Secondary structure of the 3' terminus of hepatitis C virus minus-strand RNA

The 3'-terminal ends of both the positive and negative strands of the hepatitis C virus (HCV) RNA, the latter being the replicative intermediate, are most likely the initiation sites for replication by the viral RNA-dependent RNA polymerase, NS5B. The structural features of the very conserved 3' plus [(+)] strand untranslated region [3' (+) UTR] are well established (K. J. Blight and C. M. Rice, J. Virol. 71:7345-7352, 1997). However, little information is available concerning the 3' end of the minus [(-)] strand RNA. In the present work, we used chemical and enzymatic probing to investigate the conformation of that region, which is complementary to the 5' (+) UTR and the first 74 nucleotides of the HCV polyprotein coding sequence. By combining our experimental data with computer predictions, we have derived a secondary-structure model of this region. In our model, the last 220 nucleotides, where initiation of the (+) strand RNA synthesis presumably takes place, fold into five stable stem-loops, forming domain I. Domain I is linked to an overall less stable structure, named domain II, containing the sequences complementary to the pseudoknot of the internal ribosomal entry site in the 5' (+) UTR. Our results show that, even though the (-) strand 3'-terminal region has the antisense sequence of the 5' (+) UTR, it does not fold into its mirror image. Interestingly, comparison of the replication initiation sites on both strands reveals common structural features that may play key functions in the replication process.     

Structure of the 3' terminus of the hepatitis C virus genome.

Hepatitis C virus (HCV), a positive-strand RNA virus, has been considered to have a poly(U) stretch at the 3' terminus of the genome. We previously found a novel 98-nucleotide sequence downstream from the poly(U) stretch on the HCV genome by primer extension analysis of the 5' end of the antigenomic-strand RNA in infected liver (T. Tanaka, N. Kato, M.-J. Cho, and K. Shimotohno, Biochem. Biophys. Res. Commun. 215: 744-749, 1995). Here, we show that the novel sequence is a highly conserved 3' tail of the HCV genome. We repeated primer extension analyses with four HCV-infected liver samples and found the 98-nucleotide sequence in all the samples. Furthermore, experiments in which RNA oligonucleotide was ligated to the 3' end of the HCV genome existing in infectious serum revealed nearly identical 3' termini with no extra sequence downstream from the 98-nucleotide sequence, suggesting that this sequence is the tail of the HCV genome. This tail sequence was highly conserved among individuals and even between the two most genetically distant HCV types, II/1b and III/2a. Computer modeling predicted that the tail sequence can form a conserved stem-and-loop structure. These results suggest that the novel 3' tail is a common structure of the HCV genome that plays an important role in initiation of genomic replication.     

Structural characterization of the highly conserved 98-base sequence at the 3' end of HCV RNA genome and the complementary sequence located at the 5' end of the replicative viral strand

Oligoribonucleotides that corresponded to the X regions of the (+) and (−) polarity strands of HCV RNA, as well as several shorter oligomers comprising defined stem-loop motifs of their predicted secondary structure models, were analyzed by Pb²⁺-induced cleavage, partial digestion with specific nucleases and chemical modification. Patterns characteristic of the motifs were compared with those obtained for the full-length molecules and on the basis of such ‘structural fingerprinting’ conclusions concerning folding of regions X were formulated. It turned out that the secondary structure model of X(+) RNA proposed earlier, the three-stem-loop model composed of hairpins SL1, SL2 and SL3, was only partially consistent with our experimental data. We confirmed the presence of SL1 and SL3 motifs and showed that the single-stranded stretch adjacent to the earlier proposed hairpin SL2 contributed to the folding of that region. It seemed to be arranged into two hairpins, which might form a hypothetical pseudoknot by changing their base-pairing systems. These data were discussed in terms of their possible biological significance. On the other hand, analysis of the X(−) RNA and its sub-fragments supported a three-stem-loop secondary structure model for this RNA.     

Kissing-loop interaction in the 3' end of the hepatitis C virus genome essential for RNA replication

The hepatitis C virus (HCV) is a positive-strand RNA virus belonging to the Flaviviridae. Its genome carries at either end highly conserved nontranslated regions (NTRs) containing cis-acting RNA elements that are crucial for replication. In this study, we identified a novel RNA element within the NS5B coding sequence that is indispensable for replication. By using secondary structure prediction and nuclear magnetic resonance spectroscopy, we found that this RNA element, designated 5BSL3.2 by analogy to a recent report (S. You, D. D. Stump, A. D. Branch, and C. M. Rice, J. Virol. 78:1352-1366, 2004), consists of an 8-bp lower and a 6-bp upper stem, an 8-nucleotide-long bulge, and a 12-nucleotide-long upper loop. Mutational disruption of 5BSL3.2 structure blocked RNA replication, which could be restored when an intact copy of this RNA element was inserted into the 3' NTR. By using this replicon design, we mapped the elements in 5BSL3.2 that are critical for RNA replication. Most importantly, we discovered a nucleotide sequence complementarity between the upper loop of this RNA element and the loop region of stem-loop 2 in the 3' NTR. Mismatches introduced into the loops inhibited RNA replication, which could be rescued when complementarity was restored. These data provide strong evidence for a pseudoknot structure at the 3' end of the HCV genome that is essential for replication.     

The nucleotide sequence of an untranslated but conserved domain at the 3' end of the avian sarcoma virus genome.

The genomes of numerous avian retroviruses contain at their 3' termini a conserved domain denoted "c". The precise boundaries and function of "c" have been enigmas. In an effort to resolve these issues, we determined the sequence of over 900 nucleotides at the 3' end of the genome of the Schmidt-Ruppin subgroup A strain of avian sarcoma virus (ASV). We obtained the sequence from a suitable fragment of ASV DNA that had cloned into the single-stranded DNA phage M13mp2. Computer-assisted analysis of the sequence revealed the following structural features: i) the length of "c" - 473 nucleotides; ii) the 3' terminal domain of src, ending in an amber codon at the 5'boundary of "c"; iii) terminator codons that preclude continuous translation from "c"; iv) suitably located sequences that may serve as signals for the initiation of viral RNA synthesis and for the processing and/or polyadenylation of viral mRNA; v) a repeated sequence that flanks src and that could facilitate deletion of this gene; vi) repeated sequences within "c"; and vii) unexplained homologies between sequences in "c" and sequences in several other nucleic acids, including the 5' terminal domain of the ASV genome, tRNATrp and its inversion, the complement of tRNATrp and its inversion, and the 18S RNA of eukaryotic ribosomes. We conclude that "c" probably does not encode a protein, but its sequence may nevertheless serve several essential functions in viral replication.     

Biochemical and genetic evidence for a pseudoknot structure at the 3' terminus of the poliovirus RNA genome and its role in viral RNA amplification.

The sequences in the plus-stranded poliovirus RNA genome that dictate the specific amplification of viral RNA in infected cells remain unknown. We have analyzed the structure of the 3' noncoding region of the viral genome by thermodynamic-based structure calculation and by chemical and enzymatic probing of in vitro-synthesized RNAs and provide evidence for the existence of an RNA pseudoknot structure in this region. To explore the functional significance of this structure, revertants of a mutant bearing a lesion in the proposed pseudoknot and exhibiting a temperature-sensitive defect in viral RNA synthesis were isolated and mapped. The results of this genetic analysis established a correlation between the structure of the 3' terminus of the viral RNA and its function in vivo in RNA amplification. Furthermore, phylogenetic analysis indicated that a similar structure could be formed in coxsackievirus B1, a related enterovirus, which further supports a role for the pseudoknot structure in viral RNA amplification in infected cells.     

3' nontranslated RNA signals required for replication of hepatitis C virus RNA

We describe a mutational analysis of the 3' nontranslated RNA (3'NTR) signals required for replication of subgenomic hepatitis C virus (HCV) RNAs. A series of deletion mutants was constructed within the background of an HCV-N replicon that induces the expression of secreted alkaline phosphatase in order to examine the requirements for each of the three domains comprising the 3'NTR, namely, the highly conserved 3' terminal 98-nucleotide (nt) segment (3'X), an upstream poly(U)-poly(UC) [poly(U/UC)] tract, and the variable region (VR) located at the 5' end of the 3'NTR. Each of these domains was found to contribute to efficient replication of the viral RNA in transiently transfected hepatoma cells. Replication was not detected when any of the three putative stem-loop structures within the 3'X region were deleted. Similarly, complete deletion of the poly(U/UC) tract abolished replication. Replacement of a minimum of 50 to 62 nt of poly(U/UC) sequence was required for detectable RNA replication when the native sequence was restored in a stepwise fashion from its 3' end. Lengthier poly(U/UC) sequences, and possibly pure homopolymeric poly(U) tracts, were associated with more efficient RNA amplification. Finally, while multiple deletion mutations were tolerated within VR, each led to a partial loss of replication capacity. The impaired replication capacity of the deletion mutants could not be explained by reduced translational activity or by decreased stability of the RNA, suggesting that each of these mutations may impair recognition of the RNA by the viral replicase during an early step in negative-strand RNA synthesis. The results indicate that the 3'-most 150 nt of the HCV-N genome [the 3'X region and the 3' 52 nt of the poly(U/UC) tract] contain RNA signals that are essential for replication, while the remainder of the 3'NTR plays a facilitating role in replication but is not absolutely required.     

The tRNA-like structure at the 3' terminus of turnip yellow mosaic virus RNA. Differences and similarities with canonical tRNA.

The 3' terminus of TYMV RNA, which possesses tRNA-like properties, has been studied. A 3' terminal fragment of 112 nucleotides was obtained by cleavage with RNase H after hybridization of a synthetic oligodeoxynucleotide to the viral RNA. The accessibility of cytidine and adenosine residues was probed with chemical modification. Enzymatic digestion studies were performed with RNase T1, nuclease S1 and the double-strand specific RNase from the venom of the cobra Naja naja oxiana. A model is proposed for the secondary structure of the 3' terminal region of TYMV RNA comprising 86 nucleotides. The main feature of this secondary structure is the absence of a conventional acceptor stem as present in canonical tRNA. However, the terminal 42 nucleotides can be folded in a tertiary structure which bears strong resemblance with the acceptor arm of canonical tRNA. Comparison of this region of TYMV RNA with that of other RNAs from both the tymovirus group and the tobamovirus group gives support to our proposal for such a three-dimensional arrangement. The consequences for the recognition by TYMV RNA of tRNA-specific enzymes is discussed.     

Molecular characterization of the 3' terminus of the simian hemorrhagic fever virus genome.

The 3' end of the simian hemorrhagic fever virus (SHFV) single-stranded RNA genome was cloned and sequenced. Adjacent to the 3' poly(A) tract, we identified a 76-nucleotide noncoding region preceded by two overlapping reading frames (ORFs). The ultimate 3' ORF of the viral genome encodes the capsid protein, and the penultimate ORF encodes the smallest SHFV envelope protein. These two ORFs overlap each other by 26 nucleotides. Northern (RNA) blot hybridization analyses of cytoplasmic RNA extracts from SHFV-infected MA-104 cells with gene-specific probes revealed the presence of full-length genomic RNA as well as six subgenomic SHFV-specific mRNA species. The subgenomic mRNAs are 3' coterminal. In its virion morphology and size, genome structure and length, and replication strategy, SHFV is most similar to lactate dehydrogenase-elevating virus, equine arteritis virus, and porcine reproductive and respiratory syndrome virus.     

Base pairing between hepatitis C virus RNA and microRNA 122 3' of its seed sequence is essential for genome stabilization and production of infectious virus

MicroRNA 122 (miR-122) facilitates hepatitis C virus (HCV) replication by recruiting an RNA-induced silencing complex (RISC)-like complex containing argonaute 2 (Ago2) to the 5' end of the HCV genome, thereby stabilizing the viral RNA. This requires base pairing between the miR-122 "seed sequence" (nucleotides [nt] 2 to 8) and two sequences near the 5' end of the HCV RNA: S1 (nt 22 to 28) and S2 (nt 38 to 43). However, recent reports suggest that additional base pair interactions occur between HCV RNA and miR-122. We searched 606 sequences from a public database (genotypes 1 to 6) and identified two conserved, putatively single-stranded RNA segments, upstream of S1 (nt 2 and 3) and S2 (nt 30 to 34), with potential for base pairing to miR-122 (nt 15 and 16 and nt 13 to 16, respectively). Mutagenesis and genetic complementation experiments confirmed that HCV nt 2 and 3 pair with nt 15 and 16 of miR-122 bound to S1, while HCV nt 30 to 33 pair with nt 13 to 16 of miR-122 at S2. In genotype 1 and 6 HCV, nt 4 also base pairs with nt 14 of miR-122. These 3' supplementary base pair interactions of miR-122 are functionally important and are required for Ago2 recruitment to HCV RNA by miR-122, miR-122-mediated stabilization of HCV RNA, and production of infectious virus. However, while complementary mutations at HCV nt 30 and 31 efficiently rescued the activity of a 15C,16C miR-122 mutant targeting S2, similar mutations at nt 2 and 3 failed to rescue Ago2 recruitment at S1. These data add to the current understanding of miR-122 interactions with HCV RNA but indicate that base pairing between miR-122 and the 5' 43 nt of the HCV genome is more complex than suggested by existing models.     

Analysis of the RNA chaperoning activity of the hepatitis C virus core protein on the conserved 3'X region of the viral genome

The core protein of hepatitis c virus (HCV) is a structural protein with potent RNA chaperoning activities mediated by its hydrophilic N-terminal domain D1, which is thought to play a key role in HCV replication. To further characterize the core chaperoning properties, we studied the interactions between core D1 and the conserved HCV 3'X genomic region required for genome replication. To this end, we monitored the real-time annealing kinetics of native and mutated fluorescently labelled 16-nt palindromic sequence (DLS) and 27-nt Stem Loop II (SL2) from X with their respective complementary sequences. Core D1 and peptides consisting of the core basic domains were found to promote both annealing reactions and partly switch the loop-loop interaction pathway, which predominates in the absence of peptide, towards a pathway involving the stem termini. The chaperone properties of the core D1 peptides were found to be mediated through interaction of their basic clusters with the oligonucleotide phosphate groups, in line with the absence of high affinity site for core on HCV genomic RNA. The core ability to facilitate the interconversion between different RNA structures may explain how this protein regulates RNA structural transitions during HCV replication.     

cis-acting RNA signals in the NS5B C-terminal coding sequence of the hepatitis C virus genome

The cis-replicating RNA elements in the 5' and 3' nontranslated regions (NTRs) of the hepatitis C virus (HCV) genome have been thoroughly studied before. However, no cis-replicating elements have been identified in the coding sequences of the HCV polyprotein until very recently. The existence of highly conserved and stable stem-loop structures in the RNA polymerase NS5B coding sequence, however, has been previously predicted (A. Tuplin, J. Wood, D. J. Evans, A. H. Patel, and P. Simmonds, RNA 8:824-841, 2002). We have selected for our studies a 249-nt-long RNA segment in the C-terminal NS5B coding region (NS5BCR), which is predicted to form four stable stem-loop structures (SL-IV to SL-VII). By deletion and mutational analyses of the RNA structures, we have determined that two of the stem-loops (SL-V and SL-VI) are essential for replication of the HCV subgenomic replicon in Huh-7 cells. Mutations in the loop and the top of the stem of these RNA elements abolished replicon RNA synthesis but had no effect on translation. In vitro gel shift and filter-binding assays revealed that purified NS5B specifically binds to SL-V. The NS5B-RNA complexes were specifically competed away by unlabeled homologous RNA, to a small extent by 3' NTR RNA, and only poorly by 5' NTR RNA. The other two stem-loops (SL-IV and SL-VII) of the NS5BCR domain were found to be important but not essential for colony formation by the subgenomic replicon. The precise function(s) of these cis-acting RNA elements is not known.     

Alphavirus RNA genome repair and evolution: molecular characterization of infectious sindbis virus isolates lacking a known conserved motif at the 3' end of the genome

The 3' nontranslated region of the genomes of Sindbis virus (SIN) and other alphaviruses carries several repeat sequence elements (RSEs) as well as a 19-nucleotide (nt) conserved sequence element (3'CSE). The 3'CSE and the adjoining poly(A) tail of the SIN genome are thought to act as viral promoters for negative-sense RNA synthesis and genome replication. Eight different SIN isolates that carry altered 3'CSEs were studied in detail to evaluate the role of the 3'CSE in genome replication. The salient findings of this study as it applies to SIN infection of BHK cells are as follows: i) the classical 19-nt 3'CSE of the SIN genome is not essential for genome replication, long-term stability, or packaging; ii) compensatory amino acid or nucleotide changes within the SIN genomes are not required to counteract base changes in the 3' terminal motifs of the SIN genome; iii) the 5' 1-kb regions of all SIN genomes, regardless of the differences in 3' terminal motifs, do not undergo any base changes even after 18 passages; iv) although extensive addition of AU-rich motifs occurs in the SIN genomes carrying defective 3'CSE, these are not essential for genome viability or function; and v) the newly added AU-rich motifs are composed predominantly of RSEs. These findings are consistent with the idea that the 3' terminal AU-rich motifs of the SIN genomes do not bind directly to the viral polymerase and that cellular proteins with broad AU-rich binding specificity may mediate this interaction. In addition to the classical 3'CSE, other RNA motifs located elsewhere in the SIN genome must play a major role in template selection by the SIN RNA polymerase.     

The structure of a rigorously conserved RNA element within the SARS virus genome

We have solved the three-dimensional crystal structure of the stem-loop II motif (s2m) RNA element of the SARS virus genome to 2.7-Å resolution. SARS and related coronaviruses and astroviruses all possess a motif at the 3′ end of their RNA genomes, called the s2m, whose pathogenic importance is inferred from its rigorous sequence conservation in an otherwise rapidly mutable RNA genome. We find that this extreme conservation is clearly explained by the requirement to form a highly structured RNA whose unique tertiary structure includes a sharp 90° kink of the helix axis and several novel longer-range tertiary interactions. The tertiary base interactions create a tunnel that runs perpendicular to the main helical axis whose interior is negatively charged and binds two magnesium ions. These unusual features likely form interaction surfaces with conserved host cell components or other reactive sites required for virus function. Based on its conservation in viral pathogen genomes and its absence in the human genome, we suggest that these unusual structural features in the s2m RNA element are attractive targets for the design of anti-viral therapeutic agents. Structural genomics has sought to deduce protein function based on three-dimensional homology. Here we have extended this approach to RNA by proposing potential functions for a rigorously conserved set of RNA tertiary structural interactions that occur within the SARS RNA genome itself. Based on tertiary structural comparisons, we propose the s2m RNA binds one or more proteins possessing an oligomer-binding-like fold, and we suggest a possible mechanism for SARS viral RNA hijacking of host protein synthesis, both based upon observed s2m RNA macromolecular mimicry of a relevant ribosomal RNA fold.     

Identification of a novel sequence at the 3' end of the GB virus B genome

GB virus B (GBV-B) is a virus of the family Flaviviridae that infects small primates (Saguinus sp. [tamarins]) and shows similarities to hepatitis C virus (HCV) in genome organization, protein function, tissue tropism, and pathogenicity. This suggests the possibility of using tamarins infected by GBV-B or GBV-B/HCV chimeric viruses as a surrogate animal model of HCV infection. To achieve the construction of such chimeric viruses, it is essential to produce a complete and infectious GBV-B genomic RNA. We have identified a novel sequence at the 3' end of the GBV-B genome and show that it can be arranged in a secondary structure resembling that of the 3' end of the HCV genome, which is known to be essential for infectivity.