Multivalent engineered RNA molecules that interfere with hepatitis C virus translation and replication Cristina Romero-López 1, *, Thomas Lahlali 2 , Beatriz Berzal-Herranz 1 and Alfredo Berzal-Herranz 1 1 Instituto de Parasitología y Biomedicina López-Neyra, IPBLN-CSIC, PTS Granada, Av. Conocimiento 17, 18016, Armilla-Granada, Spain. 2 INSERM U1052, Cancer Research Center of Lyon (CRCL), Université Claude-Bernard (UCBL), UMR_S1052, UCBL, 69008, Lyon, France. * Corresponding author: cristina_romero@ipb.csic.es 1
MULTIVALENT ENGINEERED RNA MOLECULES THAT INTERFERE WITH HEPATITIS C VIRUS TRANSLATION AND REPLICATION HCV IRES IIIb II IIIa IIIc IIId 5’ ORF I Translation ↓↓ 80% IV AUG Replication ↓↓ 80% Graphical abstract 2
Abstract: The design of novel and efficient compounds fighting against the highly variable RNA viruses, such as hepatitis C virus (HCV), is a major goal. Engineering different antiviral RNAs into a single molecule yields the so-called multivalent compounds, which are promising candidates for the development of new therapeutic strategies. In this work, the previously developed chimeric inhibitor RNA HH363-10 was used as archetype for the development of improved anti-HCV inhibitors. HH363- 10 consists of a hammerhead ribozyme domain, targeting the essential internal ribosome entry site (IRES) region; and an aptamer RNA molecule, directed against the highly conserved IIIf domain of the IRES. Following to the application of an in vitro selection process, new multivalent optimized chimeric anti-HCV RNA molecules derived from HH363-10 were isolated. The aptamer RNA domain was evolved to contain two binding sites: the one mapping the IIIf domain, and a newly acquired targeting site, either to the IRES domain IV (which contains the translation start codon) or the essential linker region between the IRES domains I and II. These chimeric molecules efficiently and specifically interfered with HCV IRES-dependent translation in vitro (with IC 50 values in the low µM range). They also inhibited both viral translation and replication in cell culture. These findings highlight the feasibility of using in vitro selection strategies for obtaining improved, multivalent RNA molecules with potential clinical applications. Keywords: RNA aptamer; hepatitis C virus; IRES; RNA targeting 3
THERAPEUTIC NUCLEIC ACIDS The structural flexibility of nucleic acids, particularly RNA, results in their great functional versatility. This has led to propose the use and engineering of nucleic acids as new therapeutic drugs targeting the genetic information of viruses. Moreover, advances in chemical synthesis techniques have improved the possibilities of developing therapeutic RNA molecules. RIBOZYMES APTAMERS Small catalytic RNAs are all naturally Aptamers are oligonucleotides able to efficiently bind involved in the replication process of to a wide variety of ligands. Aptamers are isolated by RNA genomes in which they are a SELEX (Systematic Evolution of Ligands by contained. Potentially therapeutic Exponential enrichment) process, which consists on ribozymes includes the hammerhead and iterative cycles of synthesis, binding, positive the hairpin ribozyme. selection and amplification steps over a randomized oligonucleotide pool. The combination of different RNA molecules with proven antiviral activity renders the so-called multivalent compounds, chimeric entities with enhanced therapeutic properties. Introduction 4
THE HCV GENOME The isolation of RNA molecules directed against different targets of the hepatitis C virus (HCV) genome has been largely described. The HCV genome is a (+)ssRNA molecule encoding a single open reading frame (ORF) flanked by untranslated regions (UTRs), which are essential for viral replication, translation and infectivity. Viral translation initiation is mediated by a highly structured motif mainly located at the 5’UTR, which acts as an internal ribosome entry site (IRES). The presence of structural elements at the 3’ end of the viral genome may also modulate the initiation and elongation steps involved in HCV translation. This might be mediated via the HCV genomic RNA, assuming a circular topology resembling the closed-loop structure adopted by cellular cap-mRNAs. Such circular formations depend on the existence of a direct, long-range RNA-RNA interaction, involving subdomain IIId of the IRES element and the essential stem-loop 5BSL3.2 of the CRE ( cis -acting replication element) region at the 3’ end of the viral protein coding sequence. CRE IIIb II IIIa 5BSL3.2 IIIc IIId 5BSL3.1 5BSL3.3 3’X-tail PK2 3’SLIII 5’ Alt PolyU/UC PK1 3’ ORF I IV 3’SLII Stop codon AUG start codon HV 3’SLI IRES 3’UTR Introduction 5
THE HCV IRES REGION AND THE PARENTAL HH363-10 RNA INHIBITOR The minimum HCV IRES element required for translation initiation folds into a IIIb U U C G 200 well-defined structure composed of two major domains (II and III) and a short U G IRES U A U U stem-loop (domain IV) containing the initiation codon (enlarge lettering in the CC U A U A figure). Domains II and III are aligned at both sides of a complex double G C G C G C pseudoknot structure allowing the correct positioning of the start codon (PK1 C G C C A U G C and PK2). Importantly, many of the crucial structural regions required for IRES- A C A U II A dependent translation are located in the highly branched domain III. The G G C CC G C 180 G A A cleavage site of the HH363-10 inhibitor RNA is marked by an arrow. The A U 220 U U 80 C G C G C G eIF3 U G nucleotides that interact with the aptamer domain of HH363-10 are shown in G A G C U G C G IIIa U A binding site G U red. A U A U A U A A A U C A CC G G G U A G G GG U C C C HH363-10 U U A GG G G G A G A I A A CCC U C A U 160 A G IIIc C G C U G U G A G C 240 C C G C G U 100 G C A U A C A C G U A G U UU U 40S C G U A C C U A U G G C G C G 60 binding site G U U A U 60 U U G G G C G G C A 260 40 U A A U C C G AG I U U A UU 80 A G G C G CC U AG G U U A A G G C G -- I C A G 3´ C U A U C GG A A GC G C A G A G A C U U G C C G C C U G C 280 IIId GG U UU U U CUGA U A 140 A U G U 5´ -- G U A C G G U C A U C G U G G C G G U UU CU A U A A A A G 120 C G A C G C PK2 C G I A U U G 5’ GCC U A GCC G A G A C A C U C C A CC A U G AA U C A C C U C A CCCCCCC CCC U GGG A C I G A G G GG CCC U GG AU C G G G G C C 40 U I 20 IIIe C G G C G C C 300 U U C 20 G A G U G U IIIf C G U G C A G C G G C PK1 U U G G A The RNA inhibitor HH363-10 was isolated by an in vitro selection U I A 3’ UAAACCUC AA A GAAAAACC… C G C I method. The catalytic hammerhead domain, HH363, is shadowed. A U 360 A C A HH363-10 C G Tertiary contacts are indicated by dotted lines. Residues in the aptamer G C cleavage site U A G C domain responsible for the interaction with domain IIIf of the IRES are C G A A 340 C G shown in red. C A U IV Introduction 6
IN VITRO SELECTION OF ANTI-HCV MULTIVALENT COMPOUNDS AGAINST THE IRES REGION OF THE HCV GENOME The chimeric inhibitor HH363-10 was subjected to an in vitro DNA library Random region Pri2is selection procedure, with the aim of identifying inhibitory RNA T7p S B 5’ 3’ 5’ 3’ molecules that act against the IRES region of the HCV genome, T7p PBS Pri1is 5’ and that showed improved aptamer and catalytic properties. For 3’ HH363 this, an RNA pool was designed, based on the HH363-10 inhibitor, 5’HCV-356 Transcription RT-PCR adhering to the following criteria: i) the maintenance of the RNA library sequence motif responsible for the aptamer's interaction with the In vitro 3’ 5’ domain IIIf of the IRES; ii) randomization of the nucleotides selection method flanking the interacting sequence with the aim of isolating RNA 5’ molecules bearing secondary anchoring sites targeting additional 3’ sites of the HCV IRES region besides the primary domain IIIf. First Active molecules S B for cleavage are selection eluted bound to the step for 5’ 5’ product of the association reaction 3’ N G R A C K U RNA pool N U Discard Washing with TMN R N buffer at room unbound N N N N molecules temperature N N 95 ºC N U 60 5’ N G denaturing 40 N A C G 80 A G G N G U A -- I S B 3´ U C C U A 3’ 5’ 3’ G A C U C C GG CUGA U UU N N 5´ -- G U A U Second A U G G Active A UU CU selection A G molecules for G C step for U A association C G cleavage 5’ C G 20 G A U G S B 3’ 5’HCV-691 Results and Discussion 7
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