Viral IRES RNA structures and ribosome interactions

Abstract

In eukaryotes, protein synthesis initiates primarily by a mechanism that requires a modified nucleotide 'cap' on the mRNA and also proteins that recruit and position the ribosome. Many pathogenic viruses use an alternative, cap-independent mechanism that substitutes RNA structure for the cap and many proteins. The RNAs driving this process are called internal ribosome-entry sites (IRESs) and some are able to bind the ribosome directly using a specific 3D RNA structure. Recent structures of IRES RNAs and IRES-ribosome complexes are revealing the structural basis of viral IRES' 'hijacking' of the protein-making machinery. It now seems that there are fundamental differences in the 3D structures used by different IRESs, although there are some common features in how they interact with ribosomes.

Figure 1

Structural features of ribosomes and ribosome-containing complexes. (a) The crystal structure of the bacterial 70S ribosome from Thermus thermophilus with three tRNAs bound is shown with the 50S (large) subunit in cyan and the 30S (small) subunit in yellow [58]. This view is sometimes called a ’top’ view. The A-, P- and E-site tRNAs are labeled and shown in blue, red and green, respectively. Only a portion of the A-site tRNA is shown because the rest of this tRNA is not visible in the crystal structure. The approximate pathway of mRNA through the decoding groove on the 30S subunit and out of the exit tunnel is shown with a black line and the L1 stalk of the 50S subunit is indicated. (b) Structure of the 30S subunit from the same structure shown in (a) and using the same coloring scheme but turned to reveal the inter-subunit face of the 30S subunit and the positions of the three tRNAs along the decoding groove. The mRNA entry and exit positions are shown. (c) Cryo-EM reconstruction of a human 80S ribosome in the same orientation as the bacterial ribosome in (a). This reconstruction is from a ribosome bound to the CrPV IRES [29]; here, the IRES density has been removed computationally. The 60S subunit is cyan, the 40S subunit is yellow, the path of the mRNA is shown with a black line and the L1 stalk is indicated. (d) Cryo-EM reconstruction of the 40S subunit portion of the ribosome in (c), rotated to show the intersubunit face. The A-, P- and E-sites in the decoding groove are indicated and the approximate pathway of mRNA is shown with a broken line. (e) 60S subunit of the cryo-EM reconstruction shown in (c), rotated to show the intersubunit face and the location of the L1 stalk.

Figure I

Comparison of cap-dependent translation and four IRES groups.

Figure 2

Structures of the Dicistroviridae intergenic region (IGR) IRES RNAs. (a) Proposed secondary structure of the Plautia stali intestine virus (PSIV) intergenic region IRES, which includes three pseudoknots and two conserved stem-loops. Important structural elements are labeled: SL, stem loop; PK, pseudoknot; L, loop; P, paired (helix). Regions 1 + 2 are boxed in blue; these regions fold together to comprise the ribosome-binding domain of the IRES. Domain 3 is the part of the IRES that docks into the P-site of the ribosome and is boxed in red. The start codon for protein synthesis is shown; in these IRESs, it is not an AUG codon as in canonical translation initiation. (b) Representation of the secondary structures of the two independently folded domains that make up the IGR IRES and for which crystal structures have been solved. On the left, regions 1 and 2 are boxed in blue as in (a), with secondary structural elements colored and labeled. Portions of the structures that were not visible in the crystal structure are shown with broken gray lines. On the right is domain 3, boxed in red as in (a). Parts of the structure that are involved in crystal packing in such a way that they are not relevant biologically are shown with dashed gray. (c) Ribbon representation of the crystal structure of the ribosome-binding domain (regions 1 + 2) of the PSIV IGR IRES [26]. The structure is color coded to match (b), with important secondary structure elements and pseudoknots labeled. (d) Ribbon representation of the crystal structure of domain 3 of the CrPV IGR IRES colored to match (b) [28]. To the right of the domain 3 structure is the crystal structure of a P-site tRNA–mRNA interaction [58] with the anticodon loop in red and the mRNA in cyan. Comparing these structures shows the mimicry between the tRNA–mRNA interaction and domain 3. (e) Cryo-EM difference density of the CrPV IRES bound to the 80S ribosome, at 7.3Å resolution from which the ribosome density has been removed computationally [29]. The crystal structures of the ribosome-binding domain from PSIV [26] and domain 3 from CrPV [28] are shown docked into the structure. The good fit indicates that the unbound structure is similar to the bound form, although differences between the cryo-EM density and the docked crystal structures reveal probable local changes in structure when the IRES binds to the ribosome. The docked structure enables predictions of what IGR IRES RNA structural features interact with specific ribosomal proteins or rRNA [–29]. The putative interactions are shown, with the relevant rRNA helices, ribosomal proteins and key IRES features labeled. Although rpS25 crosslinks to the PSIV IRES SL IV, its precise location is not known, therefore it is not shown in this figure. Ribosomal proteins or rRNA from the 60S subunit are in cyan and from the 40S subunit are in yellow. Note that, in this view, the ribosome-binding domain is flipped 180 degrees around the horizontal axis relative to panel (c). (f) Cryo-EM reconstruction of the CrPV IGR IRES bound to a human 80S ribosome [27], with the IRES density in magenta, the 60S subunit in cyan and the 40S subunit in yellow. The location of the L1 stalk and rpS5 are indicated. The view is from the ’top’ of the ribosome, as in Figure 1c. From this perspective, the view is down the length of the IRES structure in (e), looking at the L1.1 end.

Figure 3

Structures of the hepatitis C virus (HCV) IRES RNA. (a) Proposed secondary structure of the HCV IRES, with its stem-loops labeled. Stem-loop I and the portion of the RNA that is shaded can be removed without affecting IRES function. The extended stem-loop structure that contains internal loops IIa and IIb is generally referred to as ‘domain II’. In the HCV IRES, the AUG start codon is located in a stem-loop structure (IV), however, this stem-loop is not found in all related IRESs (such as that of CSFV). (b) At the left is the secondary structure of the HCV IRES surrounded by structures of secondary-structure elements solved by NMR and X-ray crystallography [–47]. The location of each element in the structure is shown with arrows and boxes that correspond to the color of the structure. (c) Secondary structure of domain II of the HCV IRES and its 3D structure as solved by NMR [41]. Blue denotes the apical loop that is placed into the 40S subunit's E-site, red denotes internal loops IIa and IIb and green denotes bulged bases. These features cause the RNA to adopt an overall bent conformation. (d) HCV IRES RNA bound to a human 80S ribosome, with the IRES density in magenta, the 60S subunit in cyan and the 40S subunit in yellow. The location of the L1 stalk and rpS5 are indicated [50]. The asterisk denotes a putative interaction between domain I (which is not needed for IRES function) of the HCV IRES and the base of the L1 stalk.

Figure 4

Comparison of cryo-EM reconstructions of the CrPV IGR IRES and the HCV IRES. (a) Cryo-EM reconstructions of IRES-containing complexes studied to date. In all reconstructions, the IRES RNA is shown in magenta, the 60S subunit in cyan and the 40S subunit in yellow. At top left is the CrPV IGR IRES RNA bound to a human 40S subunit and at top right is the CrPV IGR IRES bound to the 80S ribosome [27]. The structure of the CrPV IGR IRES bound to a yeast 80S ribosome at higher resolution is not shown [29]. In the middle are structures of the HCV IRES bound to 40S subunits from rabbit, a model of the 40S subunit–eIF3–HCV IRES complex constructed from other reconstructions (eIF3 in green) [54] and the HCV IRES bound to a human 80S ribosome [50]. Cryo-EM reconstructions of members of the group 3 or 4 IRESs do not yet exist but will probably be a goal of future efforts. (b) A close up view of the CrPV IRES bound to a human 80S ribosome, with the L1 stalk and loop L1.1 of the IRES labeled [27]. This view is rotated 90 degrees around both the horizontal axis relative to Figure 2f, with the view looking into the E-site over the L1 stalk. (c) Close-up view of the HCV IRES bound to the 80S ribosome, with the IRES mostly outside the intersubunit space, but reaching into the E-site within domain II. The location of the L1 stalk is labeled. The view is rotated 90 degrees around both the horizontal and vertical axes, relative to Figure 3d.