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Targeted inhibition of the hepatitis C internal ribosomal entry site genomic RNA with oligonucleotide conjugates.

Guerniou V, Gillet R, Berrée F, Carboni B, Felden B - Nucleic Acids Res. (2007)

Bottom Line: All these molecules inhibit, in a dose-dependent manner, the 'IRES-dependent' translation in vitro.The 5'-coupled imidazole conjugate reduces viral protein synthesis by half at a 300 nM concentration (IC50), corresponding to a 4-fold increase of activity when compared to the naked oligonucleotide.These new conjugates are now being tested for activity on infected hepatic cell lines.

View Article: PubMed Central - PubMed

Affiliation: Biochimie Pharmaceutique, Inserm U835, Upres JE 2311, Université de Rennes 1, France.

ABSTRACT
Hepatitis C is a major public health concern, with an estimated 170 million people infected worldwide and an urgent need for new drug development. An attractive therapeutic approach is to prevent the 'cap-independent' translation initiation of the viral proteins by interfering with both the structure and function of the hepatitis C viral internal ribosomal entry site (HCV IRES). Towards this goal, we report the design, synthesis and purification of novel bi-functional molecules containing DNA or RNA antisenses attached to functional groups performing RNA hydrolysis. These 5' or 3'-coupled conjugates bind the HCV IRES with affinity and specificity and elicit targeted hydrolysis of the viral genomic RNA after short (1 h) incubation at low (500 nM) concentration at 37 degrees C in vitro. Additional secondary cleavage sites are induced and their mapping within the RNA structure indicates that functional domains IIIb-e are excised from the IRES that, based on cryo-EM studies, becomes incapable of binding the small ribosomal subunit and initiation factor 3 (eIF3). All these molecules inhibit, in a dose-dependent manner, the 'IRES-dependent' translation in vitro. The 5'-coupled imidazole conjugate reduces viral protein synthesis by half at a 300 nM concentration (IC50), corresponding to a 4-fold increase of activity when compared to the naked oligonucleotide. These new conjugates are now being tested for activity on infected hepatic cell lines.

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Location of the cleavages induced by the DNA conjugates onto the HCV IRES secondary and tertiary structures in complex with the 40S subunit and eIF3 derived from cryo-EM data (16). (a) Schematic secondary structure of domains II to IV from the HCV IRES with the location of the cleavage sites (arrow and numbered stars). The four cleavage sites are visualized in the context of the binary and the ternary complexes containing ‘HCV IRES-40S subunit’ (b) or ‘HCV IRES-40S subunit-eIF3’ (c), respectively. Note that the cleavages of the IRES triggered by the conjugates remove an RNA segment (IIIb-e) essential for the recognition by eIF3.
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Figure 6: Location of the cleavages induced by the DNA conjugates onto the HCV IRES secondary and tertiary structures in complex with the 40S subunit and eIF3 derived from cryo-EM data (16). (a) Schematic secondary structure of domains II to IV from the HCV IRES with the location of the cleavage sites (arrow and numbered stars). The four cleavage sites are visualized in the context of the binary and the ternary complexes containing ‘HCV IRES-40S subunit’ (b) or ‘HCV IRES-40S subunit-eIF3’ (c), respectively. Note that the cleavages of the IRES triggered by the conjugates remove an RNA segment (IIIb-e) essential for the recognition by eIF3.

Mentions: Interestingly, additional cleavages, away from the oligonucleotide recognition sequence and also distant one another onto the IRES secondary structure (Figure 1, stars), were consistently observed. These cleavages can be weakly observed with the naked antisense oligonucleotide AS. They are significantly increased with both the 5′- and the 3′-imidazole conjugates (Figure 4). These three sites were mapped by partial RNA sequencing between U297 and A298 in domain IIIe (1*) between G171 and A172 (2*) between domains IIIa and IIIb and between G181 and A182 (3*) in domain IIIb. These additional cuts remove the essential domains IIIb to IIIe from the IRES (Figure 1), most likely affecting the recognition of the IRES by eIF3 (Figure 6c) and possibly also its interaction with the 40S subunit (Figure 6b). These data are a reasonable explanation as to why a dose-dependent IRES-mediated translation inhibition is detected in vitro. A cryo-EM structural model of the IRES locked on a translation initiation state in complex with eIF3 and the 40S subunit (13) shows that the additional cleavage sites are far away when looking at the primary sequence but are clustered around the nucleotide recognition sequence on the IRES three-dimensional structure derived from cryo-EM (Figure 6c, blue stars). In the context of the naked IRES tertiary structure, those three cleavages may even be closer one another. Upon binding of the 5′ or the 3′ complementary oligonucleotide, the IRES structure may adopt alternate conformation(s) that, in turn, become(s) hydrolysed at additional secondary sites. This conformational change is specific since only three additional cleavages sites are observed. The respective contribution of the RNA hydrolysis versus the trigger of an inactive conformation of the IRES in the overall translation inhibition observed in vitro is unknown. Both the sequence and structure of domain III from the IRES are essential for the binding of eIF3 (3,16,41,42) and of ribosomal protein S9 (43). Interestingly, this is particularly true for domains IIIb and IIId (44), which are both cleaved by conjugate 1.Figure 6.


Targeted inhibition of the hepatitis C internal ribosomal entry site genomic RNA with oligonucleotide conjugates.

Guerniou V, Gillet R, Berrée F, Carboni B, Felden B - Nucleic Acids Res. (2007)

Location of the cleavages induced by the DNA conjugates onto the HCV IRES secondary and tertiary structures in complex with the 40S subunit and eIF3 derived from cryo-EM data (16). (a) Schematic secondary structure of domains II to IV from the HCV IRES with the location of the cleavage sites (arrow and numbered stars). The four cleavage sites are visualized in the context of the binary and the ternary complexes containing ‘HCV IRES-40S subunit’ (b) or ‘HCV IRES-40S subunit-eIF3’ (c), respectively. Note that the cleavages of the IRES triggered by the conjugates remove an RNA segment (IIIb-e) essential for the recognition by eIF3.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2175329&req=5

Figure 6: Location of the cleavages induced by the DNA conjugates onto the HCV IRES secondary and tertiary structures in complex with the 40S subunit and eIF3 derived from cryo-EM data (16). (a) Schematic secondary structure of domains II to IV from the HCV IRES with the location of the cleavage sites (arrow and numbered stars). The four cleavage sites are visualized in the context of the binary and the ternary complexes containing ‘HCV IRES-40S subunit’ (b) or ‘HCV IRES-40S subunit-eIF3’ (c), respectively. Note that the cleavages of the IRES triggered by the conjugates remove an RNA segment (IIIb-e) essential for the recognition by eIF3.
Mentions: Interestingly, additional cleavages, away from the oligonucleotide recognition sequence and also distant one another onto the IRES secondary structure (Figure 1, stars), were consistently observed. These cleavages can be weakly observed with the naked antisense oligonucleotide AS. They are significantly increased with both the 5′- and the 3′-imidazole conjugates (Figure 4). These three sites were mapped by partial RNA sequencing between U297 and A298 in domain IIIe (1*) between G171 and A172 (2*) between domains IIIa and IIIb and between G181 and A182 (3*) in domain IIIb. These additional cuts remove the essential domains IIIb to IIIe from the IRES (Figure 1), most likely affecting the recognition of the IRES by eIF3 (Figure 6c) and possibly also its interaction with the 40S subunit (Figure 6b). These data are a reasonable explanation as to why a dose-dependent IRES-mediated translation inhibition is detected in vitro. A cryo-EM structural model of the IRES locked on a translation initiation state in complex with eIF3 and the 40S subunit (13) shows that the additional cleavage sites are far away when looking at the primary sequence but are clustered around the nucleotide recognition sequence on the IRES three-dimensional structure derived from cryo-EM (Figure 6c, blue stars). In the context of the naked IRES tertiary structure, those three cleavages may even be closer one another. Upon binding of the 5′ or the 3′ complementary oligonucleotide, the IRES structure may adopt alternate conformation(s) that, in turn, become(s) hydrolysed at additional secondary sites. This conformational change is specific since only three additional cleavages sites are observed. The respective contribution of the RNA hydrolysis versus the trigger of an inactive conformation of the IRES in the overall translation inhibition observed in vitro is unknown. Both the sequence and structure of domain III from the IRES are essential for the binding of eIF3 (3,16,41,42) and of ribosomal protein S9 (43). Interestingly, this is particularly true for domains IIIb and IIId (44), which are both cleaved by conjugate 1.Figure 6.

Bottom Line: All these molecules inhibit, in a dose-dependent manner, the 'IRES-dependent' translation in vitro.The 5'-coupled imidazole conjugate reduces viral protein synthesis by half at a 300 nM concentration (IC50), corresponding to a 4-fold increase of activity when compared to the naked oligonucleotide.These new conjugates are now being tested for activity on infected hepatic cell lines.

View Article: PubMed Central - PubMed

Affiliation: Biochimie Pharmaceutique, Inserm U835, Upres JE 2311, Université de Rennes 1, France.

ABSTRACT
Hepatitis C is a major public health concern, with an estimated 170 million people infected worldwide and an urgent need for new drug development. An attractive therapeutic approach is to prevent the 'cap-independent' translation initiation of the viral proteins by interfering with both the structure and function of the hepatitis C viral internal ribosomal entry site (HCV IRES). Towards this goal, we report the design, synthesis and purification of novel bi-functional molecules containing DNA or RNA antisenses attached to functional groups performing RNA hydrolysis. These 5' or 3'-coupled conjugates bind the HCV IRES with affinity and specificity and elicit targeted hydrolysis of the viral genomic RNA after short (1 h) incubation at low (500 nM) concentration at 37 degrees C in vitro. Additional secondary cleavage sites are induced and their mapping within the RNA structure indicates that functional domains IIIb-e are excised from the IRES that, based on cryo-EM studies, becomes incapable of binding the small ribosomal subunit and initiation factor 3 (eIF3). All these molecules inhibit, in a dose-dependent manner, the 'IRES-dependent' translation in vitro. The 5'-coupled imidazole conjugate reduces viral protein synthesis by half at a 300 nM concentration (IC50), corresponding to a 4-fold increase of activity when compared to the naked oligonucleotide. These new conjugates are now being tested for activity on infected hepatic cell lines.

Show MeSH
Related in: MedlinePlus