An essential RNA element resides in a central region of hepatitis E virus ORF2

Abstract

Hepatitis E virus (genus Hepevirus, family Hepeviridae) is one of the most important causes of acute hepatitis in adults, particularly among pregnant women, throughout Asia and Africa where mortality rates can be 20-30 %. Hepatitis E virus has a single-stranded positive-sense RNA genome that contains three translated ORFs. The two 3' ORFs are translated from a subgenomic RNA. Functional RNA elements have been identified in and adjacent to the genomic 5' and 3' UTRs and in and around the intergenic region. Here we describe an additional RNA element that is located in a central region of ORF2. The RNA element is predicted to fold into two highly conserved stem-loop structures, ISL1 and ISL2. Mutations that disrupt the predicted structures, without altering the encoded amino acid sequence, result in a drastic reduction in capsid protein synthesis. This indicates that the RNA element plays an important role in one of the early steps of virus replication. The structures were further investigated using a replicon that expresses Gaussia luciferase in place of the capsid protein. Single mutations in ISL2 severely reduced luciferase expression, but a pair of compensatory mutations that were predicted to restore the ISL2 structure, restored luciferase expression to near-WT levels, thus lending experimental support to the predicted structure. Nonetheless the precise role of the ISL1+ISL2 element remains unknown.

Fig. 1.

HEV genome organization. (a) Map of the ~7.2 kb genome. ORF1 is translated from the genomic RNA and encodes the replication protein domains including the RNA-dependent RNA polymerase. ORF2 and ORF3 are translated from a sgRNA. ORF2 encodes the capsid protein. (b) Conservation at synonymous sites in alignments of 185 ORF1 and 205 ORF2 nucleotide sequences, using a 5-codon sliding window. The putative packaging signal is according to Surjit et al. (2004). The lower panels show the ratio of the observed number of substitutions to the number expected under a null model of neutral evolution at synonymous sites, while the upper panels show the corresponding P-values. In order to map the conservation statistic onto the coordinates of a specific sequence in the alignment, all alignment columns with gaps in a chosen reference sequence (viz. NC_001434) were removed (note that the ORF2 alignment had no gaps in the reference sequence). Note, as expected, the extreme reduction in ORF2-frame synonymous substitutions in the region where ORF2 overlaps with ORF3. (c) Zoom-in of four regions that show particularly pronounced conservation at ORF1- or ORF2-frame synonymous sites. The dashed line indicates a P = 0.05 threshold after applying a rough correction for multiple testing (viz. 0.05/[2355 codons/5-codon window] ~ 1.1×10⁻⁴).

Fig. 2.

Identification of potential RNA secondary structure in the central conserved region in ORF2. (a) Extracts from representative sequences showing the predicted ISL1 (yellow) and ISL2 (green) stem–loop structures in the conserved region. Predicted base pairings are indicated with parentheses. Single substitutions that preserve the predicted base pairings (e.g. G-C to G-U) are indicated with cyan. Paired substitutions that preserve the predicted base pairings are indicated with pink. GenBank accession numbers are shown at left. Genotype and isolation source species are shown at right. See Johne et al. (2012) for a phylogenetic tree. Numbers 380 and 413 below indicate codon positions in ORF2. Note that, in some isolates, ISL1 and ISL2 can be basally extended by, respectively, another one or two base pairings. The corresponding synonymous site conservation P-values from Fig. 1 are shown at bottom. See also Fig. S1 for the ISL1, ISL2 and flanking sequences from all 205 ORF2 sequences used. (b) Predicted secondary structure of the ISL1+ISL2 and flanking region from isolate Kp6. Structures ISL1 and ISL2 (left) are based on comparative genomics. In Kp6 and some other isolates, however, the minimum free energy fold (right) contains ISL2 and the peripheral base pairings (indicated with orange/salmon), but ISL1 is disrupted and replaced by a new stem–loop, ISL3. Nucleotides that were altered in the M1 mutant are indicated in bold. Nucleotide coordinates refer to GenBank accession JQ679013. Nucleotides 6508 to 6582 correspond to the region inserted into the HVR. (c) In the M1 mutant, many synonymous mutations (cyan) were introduced within the two regions showing highest conservation (corresponding to ISL1 and ISL2), and additional mutations were introduced to also disrupt the potential ISL3. The colour coding in the WT sequence corresponds to the structure predictions in Fig. 2b. Numbers at left indicate genomic coordinates of the first nucleotide in each line.

Fig. 3.

Analysis of ISL1+ISL2 mutants. (a) Representative example of IF microscopy of S10-3 cells stained for capsid protein (green) and nuclei (blue) 4 days after transfection with WT or M1 genomes. (b) Schematic map of WT virus and the GLuc construct indicating the position of the hypervariable region (HVR) and the inserted Gaussia luciferase gene relative to ISL1 and ISL2. (c) Flow cytometry of S10-3 cells immunostained for capsid protein 3 days after transfection with WT/WT or M1/WT viruses (four clones of each). Bars indicate the SD of three measurements.

Fig. 4.

Analysis of virus genomes expressing luciferase. (a) S10-3 cells were transfected with the indicated virus genomes expressing Gaussia luciferase and aliquots of medium were tested for luciferase activity at 2 days post-transfection, at which point the harvested medium had been on the cells for 24 h. Bars indicate the SD of three transfections, representing the mean of three aliquots per transfection. WT and M1 refer to the WT and mutant sequences in Fig. 2(c) and GAD is a polymerase mutant. (b) Schematic diagram indicating single substitutions (pink) that, separately, are predicted to disrupt stem–loop ISL2 (M3 and M5 mutants) and, when combined, are predicted to restore the stem–loop structure but with a reversed apical base pairing (M35 mutant). (c) S10-3 cells were transfected with the indicated virus genomes expressing luciferase and aliquots of medium harvested at 5 days post-transfection were assayed for luciferase activity, at which point the harvested medium had been on the cells for 72 h. Bars indicate the SD of three transfections, representing the mean of three aliquots per transfection.