Viral internal ribosome entry site structures segregate into two distinct morphologies

Abstract

An increasing number of viruses have been shown to initiate protein synthesis by a cap-independent mechanism involving internal ribosome entry sites (IRESs). Predictions of the folding patterns of these RNA motifs have been based primarily on sequence and biochemical analyses. Biophysical confirmation of the models has been achieved only for the IRES of hepatitis C virus (HCV), which adopts an open structure consisting of two major stems. We have conducted an extensive comparison of flavivirus and picornavirus IRES elements by negative stain transmission electron microscopy. All of the flavivirus IRESs we examined (those of GB virus-B, GB virus-C, and classical swine fever virus) fold to give a structure similar to that of the HCV IRES, as does an IRES recently found on mRNA encoded by human herpesvirus 8. The larger picornavirus IRESs (those of foot-and-mouth disease virus, rhinovirus, encephalomyocarditis virus, and hepatitis A virus) are morphologically similar, comprising a backbone with two protruding stems, and distinct from the flavivirus IRESs.

FIG. 1.

Proposed secondary structures of virus IRES elements from (a) HRV (type I picornavirus IRES) (4), (b) FMDV (type II picornavirus IRES) (24), (c) HAV (type III picornavirus IRES) (7), (d) HCV (12), (e) GBV-B (28), (f) GBV-C (28), (g) CSFV (10), and (h) HHV8 (3). The numbering of nucleotides on FMDV (b) and HHV8 (h) models is from the initiation codon. In all other models, numbering is from the 5′ end of the corresponding viral genome.

FIG. 2.

Polyacrylamide gel analysis of transcribed IRES RNAs. The electrophoresed RNA was visualized by immersing the gel in 0.025% methylene blue for 1 h followed by destaining in several changes of tap water.

FIG. 3.

Representative negative stain TEM images of flavivirus-like IRES transcripts, with diagrammatic depictions of the structures noted. (a) HCV. (b) HCV 5′ UTR nt 119 to 156 (domains III and IV). (c) GBV-B. (d) CSFV. (e) GBV-C. (f) HHV8.

FIG. 4.

Representative negative stain TEM images of picornavirus IRES transcripts, with diagrammatic depictions of the structures noted. (a) Type II. (b) Type I. (c) Type III.