Functionally conserved architecture of hepatitis C virus RNA genomes

Abstract

Hepatitis C virus (HCV) infects over 170 million people worldwide and is a leading cause of liver disease and cancer. The virus has a 9,650-nt, single-stranded, messenger-sense RNA genome that is infectious as an independent entity. The RNA genome has evolved in response to complex selection pressures, including the need to maintain structures that facilitate replication and to avoid clearance by cell-intrinsic immune processes. Here we used high-throughput, single-nucleotide resolution information to generate and functionally test data-driven structural models for three diverse HCV RNA genomes. We identified, de novo, multiple regions of conserved RNA structure, including all previously characterized cis-acting regulatory elements and also multiple novel structures required for optimal viral fitness. Well-defined RNA structures in the central regions of HCV genomes appear to facilitate persistent infection by masking the genome from RNase L and double-stranded RNA-induced innate immune sensors. This work shows how structure-first comparative analysis of entire genomes of a pathogenic RNA virus enables comprehensive and concise identification of regulatory elements and emphasizes the extensive interrelationships among RNA genome structure, viral biology, and innate immune responses.

Keywords: RNA structure; evolution; functional validation; motif discovery.

Fig. 1.

Global structural analysis of three HCV genomes. (A) SHAPE reactivities for each genome over the first 450 nt. IRES subdomains II–IV and the ORF are highlighted. Reactivities are shown relative to the global median. (B) Heat maps showing median SHAPE reactivities (51-nt windows) for each of three genomes. Regions identified as mutually structured across all three genomes are emphasized with blue bars. (C) Median Shannon entropies (51-nt windows) for each of three HCV genomes. Regions with low Shannon entropies in all three genomes are indicated above the histograms with gray bars. (D) Representative structural model for the JFH1 RNA genome. Helices are shown as arcs, colored according to base-pairing probabilities as calculated from the SHAPE-directed partition function (26). Regions with green arcs represent well-defined structures; regions with overlapping blue, yellow, and gray arcs likely sample multiple conformations. Full arc models for all three RNA genomes are provided in Figs. S1–S3.

Fig. 2.

Selective pressures on higher-order HCV RNA structures. (A) Locations of 15 regions that have conserved base pairing in the SHAPE-directed models across all three HCV genotypes. Regions are numbered relative to their position in the H77c genome. (B) Synonymous substitution rates for each of seven individual genomic alignments over all regions of conserved base pairing combined. Larger (negative) values indicate lower synonymous substitution rates, consistent with evolutionary conservation of RNA structure. (C) Differences in synonymous substitution rates for individual regions. The z-scores above 1.96 and below −1.96 (dashed lines) are significant at the P < 0.05 level. The lower synonymous substitution rates in regions 316 and 603 may reflect evolutionary constraints imposed by an alternative ORF (48). (D) Complementary coevolution between base-paired sites. Values for predicted base pairs across the entire genome and over regions with high degrees of conserved base pairing are shown at left. Values below −1.96 (dashed line) are significant at the P < 0.05 level.

Fig. 3.

Comparative HCV RNA genome structure analysis. Summary of notable regions based on each analysis class: structured regions (blue), regions of low Shannon entropy (gray), regions of conserved base pairing (brown), regions with low synonymous substitution rates (green), regions with high synonymous substitution rates (red), and regions with evidence of complementary coevolution of base pairs (orange) for all three HCV genomes. Six regions (purple) have conserved base paring in the SHAPE-directed structural models and phylogenetic support for broad conservation. These regions are annotated with the four structural elements (J750, J7880, J8640, and J8880) evaluated in HCV replication assays. SHAPE-informed secondary structure models of the JFH1 genome are shown for regions with notable features conserved across all three HCV RNA genomes. Nucleotides are colored by SHAPE reactivity.

Fig. 4.

Functional regulatory structures within the JFH1 RNA genome. (A) JFH1-QL/GLuc expression construct. The four structural elements tested in functional assays are shown. RNA secondary structure models for (B) J750, (C) J7880, (D) J8640, and (E) J8880. The positions of structure-disrupting, silent mutations are shown with asterisks. Nucleotides are colored by SHAPE reactivity (see scale). (F) Relative levels of HCV-encoded G. princeps luciferase protein, normalized to the 4-h time point, secreted by cells transfected with JFH1-QL/GLuc RNA or structure disrupting mutants. The bars show the means of triplicate measurements; error bars report SDs. NS5B-GND is a lethal (negative) control. (G) Titers of infectious virus generated 72 h posttransfection by JFH1-QL (no GLuc2A insertion), the lethal mutant NS5B-GND, and structure-disrupting mutants. Histograms show the mean of triplicate measurements; error bars report SDs.

Fig. 5.

Relationships between HCV genome structure and innate immune factor recognition features. (A) RNA helix lengths in the SHAPE-directed folding models for each of three HCV genomes. For comparison, helix lengths for models of the human 18S and yeast 28S ribosomal RNAs (49) are shown. (B) Comparison of measured RNase L cleavage sites (22, 24) with mean SHAPE reactivities randomly sampled from UU/UA dinucleotides in the H77c genome. Green bars show a bootstrap analysis of mean SHAPE reactivities for 10,000 populations of UU/UA motifs chosen at random from the H77 genome. The mean SHAPE reactivity for efficient RNase L cleavage sites (red line) lies well outside the distribution expected by chance. (C) RNA structure models for the five strongest RNase L cleavage sites (triangles) in the H77 genome. Nucleotides are colored by SHAPE reactivity.