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.
Highlights
Hepatitis C virus (HCV) is a hepatotropic RNA virus of the genus hepacivirus in the Flaviviridae family [1]
RNAcofold predicts an optimal minimal free energy (MFE) of -24.0 for a duplex model comprised of HCV internal ribosomal entry site (IRES) nt 95–110 and NS5B nt 8528–8543, and whose interaction is composed of wobble and Watson-Crick base-pairs (Figure 1, hatched boxed)
Small mutations in 50 untranslated regions (UTRs)(95–110) and NS5B(8528–8543) showed a 4.5- and 5.8-fold increase in Kd values as compared to with HCV genomic RNA (WT) HCV RNA (Figure 3 and Table 1). These findings indicate that mutations predicted to lessen WT duplex MFE (i.e., −24) greatly decreased duplex affinity (Table 1)
Summary
Hepatitis C virus (HCV) is a hepatotropic RNA virus of the genus hepacivirus in the Flaviviridae family [1]. Members of this family have a single-stranded RNA genome of positive polarity containing a single long open reading frame (ORF) flanked by highly structured 50 and 30 untranslated regions (UTRs). Following HCV entry into the host cell and cytoplasmic release of the 9.6 kb HCV RNA genome, the 50 UTR internal ribosomal entry site (IRES) initiates polyprotein synthesis from the large ORF that is cleaved co- and post-translationally into three virion structural proteins and seven nonstructural (NS) replication-essential proteins [2]. Like the RNA genomes of other flaviviruses, HCV usurps the cell’s normal mRNA structural signals by forming local or distal genomic linkages that allow the virus to replicate through initial
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