fig. 1B. (E) Control reactions to determine if the positive-strand leaderless genome is a degradation product. RT-PCR product was synthesized by the method described in Fig. 1A except RNA sample was not treated with alkaline phosphatase and tobacco acid pyrophosphatase. RT-PCR products with a length of more than 200 bp were detected. (F) Identification of negative-strand leaderless genomic RNA. Total cellular RNA was treated with tobacco acid pyrophosphatase and ligated with T4 RNA ligase I. RT-PCR product was synthesized by the method described in Fig. 1A except that primer BCV3′UTR1(−) was used for RT. RT-PCR products with a size of more than 200 bp (lanes 2–7, marked with black arrowhead) were observed. M, ds DNA size markers in nt pairs. dpi: days postinfection.</p

    Determination of <i>cis</i>-acting RNA elements between the nts −4 and −40 required for (−)- and (+)-strand RNA synthesis.

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    <p>(A) Deletion mutants of BCoV DI RNA. Dashes denote deleted sequences. (B) The relative levels of (−)-strand DI RNA synthesis. The synthesis of (−)-strand DI RNA from the deletion mutant was quantitated by qRT-PCR and was compared with that from wt BM25. Control A: total cellular RNA from mock-infected cells. Control B: total cellular RNA from BCoV-infected cells. Control C: total cellular RNA from DI RNA-transfected mock-infected cells. Control D: a mixture of BCoV-infected cellular RNA extracted at 10 hpi and 200 ng of BM25A transcript. (C) Upper panel: the synthesis of (+)-strand DI RNA as detected by Northern blot assay. Total cellular RNA was extracted at 48 hpi of VP1 and was subjected to Northern blot assay with N sgmRNA and 18S rRNA as internal controls. Middle panel: the relative levels of the (+)-strand DI RNA synthesis. Lower panel: the sequence of the BCoV DI RNA at 48 hpi of VP1 as determined by RT-PCR and sequencing analysis. The values (B) and (C) represent the mean ±SD of three individual experiments. SD: standard deviation, wt: wild type. **p<0.01.</p

    Identification of <i>cis</i>-acting RNA elements within the 3′-terminal 55 nts that are required for (−)- and (+)-strand RNA synthesis.

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    <p>(A) Constructs of deletion mutants within the 3′-terminal 55 nucleotides of BCoV DI RNA. Dashes denote deleted sequences. (B) The relative levels of (–)-strand DI RNA synthesis. Total cellular RNA was extracted from DI RNA-transfected BCoV-infected cells at 8 hpt. The synthesis of (–)-strand DI RNA from the deletion mutant was quantitated by qRT-PCR and was compared with that from wt BM25. Control A: total cellular RNA from mock-infected cells. Control B: total cellular RNA from BCoV-infected cells. Control C: total cellular RNA from DI RNA-transfected mock-infected cells. Control D: a mixture of BCoV-infected cellular RNA extracted at 10 hpi and 200 ng of BM25A transcript. (C) Upper panel: the synthesis of (+)-strand DI RNA as detected by Northern blot assay. Total cellular RNA was extracted at 48 hpi of VP1 and was subjected to Northern blot assay with N sgmRNA and 18S rRNA as internal controls. Middle panel: the relative levels of the (+)-strand DI RNA synthesis. Lower panel: the sequence of the BCoV DI RNA at 48 hpi of VP1 as determined by RT-PCR and sequencing analysis. The values (B) and (C) represent the mean ±SD of three individual experiments. SD: standard deviation, wt: wild type, mx: mixed, NA: not available. *p<0.05, **p<0.01, ***p<0.001.</p

    Analysis of the requirement of 3′-terminal 55 nts for the synthesis of (−)-strand BCoV DI RNA.

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    <p>(A) Diagram of the BCoV DI RNA BM25A with the intact 3′-terminal 55 nts and the mutant construct Δ55 with the deletion of 3′-terminal 55 nts (denotes with dashes). (B) Detection of (–)-strand BCoV DI RNA with head-to-tail ligation and RT-PCR. RT-PCR products with a size of ∼150 bp were observed from BCoV-infected BM25A-transfected cells (lanes 2–8, arrowhead) but not from BCoV-infected Δ55-transfected cells (lanes 10-16). Lanes 18–21 represent the controls for RT-PCR. C1: total cellular RNA from mock-infected cells. C2: total cellular RNA from BCoV-infected cells. C3: total cellular RNA from DI RNA-transfected mock-infected cells. C4: a mixture of BCoV-infected cellular RNA extracted at 10 hpi and 200 ng of BM25A transcript. (C) Sequence of the cDNA-cloned RT-PCR product with a size of ∼150 bp from lane 5 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098422#pone-0098422-g003" target="_blank">Fig. 3B</a>. [shown in the (+)-strand]. (D) Detection of the potential recombination between the helper virus genome and DI RNA. The primers MHV3′ UTR2(+), which anneals to the MHV 3′ UTR and was used for RT, and M3(–), which anneals to BCoV M protein gene, were used for PCR to detect potential recombination between helper virus BCoV genome and BM25A (lane 2) or Δ55 (lane 3). A recombinant DNA of 1,639 nt shown in lane 4 was created by overlap RT-PCR and was used as a size marker for the product generated with the primers MHV 3′ UTR2(+) and M3(–). (E) Left panel: the relative levels of (–)-strand DI RNA synthesis as measured by qRT-PCR. Control A: total cellular RNA from mock-infected cells. Control B: total cellular RNA from BCoV-infected cells. Control C: total cellular RNA from DI RNA-transfected mock-infected cells. Control D: a mixture of BCoV-infected cellular RNA extracted at 10 hpi and 200 ng of BM25A transcript. Right panel: the amounts of DI RNA, helper virus N sgmRNA, and 18S rRNA from DI RNA-transfected BCoV-infected cells at 8 hpt of VP0 as measured by Northern blot assay. The values (E) represent the mean ±SD of three individual experiments. SD: standard deviation. ***p<0.001.</p
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