Conserved RNA secondary structures and long-range interactions in hepatitis C viruses

Abstract

Hepatitis C virus (HCV) is a hepatotropic virus with a plus-strand RNA genome of ∼9.600 nt. Due to error-prone replication by its RNA-dependent RNA polymerase (RdRp) residing in nonstructural protein 5B (NS5B), HCV isolates are grouped into seven genotypes with several subtypes. By using whole-genome sequences of 106 HCV isolates and secondary structure alignments of the plus-strand genome and its minus-strand replication intermediate, we established refined secondary structures of the 5' untranslated region (UTR), the cis-acting replication element (CRE) in NS5B, and the 3' UTR. We propose an alternative structure in the 5' UTR, conserved secondary structures of 5B stem-loop (SL)1 and 5BSL2, and four possible structures of the X-tail at the very 3' end of the HCV genome. We predict several previously unknown long-range interactions, most importantly a possible circularization interaction between distinct elements in the 5' and 3' UTR, reminiscent of the cyclization elements of the related flaviviruses. Based on analogy to these viruses, we propose that the 5'-3' UTR base-pairing in the HCV genome might play an important role in viral RNA replication. These results may have important implications for our understanding of the nature of the cis-acting RNA elements in the HCV genome and their possible role in regulating the mutually exclusive processes of viral RNA translation and replication.

Keywords: HCV; RNA long-range interaction prediction; RNA secondary structure prediction; bioinformatics; circularization.

FIGURE 1.

Overview of previously known (black) and novel (gray) RNA stem–loop structures of the HCV RNA genome. (A) 5′ UTR and core region SLV and SLVI of the viral plus-strand. (B) CRE region and 3′ UTR of the viral plus-strand. (C) 3′ End of the viral minus-strand. (D) 5′ End of the viral minus-strand.

FIGURE 2.

(A) Alignment based secondary structure prediction of the 5′ UTR of 86 isolates. The alignment was calculated by LocARNA, the consensus sequence and structure by RNAalifold. The sequence for SLI is contained in only nine isolate sequences and therefore appears here as white. SLII is different from that described in Honda et al. (1999a), but in agreement with Zhao and Wimmer (2001) and SHAPE analysis of Pang et al. (2011) (see Supplemental Fig. 1C). Here, we show an alternative secondary structure for SLIIId with a slightly better MFE and conservation compared with the known structure. (Blue sequences) Forbidden to pair by RNAalifold in order to build the pseudoknot that is essential for the recruitment of the ribosome and therefore for viral protein translation (Berry et al. 2011). (Gray) Secondary structure different from those proposed by Honda et al. (1999a). (B) Consensus sequences and secondary structures of the CRE, VR, and X-tail in two alternative foldings. CRE and VR based on 96 isolates, X-tail based on 19 isolates. Apical regions of 5BSL1, 5BSL2, and 5BSL3.1 show up as described previously (Diviney et al. 2008). Even though it seems that the apical part of the stems can vary between isolates, the consensus secondary structure leaves little variability (see dotplot in Fig. 3). SLIV is present in all tested isolates but appears white since the stem is variable (see supplement, STK file for variable region). (Gray) Differences in secondary structures of 5BSL1 (SL9033), and 5BSL2 (SL9033) compared with data of Diviney et al. (2008), of 5BSL3.1 (SL9132) compared with data of Diviney et al. (2008) and Romero-López et al. (2014), of SLIV (Kolykhalov et al. 1996), of SLII (Kolykhalov et al. 1996; Blight and Rice 1997; Ito and Lai 1997), and of DLS (Ivanyi-Nagy et al. 2006; Shetty et al. 2010; Romero-López et al. 2014). (Rectangle) Consensus of RNAalifold to establish 5BSL3.2, instead of the interaction in Figures 3 (oval) and 7, No. 15 (Diviney et al. 2008). A detailed explanation of the base pair color code is available in the Supplemental Material.

FIGURE 3.

RNAalifold base-pairing probability matrix of the CRE and VR region, based on 96 isolates. The upper half of the matrix shows all base pair probabilities <10⁻⁶. The lower half shows the base pair probabilities of the MFE structure. (Oval) The interaction of the internal bulge of 5BSL3.2 with basal region of 5BSL2 (Fig. 7, No. 15), described previously in Diviney et al. (2008) and Tuplin et al. (2012). Dotplot and structure of CRE, VR, and X-tail region (19 isolates) without constraints are available in the Supplemental Material (Supplemental Fig. 3A,B).

FIGURE 4.

Alignment based secondary structures of the X-tail using sequences of 19 isolates. (Top) RNAalifold base-pairing probability matrix of the X-tail region. (A) Consensus secondary structure without applying any constraints. (B) Consensus secondary structure using constraints that force the postulated structure of Blight and Rice (1997). (C,D) Previously unknown secondary structures of the X-tail. All alternative structures contain SLI but show shifted hairpins compared with DLS or SLII.

FIGURE 5.

Alignment based secondary structure of the 5′ end of the minus-strand with 19 isolates. (A) Without constraints; (B) with constraints aiming at a similar structure like the mirror plus-strand; (C) suboptimal structure according to dotplot (Supplemental Fig. 5).

FIGURE 6.

Alignment based secondary structure of the 3′ end of the minus-strand with 86 isolates. (A) RNAalifold base-pairing probability matrix. The upper half of the matrix shows all base pair probabilities <10⁻⁶. The lower half shows the base pair probabilities of the MFE structure. (B) Consensus secondary structure without constraints. (C) Consensus secondary structure with constraints: The last 3 nt at the very 3′ end were forced to be unpaired (blue). The resulting structure mirrors the secondary structure of the domain SLIII from RNA plus strand. (D) Consensus secondary structure with constraints forcing the postulated structure of Smith et al. (2002). (E) Consensus secondary structure with constraints forcing the postulated structure of Dutkiewicz et al. (2008). (F) Consensus secondary structure with constraints forcing the postulated structure of Schuster et al. (2002). (Dots) Nucleotides base-pairing with an extended version of the 3′ end; for details see Schuster et al. (2002). (Connected rectangles) Further interactions calculated by LocARNA. White base-pairings are derived from isolates being sequenced only partially.

FIGURE 7.

Long-range interactions in 5′ UTR, CRE, VR, and X-tail. (A) Overview. (Gray) Known interactions (No. 13–17 from the literature, validated by our analysis for all examined isolates); (green) new interactions (from our calculations). Numbers indicate different interactions shown in detail in B. (B) Interactions for all available sequences labeled with numbers corresponding to A. Bottom numbers indicate number of sequences in the alignment used for the interaction. Interaction No. 1 can be extended for a possible circularization. (C) Possible circularization of the HCV RNA. The interaction of SLII and DLS of HCV plus-strand RNA can be extended to at least 62 bp in all available 19 isolates. (D) Corresponding interaction of minus-strand RNA.