Base pairing between hepatitis C virus RNA and 18S rRNA is required for IRES-dependent translation initiation in vivo

Abstract

Degeneracy in eukaryotic translation initiation is evident in the initiation strategies of various viruses. Hepatitis C virus (HCV) provides an exceptional example-translation of the HCV RNA is facilitated by an internal ribosome entry site (IRES) that can autonomously bind a 40S ribosomal subunit and accurately position it at the initiation codon. This binding involves both ribosomal protein and 18S ribosomal RNA (rRNA) interactions. In this study, we evaluate the functional significance of the rRNA interaction and show that HCV IRES activity requires a 3-nt Watson-Crick base-pairing interaction between the apical loop of subdomain IIId in the IRES and helix 26 in 18S rRNA. Mutations of these nucleotides in either RNA dramatically disrupted IRES activity. The activities of the mutated HCV IRESs could be restored by compensatory mutations in the 18S rRNA. The effects of the 18S rRNA mutations appeared to be specific inasmuch as ribosomes containing these mutations did not support translation mediated by the wild-type HCV IRES, but did not block translation mediated by the cap structure or other viral IRESs. The present study provides, to our knowledge, the first functional demonstration of mRNA-rRNA base pairing in mammalian cells. By contrast with other rRNA-binding sites in mRNAs that can enhance translation as independent elements, e.g., the ShineDalgarno sequence in prokaryotes, the rRNA-binding site in the HCV IRES functions as an essential component of a more complex interaction.hepatitis C virus | 18S rRNA | IRES | base pairing | translation H CV is a single-stranded RNA virus that is a major cause of severe liver disease. The RNA genome contains a large ORF and expresses a single polypeptide that is processed into smaller proteins, which are necessary for replication and assembly of viral particles. The RNA genome is uncapped, and translation does not require the eukaryotic initiation factor 4F (eIF4F) complex (1), which mediates cap-dependent translation. Instead, translation is facilitated by an internal ribosome entry site (IRES) located in the 5′ nontranslated region (2, 3). Inasmuch as the production of all HCV proteins requires the HCV IRES, the HCV IRES is a therapeutic target (4).IRESs encompass a variety of initiation mechanisms and have been extensively studied in viruses, which often exploit the translation capabilities of the host for their own use (5-7). In many cases, the IRES mechanism complements the infection strategy of the virus. In the case of the HCV IRES and other IRESs of this type, the IRES can effectively recruit the host translation machinery by directly binding to 40S ribosomal subunits (1,(8)(9)(10)(11)(12). The HCV-ribosome interaction has been shown to require an interaction with ribosomal protein S25 (13) and can also involve the eIF3 complex, which increases the stability of the interaction (14). In the presence of ternary complex, which contains the initiator Met-tRNA, a preinitiation complex can assemble at the start site.Various studies have investigated the binding of ...

Molecular architecture of the ribosome‐bound Hepatitis C Virus internal ribosomal entry site RNA

“…Subdomains IIId and IIIe have been implicated as major determinants for IRES binding by a large body of evidence (Kolupaeva et al , ; Kieft et al , ). Rationalizing recent chemical probing and mutagenesis studies (Kolupaeva et al , ; Malygin et al , ; Matsuda & Mauro, ), our model shows a three‐nucleotide mini‐helix between the 266 GGG 268 sequence in the apical loop of subdomain IIId with the complementary 1116 CCC 1118 sequence in the loop of ES7 (Fig A and E). Formation of this mini‐helix requires conformational changes in both the apical loop of IIId as defined by the NMR structure (Klinck et al , ; Lukavsky et al , ) and ES7.…”

Widespread distribution and structural diversity of Type IV IRESs in members of Picornaviridae

“…The PK-IIId-IIIe core contains some highly conserved elements, but also exhibited significant variability. The apical GGG motif in domain IIId is invariant, consistent with its function in base-pairing with the apical UCCC loop of ES7 of 18S ribosomal RNA (rRNA) (Hashem et al, 2013; Matsuda and Mauro, 2014), and its consequent importance in all Type IV IRESs (Kolupaeva et al, 2000a; Kieft et al, 2001; de Breyne et al, 2007; Easton et al, 2009; Willcocks et al, 2011; Pan et al, 2012). This apical loop occurs in ES7 of 18S rRNA in birds, fish, amphibians and reptiles as well as mammals, and it would therefore be available in ribosomes from them all for binding to Type IV IRESs.…”

“…HCV domain IIId also contains an ‘loop E’ motif (indicated by grey shading), but an equivalent structure does not occur at this position in other Type IV IRESs. The model has been annotated to show interactions of domain III of Type IV IRESs with eIF3 (green arrows) (Sizova et al, 1998; Kieft et al, 2001; Hashem et al, 2013; Sun et al, 2013) and with 18S rRNA ES7 and ribosomal protein S1e, S26e, S27e and s28e components of the 40S ribosomal subunit (red arrows) (Boehringer et al, 2005; Hashem et al, 2013; Matsuda and Mauor, 2014). The tertiary base-pairing interaction between the unpaired purine residue in helix III 1 and loop IIIe is indicated by a blue line.…”

Functional RNA structures throughout the Hepatitis C Virus genome

“…For example, the foundation for a strong IRES-40S interaction is the conserved base-pairing that forms between the 18S RNA of the 40S subunit and domain III of the IRES. Intriguingly, nucleotides involved in pairing to 18S RNA are required for IRES-mediated translation, but they are not required for canonical cellular translation [8], indicating that they have been co-opted by the virus. After the 40S subunit has been recruited, domain II reaches across the head of the 40S subunit, wedging open the mRNA binding tunnel and allow the HCV coding sequence to bind [7].…”