Genomic-Scale Interaction Involving Complementary Sequences in the Hepatitis C Virus 5'UTR Domain IIa and the RNA-Dependent RNA Polymerase Coding Region Promotes Efficient Virus Replication

Abstract

The hepatitis C virus (HCV) genome contains structured elements thought to play important regulatory roles in viral RNA translation and replication processes. We used in vitro RNA binding assays to map interactions involving the HCV 5'UTR and distal sequences in NS5B to examine their impact on viral RNA replication. The data revealed that 5'UTR nucleotides (nt) 95⁻110 in the internal ribosome entry site (IRES) domain IIa and matching nt sequence 8528⁻8543 located in the RNA-dependent RNA polymerase coding region NS5B, form a high-affinity RNA-RNA complex in vitro. This duplex is composed of both wobble and Watson-Crick base-pairings, with the latter shown to be essential to the formation of the high-affinity duplex. HCV genomic RNA constructs containing mutations in domain IIa nt 95⁻110 or within the genomic RNA location comprising nt 8528⁻8543 displayed, on average, 5-fold less intracellular HCV RNA and 6-fold less infectious progeny virus. HCV genomic constructs containing complementary mutations for IRES domain IIa nt 95⁻110 and NS5B nt 8528⁻8543 restored intracellular HCV RNA and progeny virus titers to levels obtained for parental virus RNA. We conclude that this long-range duplex interaction between the IRES domain IIa and NS5B nt 8528⁻8543 is essential for optimal virus replication.

Keywords: Flaviviridae; HCV IRES; NS5B; RNA folding; RNA stem-loop; circular RNA; long-distance RNA–RNA interaction; secondary structure.

Figure 1

Depiction of minimum free energy duplex model (top panel) and stem-loop structure plots for the IRES domain II (bottom left) and NS5B region 8490–8659 (bottom right). Domain IIa and NS5B sequence pairings (bottom, shaded circles), small mutation SM constructs (red), large mutation LM constructs (red and yellow) and adjacent stem-loop SL8670/J8640/8376 are shown [15].

Figure 2

In vitro complex between 5′UTR nt 1–290 and NS5B nt 8374–8676 RNAs. ³²P-labeled RNA probe (20 nM) was incubated with increased amounts of unlabeled RNA target (50–800 nM) in low magnesium binding buffer. Complexes were separated in a native 5% acrylamide gel buffered with TBM. Plots of % probe complex versus target concentration ± SE were derived from three separate experiments. See Materials and Methods Section 2.5 RNA Binding Assays for derivation of K_d and B_max values.

Figure 3

In vitro complex between 5′UTR nt 1–290 and NS5B nt 8374-8676 RNAs containing six and five mutations (LM) or two mutations (SM) in the 5′UTR nt 95–110 (•) or NS5B nt 8528-8543 (◦). ³²P-labeled RNA probe (20 nM) was incubated with two-fold increasing amounts of unlabeled RNA target (six lane gels = 0, 50, 100, 200, 400, 1000 nM) or (five lane gels = 0, 50, 100, 200, 400 nM) in low magnesium binding buffer. Plots of % probe complex versus target concentration ± SE were derived from three separate experiments and used to calculate K_d and B_max values as summarized in Table 1.

Figure 4

Disruption in the 5′UTR nt95–110:NS5B nt8528–8543 duplex reduces intracellular HCV RNA levels. (A) Huh-7.5 cells transfected with HCV genomic RNA (WT) or HCV genomic RNA containing mutations at 5′UTR nt 95–110 and NS5B nt 8528–8543 express NS5B antigen 24 h post-transfection. Scale bar = 50 µm. (B) Plots represent the average number of HCV RNA genome copies ± SE in 1 μg of cellular RNA at 48 h post-transfection. p-values ≤ 0.05 (*) or ≤ 0.01 (**) were determined by the Student’s t-test and represent four or more independent experiments.

Figure 5

Disruption in the 5′UTR nt 95–110:NS5B nt 8528–8543 duplex reduced progeny virus titers. Huh-7.5 cells were infected with HCV parental virus (WT) or HCV containing mutations at 5′UTR nt 95–110 and NS5B nt 8528–8543. Plots represent the average number of infectious virus titers as focus-forming units (FFU)/mL or HCV RNA genome copies ± SE in 1 μg of cellular RNA taken at 48 h post-infection. p-values ≤ 0.05 (*) or ≤ 0.01 (**) were determined by the Student’s t-test and represent six independent experiments.

Figure 6

Secondary structural elements located in the 5′UTR-Core and the NS5B-3′UTR. Tertiary interactions between 5′UTR IIa nt 95–110 and NS5B SL8490 nt 8528–8543 are indicated by the red line. Tertiary interactions of SL9326 with the 3′UTR X-tail SL2 are indicated by the green line. SL9326 interaction with NS5B SL9074 and the 3′UTR Variable Region (VR, VSL2) is indicated by the blue and turquoise lines, respectively. SL9326 interaction with the IRES IIId apical loop is indicated by the yellow line. Listed NS5B-3′UTR stem-loop nomenclature is based on Adams RL et al. 2017, Figure 1 [15].