Wild-type is the optimal sequence of the HDV ribozyme under cotranscriptional conditions

Abstract

RNA viruses are responsible for a variety of human diseases, and the pathogenicity of RNA viruses is often attributed to a high rate of mutation. Self-cleavage activity of the wild-type hepatitis delta virus (HDV) ribozyme as measured in standard divalent ion renaturation assays is biphasic and mostly slow and can be improved by multiple rational changes to ribozyme sequence or by addition of chemical denaturants. This is unusual in the sense that wild type is the most catalytically active sequence for the majority of protein enzymes, and RNA viruses are highly mutable. To see whether the ribozyme takes advantage of fast-reacting sequence changes in vivo, we performed alignment of 76 genomic and 269 antigenomic HDV isolates. Paradoxically, the sequence for the ribozyme was found to be essentially invariant in nature. We therefore tested whether three ribozyme sequence changes that improve self-cleavage under standard divalent ion renaturation assays also improve self-cleavage during transcription. Remarkably, wild type was as fast, or faster, than these mutants under cotranscriptional conditions. Slowing the rate of transcription or adding the hepatitis delta antigen protein only further stimulated cotranscriptional self-cleavage activity. Thus, the relative activity of HDV ribozyme mutants depends critically on whether the reaction is assayed under in vivo-like conditions. A model is presented for how wild-type ribozyme sequence and flanking sequence work in concert to promote efficient self-cleavage during transcription. Wild type being the optimal ribozyme sequence under in vivo-like conditions parallels the behavior of most protein enzymes.

FIGURE 1.

Secondary structures present in HDV ribozyme-containing transcripts. The structures are color coded by native pairings, which are based on the crystal structure of the cleaved form of the ribozyme (Ferre-D'Amare et al. 1998) and extensive mutagenesis experiments (Wadkins et al. 1999; Chadalavada et al. 2000, 2002; Diegelman-Parente and Bevilacqua 2002; Brown et al. 2004). Black arrowheads denote 5′ to 3′ directionality. (A,B) Native secondary structures of constructs studied. These transcripts begin at −54, which allows formation of the P(−1) pairing that facilitates native secondary structure (Chadalavada et al. 2000). P(−1) has the L(−1) hairpin loop at its apex and is joined to the ribozyme by the J(−1/1) single-stranded joining region. Pairings P1, P2, P3, P4, and P1.1 are contained within the ribozyme, and the site of cleavage between −1 and 1 is denoted with an arrow. For the transcript in B, increasing length of downstream sequence is present, which results in formation of P5 and the attenuator. Pairing register of the attenuator, which is mutually exclusive with much of the ribozyme secondary structure, is denoted by an extension (dashed line). (C) Secondary structure of the −30/99 RNA showing some of the known alternative pairings. Alt 1 and Alt 2 involve interactions of upstream flanking sequence with the ribozyme, while Alt 3 involves ribozyme–ribozyme pairing (Chadalavada et al. 2000, 2002). Other alternative pairings not shown include Alt X and Alt Y, which disfavor native RNA folding (Brown et al. 2004). (D) Sequence of wild-type ribozyme (Chadalavada et al. 2002).

FIGURE 2.

Nucleotide conservation of the HDV ribozyme and flanking RNA. Numbering is relative to the cleavage site between nucleotides −1 and +1. The −54 to 140 region of 76 genomic HDV RNA isolates was aligned using nucleotide–nucleotide BLAST (blastn). Information content, R _sequence(L), in bits per base at various positions (L) was determined as described in Materials and Methods. To enhance visual clarity, data for genomic sequence have been divided into upstream flanking sequence (A), ribozyme sequence (B), and downstream flanking sequence (C). Panel D shows data for the antigenomic ribozyme sequence. A score of 0–2 was given for the information content in bits. Positions with variability in R _sequence(L) values are emphasized by the placement of open circles on the plot. Regions with helical nucleotides are shaded gray, while regions with nonpaired nucleotides are white. The ribozyme and downstream sequences have a few pairing regions that are contiguous; in these cases, a dashed line was used to partition the two helices. The lower half of each panel enumerates the actual nucleotide sequence, with the letter height proportional to the nucleotide frequency. Nucleotides with an occurrence of less than 3% are not depicted.

FIGURE 3.

Covariation in the P5 helix and flanking regions. Nucleotide sequence corresponding to the 75–140 region of the genomic HDV RNA was folded using mfold v3.2 (Mathews et al. 1999; Zuker 2003). Numbering is relative to the cleavage site between nucleotides −1 and +1. Sequence variability information was obtained from blastn analysis of the 76 genomic HDV RNA isolates. Positions that vary are shaded gray and boxed for base pairs and circled for single-stranded nucleotides. Nonconserved nucleotides that base-pair were observed to covary so as to retain base-pairing. The number of isolates for a given base pair is provided below the figure. Nucleotides with an occurrence of less than 3% are not depicted. In this figure, residue 85 is a C because this is the most common sequence; however, it was G in the isolate used for wild-type, which was isolated from a patient with acute HDV (Makino et al. 1987) (accession M28267). The pairing elements aJ4/2, aP2_3′, P5, and L5 are denoted in the figure.

FIGURE 4.

Effect of ribozyme sequence on full-length transcript self-cleavage and cotranscriptional self-cleavage reactions. (A) Self-cleavage of full-length ribozymes (standard assay) in which the reaction was initiated by addition of MgCl₂. The three sequences are wild-type (•), G10C (Δ), and G11C (○). Fits are to Equation 6 and parameters are provided in Table 1. (B,C) Cotranscriptional self-cleavage for −54/99 (B) and −54/140 (C) transcripts. Points are an average of at least two trials. Time points range from 15 sec to 10 min. The four sequences are wild type (•), G10C (Δ), G11C (○), and G85C (▴). Fits for G10C, G85C, and wild type are to Equation 5, while the fit for G11C in the −54/99 background is to Equation 4; fits for closed symbols are solid lines, while fits for open symbols are dashed lines. Parameters are provided in Table 1.

FIGURE 5.

Effect of lowering the rate of transcription and of HDAg on wild-type cotranscriptional self-cleavage reactions. (A) Cotranscriptional self-cleavage in which the rate of transcription was decreased by lowering NTP concentrations. The rate of transcription was slowed by lowering the concentrations of all NTPs other than GTP in the transcription reaction to 10 μM. The three backgrounds are −54/99 (•, ○), −54/140 (■, □), and −54/155 (▲, △). Closed symbols are at 600 μM NTPs, while open symbols are at 10 μM NTPs (600 μM GTP). (B) Cotranscriptional self-cleavage in which the HDAg was added. The delta antigen was added to a final concentration of 3 μM in the background of 600 μM each NTP. The three backgrounds are −54/99 (•, ○), −54/140 (■, □), and −54/155 (▲, △). Closed symbols are without HDAg, while open symbols include 3 μM HDAg. Data for lower NTP concentrations and HDAg could not be fit to Equations 4 or 5. In these cases the lines are present only to help visualize data trends. Lines for closed symbols are solid, while fits for open symbols are dashed.

FIGURE 6.

Flanking sequence promotes efficient cotranscriptional ribozyme folding. (A) Rod model for the genomic HDV RNA, starting at −54. Ribozyme is base-paired to its complementary attenuator sequence. The site of cleavage between −1 and 1 is denoted with an arrow. The 5′-upstream nucleotides are shown forming the P(−1) pairing element since the attenuator sequence complementary to this region is not depicted. The 5′ boundary of the attenuator is defined as the region that begins base-pairing with the ribozyme (Fig. 3). The pyrimidine-rich nucleotide sequence for the upstream J(−1/1) element and P5_5′ are highlighted in yellow; these regions are complementary to the purine-rich downstream P5_3′ and anti J(−1/1) respectively, highlighted in blue. There is a small stretch of nucleotides between P2_3′ and P5_5′ that is composed of both R and Y residues (see Fig. 3). The actual nucleotide sequences for the two R-rich and Y-rich regions are shown in panel A. Ways in which flanking sequence conspires to avoid noncatalytic ribozyme pairings are illustrated in panel B and described in the text.