Abstract
The RNA genome of the hepatitis C virus (HCV) encodes a single open reading frame (ORF) containing numerous functional elements. Among these, the cis-acting replication element (CRE) at the 3′ end of the viral ORF, has become of increasing interest given its dual role as a viral translation repressor and replication enhancer. Long-range RNA-RNA contacts mediated by the CRE build the structural scaffold required for its proper functioning. The recruitment of different cellular factors, many related to the functioning of the translation machinery, might aid in the CRE-exerted downregulation of viral translation. The present data show that the CRE promotes a defect in polysome production, and hinders the assembly of the 80S complex, likely through the direct, high affinity recruitment of the 40S ribosomal subunit. This interaction involves the highly conserved 5BSL3.1 and 5BSL3.3 domains of the CRE, and is strictly dependent on RNA-protein contacts, particularly with the ribosomal proteins RPSA and RPS29. These observations support a model in which the CRE-mediated inhibition of viral translation is a multifactorial process defined by the establishment of long-range RNA-RNA interactions between the 5′ and 3′ ends of the viral genome, the sequestration of the 40S subunit by the CRE, and the subsequent stalling of polysome elongation at the 3′ end of the ORF, all governed by the highly stable hairpin domains 5BSL3.1 and 5BSL3.3. The present data thus suggest a new managerial role in HCV translation for these 5BSL3.1 and 5BSL3.3 domains.
Highlights
In positive, single-stranded RNA viruses, such as the hepatitis C virus (HCV), the genomic RNA acts as a template for both translation and replication
The ICU molecule encompasses the HCV internal ribosome entry site (IRES) region fused to the sequence coding for the FLuc protein, followed by the cis-acting replication element (CRE) and the 3′UTR; it meets the minimal requirements for controlling viral translation
The present work provides evidence that the CRE sequesters 40S ribosomal particles, which might be related to the negative regulation exerted by it on IRES-dependent translation, and the concomitant increase in viral RNA replication
Summary
Single-stranded RNA viruses, such as the hepatitis C virus (HCV), the genomic RNA acts as a template for both translation and replication. The mechanism by which the CRE interferes with HCV protein synthesis is not completely understood It is known, that the 5BSL3.2 domain governs a complex network of contacts that organise the conformation of the HCV genome at two levels[19]: (i) by promoting the local structural tuning of the 3′ end of the viral RNA genome[20], which seems to be essential for replication[16,21]; and (ii) by allowing the circularization of the HCV RNA via the formation of long-range RNA-RNA interactions with subdomain IIId of the IRES region[11,22,23]. Our group has previously isolated ribosomal proteins bound to the CRE region[24] These observations prompted the proposal that the well-known RNA-RNA interaction network involved in the control of viral translation might be aided via the direct recruitment, or sequestration, of the cellular translation machinery. The present results support a model in which the CRE region sequesters 40S subunits, preventing efficient ribosome reloading at the 5′ end of the mRNA genome, repressing translation
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