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The folding of the hepatitis C virus internal ribosome entry site depends on the 3'-end of the viral genome.

Romero-López C, Barroso-Deljesus A, García-Sacristán A, Briones C, Berzal-Herranz A - Nucleic Acids Res. (2012)

Bottom Line: Importantly, many of the observed changes involved significant decreases in the dimethyl sulfate or N-methyl-isatoic anhydride reactivity profiles at subdomains IIIb and IIId, while domain IV appeared as a more flexible element.These observations were additionally confirmed in a replication-competent RNA molecule.Our results suggest that a complex, direct and long-distance RNA-RNA interaction network plays an important role in the regulation of HCV translation and replication, as well as in the switching between different steps of the viral cycle.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Biología Molecular, Instituto de Parasitología y Biomedicina López-Neyra, IPBLN-CSIC, Parque Tecnológico de Ciencias de la Salud, Avda. del Conocimiento s/n, Armilla, 18100 Granada, Spain. cristina_romero@ipb.csic.es

ABSTRACT
Hepatitis C virus (HCV) translation initiation is directed by an internal ribosome entry site (IRES) and regulated by distant regions at the 3'-end of the viral genome. Through a combination of improved RNA chemical probing methods, SHAPE structural analysis and screening of RNA accessibility using antisense oligonucleotide microarrays, here, we show that HCV IRES folding is fine-tuned by the genomic 3'-end. The essential IRES subdomains IIIb and IIId, and domain IV, adopted a different conformation in the presence of the cis-acting replication element and/or the 3'-untranslatable region compared to that taken up in their absence. Importantly, many of the observed changes involved significant decreases in the dimethyl sulfate or N-methyl-isatoic anhydride reactivity profiles at subdomains IIIb and IIId, while domain IV appeared as a more flexible element. These observations were additionally confirmed in a replication-competent RNA molecule. Significantly, protein factors are not required for these conformational differences to be made manifest. Our results suggest that a complex, direct and long-distance RNA-RNA interaction network plays an important role in the regulation of HCV translation and replication, as well as in the switching between different steps of the viral cycle.

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The SHAPE pattern of the HCV IRES is influenced by the 3′-end of the viral genome. (A) Mean NMIA reactivity values for IRES domains III and IV of the transcripts I, IU, IC and ICU calculated from, at least, five independent experiments and represented in a line graph according to color code indicated in Figure 3A. Nucleotides with mean reactivity values <0.3 were considered non-reactive. The gray box indicates noisy positions. Lower panel: detailed views of the SHAPE profile for subdomains IIIb (nucleotides 180–219) and IIId (nucleotides 253–280) and domain IV (nucleotides 331–360). (B) Graphs show significant variations (P ≤ 0.05) in the SHAPE pattern detected at subdomains IIIb, IIId and IV for molecules IU, IC and ICU with respect to RNA I at positions with reactivity values of ≥0.3. Reactivity changes are qualitatively indicated by (+) (increase) or (−) (decrease). Nucleotide numbering is indicated according to Figure 1A. Color code is indicated as in Figure 3.
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gks927-F4: The SHAPE pattern of the HCV IRES is influenced by the 3′-end of the viral genome. (A) Mean NMIA reactivity values for IRES domains III and IV of the transcripts I, IU, IC and ICU calculated from, at least, five independent experiments and represented in a line graph according to color code indicated in Figure 3A. Nucleotides with mean reactivity values <0.3 were considered non-reactive. The gray box indicates noisy positions. Lower panel: detailed views of the SHAPE profile for subdomains IIIb (nucleotides 180–219) and IIId (nucleotides 253–280) and domain IV (nucleotides 331–360). (B) Graphs show significant variations (P ≤ 0.05) in the SHAPE pattern detected at subdomains IIIb, IIId and IV for molecules IU, IC and ICU with respect to RNA I at positions with reactivity values of ≥0.3. Reactivity changes are qualitatively indicated by (+) (increase) or (−) (decrease). Nucleotide numbering is indicated according to Figure 1A. Color code is indicated as in Figure 3.

Mentions: Antisense oligonucleotide microarray analysis and DMS probing can provide accurate information on the secondary and, eventually, higher-order structure of a target RNA molecule. However, in many cases, the folding features of a target molecule do not necessarily depend on base-pair interactions, but on the conformation of the ribose-phosphate backbone. This may ultimately define the flexibility and overall geometry of an RNA molecule. Selective 2′-hydroxyl acylation and primer extension (SHAPE) (53,54) analysis was therefore performed on transcripts I, IU, IC and ICU using NMIA as the acylating reagent. The formation of 2′-O-adducts was monitored by primer extension with two sets of fluorescently labeled DNA oligonucleotides and analyzed using ShapeFinder software as described earlier (see also ‘Materials and Methods’ section). The mean relative reactivity and SD (for at least five independent experiments) was calculated for each nucleotide position. This allowed the construction of the corresponding SHAPE profile of the studied region (Figure 4A and Supplementary Figure S4A).Figure 4.


The folding of the hepatitis C virus internal ribosome entry site depends on the 3'-end of the viral genome.

Romero-López C, Barroso-Deljesus A, García-Sacristán A, Briones C, Berzal-Herranz A - Nucleic Acids Res. (2012)

The SHAPE pattern of the HCV IRES is influenced by the 3′-end of the viral genome. (A) Mean NMIA reactivity values for IRES domains III and IV of the transcripts I, IU, IC and ICU calculated from, at least, five independent experiments and represented in a line graph according to color code indicated in Figure 3A. Nucleotides with mean reactivity values <0.3 were considered non-reactive. The gray box indicates noisy positions. Lower panel: detailed views of the SHAPE profile for subdomains IIIb (nucleotides 180–219) and IIId (nucleotides 253–280) and domain IV (nucleotides 331–360). (B) Graphs show significant variations (P ≤ 0.05) in the SHAPE pattern detected at subdomains IIIb, IIId and IV for molecules IU, IC and ICU with respect to RNA I at positions with reactivity values of ≥0.3. Reactivity changes are qualitatively indicated by (+) (increase) or (−) (decrease). Nucleotide numbering is indicated according to Figure 1A. Color code is indicated as in Figure 3.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3526292&req=5

gks927-F4: The SHAPE pattern of the HCV IRES is influenced by the 3′-end of the viral genome. (A) Mean NMIA reactivity values for IRES domains III and IV of the transcripts I, IU, IC and ICU calculated from, at least, five independent experiments and represented in a line graph according to color code indicated in Figure 3A. Nucleotides with mean reactivity values <0.3 were considered non-reactive. The gray box indicates noisy positions. Lower panel: detailed views of the SHAPE profile for subdomains IIIb (nucleotides 180–219) and IIId (nucleotides 253–280) and domain IV (nucleotides 331–360). (B) Graphs show significant variations (P ≤ 0.05) in the SHAPE pattern detected at subdomains IIIb, IIId and IV for molecules IU, IC and ICU with respect to RNA I at positions with reactivity values of ≥0.3. Reactivity changes are qualitatively indicated by (+) (increase) or (−) (decrease). Nucleotide numbering is indicated according to Figure 1A. Color code is indicated as in Figure 3.
Mentions: Antisense oligonucleotide microarray analysis and DMS probing can provide accurate information on the secondary and, eventually, higher-order structure of a target RNA molecule. However, in many cases, the folding features of a target molecule do not necessarily depend on base-pair interactions, but on the conformation of the ribose-phosphate backbone. This may ultimately define the flexibility and overall geometry of an RNA molecule. Selective 2′-hydroxyl acylation and primer extension (SHAPE) (53,54) analysis was therefore performed on transcripts I, IU, IC and ICU using NMIA as the acylating reagent. The formation of 2′-O-adducts was monitored by primer extension with two sets of fluorescently labeled DNA oligonucleotides and analyzed using ShapeFinder software as described earlier (see also ‘Materials and Methods’ section). The mean relative reactivity and SD (for at least five independent experiments) was calculated for each nucleotide position. This allowed the construction of the corresponding SHAPE profile of the studied region (Figure 4A and Supplementary Figure S4A).Figure 4.

Bottom Line: Importantly, many of the observed changes involved significant decreases in the dimethyl sulfate or N-methyl-isatoic anhydride reactivity profiles at subdomains IIIb and IIId, while domain IV appeared as a more flexible element.These observations were additionally confirmed in a replication-competent RNA molecule.Our results suggest that a complex, direct and long-distance RNA-RNA interaction network plays an important role in the regulation of HCV translation and replication, as well as in the switching between different steps of the viral cycle.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Biología Molecular, Instituto de Parasitología y Biomedicina López-Neyra, IPBLN-CSIC, Parque Tecnológico de Ciencias de la Salud, Avda. del Conocimiento s/n, Armilla, 18100 Granada, Spain. cristina_romero@ipb.csic.es

ABSTRACT
Hepatitis C virus (HCV) translation initiation is directed by an internal ribosome entry site (IRES) and regulated by distant regions at the 3'-end of the viral genome. Through a combination of improved RNA chemical probing methods, SHAPE structural analysis and screening of RNA accessibility using antisense oligonucleotide microarrays, here, we show that HCV IRES folding is fine-tuned by the genomic 3'-end. The essential IRES subdomains IIIb and IIId, and domain IV, adopted a different conformation in the presence of the cis-acting replication element and/or the 3'-untranslatable region compared to that taken up in their absence. Importantly, many of the observed changes involved significant decreases in the dimethyl sulfate or N-methyl-isatoic anhydride reactivity profiles at subdomains IIIb and IIId, while domain IV appeared as a more flexible element. These observations were additionally confirmed in a replication-competent RNA molecule. Significantly, protein factors are not required for these conformational differences to be made manifest. Our results suggest that a complex, direct and long-distance RNA-RNA interaction network plays an important role in the regulation of HCV translation and replication, as well as in the switching between different steps of the viral cycle.

Show MeSH
Related in: MedlinePlus