Journal: Viruses

Article Title: Beet Necrotic Yellow Vein Virus Noncoding RNA Production Depends on a 5′→3′ Xrn Exoribonuclease Activity

doi: 10.3390/v10030137

Figure Lengend Snippet: A 55 nt fragment containing the coremin motif is sufficient to stall XRN1. ( A ) 5′ monophosphorylated radiolabeled RNA substrates containing either control sequences derived from pGEM-4 or a 101-nt RNA containing a 55 base fragment from the RNA3 segment of BNYVV (nts 1222–1277) at the 3′ end and 53 nts of pGEM-4 polylinker sequence at its 5′ end to serve as a landing site for 5′-to-3′ exonucleases) were incubated with either purified recombinant XRN1 from Kluyveromyces lactis (rXrn1 panel) or cytoplasmic extract from C6/36 Aedes albopictus cells for the times indicated. Reaction products were resolved on a 5% acrylamide gel containing urea and viewed by phosphorimaging. ( B ) Top: sequence of the 55 nts BNYVV RNA fragment. The black arrow indicates the site of XRN1 stalling. Fragments B-1, B-2, and B-3 containing BNYVV-specific sequences ranging from position 1222 to the base indicated in the figure. Bottom: 5′ monophosphorylated radiolabeled RNA substrates containing either control sequences derived from pGEM-4 or the B-1, B-2, or B-3 fragments of the RNA3 segment (nts 1222–1277) as indicated in the top part of the panel (inserted into the pGEM-4 polylinker as indicated in panel A) were incubated with purified recombinant XRN1 for the times indicated. Reaction products were resolved on a 5% acrylamide gel containing urea and viewed by phosphorimaging. Arrows indicate the positions of the RNA species stalling XRN1.

Article Snippet: As seen in A, while the presence of a 20X excess of a non-specific control RNA in the reaction had no effect on Xrn1 activity on a radiolabeled reporter RNA, the accumulation of ncRNA species due to Xrn1 stalling on either the BNYVV 55 nt RNA or the DENV 3′ UTR transcript correlated with an inhibition of the substrate degradation.

Techniques: Derivative Assay, Sequencing, Incubation, Purification, Recombinant, Acrylamide Gel Assay

Journal: Viruses

Article Title: Beet Necrotic Yellow Vein Virus Noncoding RNA Production Depends on a 5′→3′ Xrn Exoribonuclease Activity

doi: 10.3390/v10030137

Figure Lengend Snippet: Both ncRNA3sf and ncRNA3pk1 stall Xrn1 processing in vitro. ( A ) Schematic representation of the T7-driven cDNA clones constructs used to produce run-off in vitro transcripts depicted in waved lines. Double and single arrowheads correspond to the sf and pk1 structural motifs, respectively. The position of the restriction sites used and the size of the transcripts are indicated; ( B ) 5′ phosphorylated chimeric RNA3sf or RNA3pk1 and ( C ) BN3sf or BN3pk1 species were mixed with commercial Xrn1 enzyme for 6 h. Aliquots were sampled at the time indicated and RNAs species were detected by northern blot using a specific DNA probe able to reveal both full-length and ncRNA3 species.

Article Snippet: As seen in A, while the presence of a 20X excess of a non-specific control RNA in the reaction had no effect on Xrn1 activity on a radiolabeled reporter RNA, the accumulation of ncRNA species due to Xrn1 stalling on either the BNYVV 55 nt RNA or the DENV 3′ UTR transcript correlated with an inhibition of the substrate degradation.

Techniques: In Vitro, Clone Assay, Construct, Northern Blot

Journal: Viruses

Article Title: Beet Necrotic Yellow Vein Virus Noncoding RNA Production Depends on a 5′→3′ Xrn Exoribonuclease Activity

doi: 10.3390/v10030137

Figure Lengend Snippet: Exoribonucleases are responsible for noncoding RNA3 species accumulation in Saccharomyces cerevisiae . ( A ) Representation of the expression vector cassettes (solid lines) used to produce RNA3 and RNA3E species under the control of constitutive G6PDH promoter (black arrowhead). Capped (•) full-length RNAs and ncRNA species are depicted by waved grey and black lines, respectively. Wild-type (wt) ‘core’ sequence is presented in dark and mutated “core” in grey bold lines. The “coremin” motif and its antisense orientation, “nimeroc”, are depicted by black sense and antisense arrows, respectively. Drawings are not to scale and for a better representation of RNA3 species, refer to figure 2 of reference [ 27 ]. RNA sizes are presented on the right; ( B , C ) Northern blot analyses of RNA3 and ncRNA species produced in yeasts; ( B ) vectors producing wt RNA3, mutated RNA3E, or control vector (Ø) were introduced in S. cerevisiae (lanes 1–3) or Xrn1 defective strain (Δ xrn1 ) (lanes 4–5). Expression of RNA3 (lanes 6–10) was performed in ∆ xrn1 strain together with an empty vector (EV, lane 6) or vectors expressing: yeast Xrn1 (lane 7), plant ATXRN4 (XRN4, lanes 8 and 9), or a defective Xrn1 enzyme (Xrn1cat, lane 10). Total RNAs were subjected to northern blot analysis using a beet necrotic yellow vein virus (BNYVV)-specific 3′ probe complementary to nt 1277–1774; ( C ) total RNAs from yeast expressing RNA3 were differentially visualized using two probes (lanes 1 and 2). RNA3, RNA3*, ncRNA3, and ncRNA3* were revealed with a coremin-specific probe (lane 1) while RNA3* and ncRNA3* appeared only when the CYC1 terminator-specific probe was used (lane 2). Black triangles indicate the two full-length RNA3 species. Loadings were visualized by ethidium-bromide staining (rRNA).

Article Snippet: As seen in A, while the presence of a 20X excess of a non-specific control RNA in the reaction had no effect on Xrn1 activity on a radiolabeled reporter RNA, the accumulation of ncRNA species due to Xrn1 stalling on either the BNYVV 55 nt RNA or the DENV 3′ UTR transcript correlated with an inhibition of the substrate degradation.

Techniques: Expressing, Plasmid Preparation, Sequencing, Northern Blot, Produced, Staining

Journal: Viruses

Article Title: Beet Necrotic Yellow Vein Virus Noncoding RNA Production Depends on a 5′→3′ Xrn Exoribonuclease Activity

doi: 10.3390/v10030137

Figure Lengend Snippet: A 55 nt core fragment of RNA3 of BNYVV represses Xrn1. ( A ) A radiolabeled RNA containing a 5′ monophosphate (Reporter) was incubated with purified recombinant XRN1 for the times indicated. A 20X molar excess of lightly radiolabeled, 5′ monophosphorylated non-specific competitor RNA (‘Control RNA’ lanes), a competitor transcript containing the 55 nt core BNYVV RNA3 fragment (‘BNYVV-55mer’ lanes), or a competitor transcript containing the 3′ UTR of Dengue virus type 2 (‘DENV 3′ UTR’ lanes) was added to reactions. After the times indicated, reaction products were analyzed on 5% polyacrylamide gels containing urea and visualized by phosphorimaging. ( B ) Graphical presentation of the effect of the various competitor RNAs on Xrn1 activity on the Reporter transcript. Results shown are from three independent experiments. The asterisk represents a p value of

Article Snippet: As seen in A, while the presence of a 20X excess of a non-specific control RNA in the reaction had no effect on Xrn1 activity on a radiolabeled reporter RNA, the accumulation of ncRNA species due to Xrn1 stalling on either the BNYVV 55 nt RNA or the DENV 3′ UTR transcript correlated with an inhibition of the substrate degradation.

Techniques: Incubation, Purification, Recombinant, Activity Assay

Journal: Viruses

Article Title: Beet Necrotic Yellow Vein Virus Noncoding RNA Production Depends on a 5′→3′ Xrn Exoribonuclease Activity

doi: 10.3390/v10030137

Figure Lengend Snippet: Characterization of the minimal RNA3 3′ domain required for the efficient stalling of the Xrn1 enzyme. ( A ) Representation of the fragments obtained using T7 promoter containing primer (T7-RNA3F) and reverse primers (positions specified by blue boxes) cloned into pUC57 that served as templates for the production of 1R to 9R transcripts, ranging from 989 to 558 nt, possessing the same 5′ extremity with decreasing 3′ end length. The arrowhead locates the 5′ position of the ncRNA3 species (nt 1234). The expected product size after RppH and Xrn1 treatments are specified. Construct 6R was not obtained; ( B ) after 6 h of RppH/Xrn1 treatment, RNAs were analyzed by a 24-cm long run on 1.5% denaturing-agarose gel followed by northern blot using a radiolabeled DNA oligomer probe complementary to the “coremin” sequence. The ncRNA3 transcripts and 1R transcripts were treated with RppH alone or with RppH and Xrn1. The lengths of some RNA species are specified on the left side.

Article Snippet: As seen in A, while the presence of a 20X excess of a non-specific control RNA in the reaction had no effect on Xrn1 activity on a radiolabeled reporter RNA, the accumulation of ncRNA species due to Xrn1 stalling on either the BNYVV 55 nt RNA or the DENV 3′ UTR transcript correlated with an inhibition of the substrate degradation.

Techniques: Clone Assay, Construct, Agarose Gel Electrophoresis, Northern Blot, Sequencing

Journal: Viruses

Article Title: Beet Necrotic Yellow Vein Virus Noncoding RNA Production Depends on a 5′→3′ Xrn Exoribonuclease Activity

doi: 10.3390/v10030137

Figure Lengend Snippet: Accumulation of noncoding RNA3sf from chimeric RNA3sf inhibits Xrn1 and AtXRN4 exoribonucleases in Saccharomyces cerevisiae . ( A ) The ‘core’ sequence was replaced by a wt (sf) or mutated (pk1) flavivirus sequence to produce RNA3sf and RNA3pk1, respectively. PK1 pseudoknot involving stem-loop II (SL-II) is shown; ( B ) northern blot analyses of RNA3 and ncRNA species produced in yeasts. Plasmids allowing the expression of RNA species indicated that RNA3, RNA3E, RNA3sf, RNA3pk1, or empty vector (Ø) were introduced in wt yeasts (lanes 1–5) or Xrn1-defective yeasts (Δ xrn1 , lanes 6–9) complemented with a vector allowing for the production of yeast Xrn1 (lanes 6, 8, and 9) or plant XRN4 (lane 7). The RNA3* and ncRNA3* are similar to RNA3 and ncRNA3, respectively, but possess a CYC1 terminator sequence followed by a polyA tail (see Figure 1 and text for details). Positions of the RNA species are indicated on the right. Total RNAs were subjected to northern blot analysis using a BNYVV-specific 3′ probe complementary to nt 1277–1774. The partial complementarity of the probe with the ncRNA3sf species does not allow for quantitative comparisons. Loading was visualized by ethidium-bromide staining (rRNA). No sample was loaded between lanes 8 and 9.

Article Snippet: As seen in A, while the presence of a 20X excess of a non-specific control RNA in the reaction had no effect on Xrn1 activity on a radiolabeled reporter RNA, the accumulation of ncRNA species due to Xrn1 stalling on either the BNYVV 55 nt RNA or the DENV 3′ UTR transcript correlated with an inhibition of the substrate degradation.

Techniques: Sequencing, Northern Blot, Produced, Expressing, Plasmid Preparation, Staining

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Positive epistasis between viral polymerase and the 3′ untranslated region of its genome reveals the epidemiologic fitness of dengue virus

doi: 10.1073/pnas.1919287117

Figure Lengend Snippet: PR2B sfRNA is packaged inside E protein-containing infectious particles and transmitted into new susceptible cells. ( A ) Quantification of sfRNA:gRNA ratios in supernatant of A549 cells at 24 hpi by qPCR. ( B ) Quantification of gRNA:sfRNA ratios in supernatant of A549 cells on knockdown of CD9, CD63, CD81, and CD151. NTC served as a reference point for assessment of significance. ( C – E ) Quantification of gRNA copy numbers ( C ), sfRNA copy numbers ( D ), and sfRNA:gRNA ratios ( E ) from 4G2 pull-down precipitate. LOD, limit of detection; ND, no detection. PR1 served as a reference point for assessment of significance. ( F ) Western blots of CD63 and 4G2 in pull-down precipitate. ( G – I ) Quantification of gRNA ( G ), sfRNA ( H ), and sfRNA:gRNA ratios ( I ) in A549 cells at 2 hpi, after XRN1 knockdown. ( J ) Western blots of phosphorylated IRF3, IRF3, and LAMP-1 at 6 hpi with MOIs of 1 and 10 of mock, PR1, and PR1NS53UTR and cotreatment of NITD008. ( K ) Level of infection with MOIs of 1 and 10 of mock, PR1, and PR1NS53UTR quantified by immunofluorescence assay. Data are presented as mean ± SD. * P

Article Snippet: The denaturing condition was applied for XRN1 (Bethyl; 1:2,500), CD63 (Abcam; 1:5,000), phosphorylated IRF3 (Cell Signaling Technology; 1:1,000), IRF3 (Cell Signaling Technology; 1:1,000), β-actin (1:5,000), and LAMP-1 (1:5,000), and the nondenaturing condition was applied for CD9 (Abcam; 1:500), CD81 (Abcam; 1:250), and CD151 (Thermo Fisher Scientific; 1:1,000).

Techniques: Real-time Polymerase Chain Reaction, Western Blot, Infection, Immunofluorescence