3' RNA elements in hepatitis C virus replication: kissing partners and long poly(U)

The hepatitis C virus (HCV) genomic RNA possesses conserved structural elements that are essential for its replication. The 3′ nontranslated region (NTR) contains several of these elements: a variable region, the poly(U/UC) tract, and a highly conserved 3′ X tail, consisting of stem-loop 1 (SL1), SL2, and SL3. Studies of drug-selected, cell culture-adapted subgenomic replicons have indicated that an RNA element within the NS5B coding region, 5BSL3.2, forms a functional kissing-loop tertiary structure with part of the 3′ NTR, 3′ SL2. Recent advances now allow the efficient propagation of unadapted HCV genomes in the context of a complete infectious life cycle (HCV cell culture [HCVcc]). Using this system, we determine that the kissing-loop interaction between 5BSL3.2 and 3′ SL2 is required for replication in the genotype 2a HCVcc context. Remarkably, the overall integrity of the 5BSL3 cruciform is not an absolute requirement for the kissing-loop interaction, suggesting a model in which trans-acting factor(s) that stabilize this interaction may interact initially with the 3′ X tail rather than 5BSL3. The length and composition of the poly(U/UC) tract were also critical determinants of HCVcc replication, with a length of 33 consecutive U residues required for maximal RNA amplification. Interrupting the U homopolymer with C residues was deleterious, implicating a trans-acting factor with a preference for U over mixed pyrimidine nucleotides. Finally, we show that both the poly(U) and kissing-loop RNA elements can function outside of their normal genome contexts. This suggests that the poly(U/UC) tract does not function simply as an unstructured spacer to position the kissing-loop elements.     

Hsp90 Interacts Specifically with Viral RNA and Differentially Regulates Replication Initiation of Bamboo mosaic virus and Associated Satellite RNA

Host factors play crucial roles in the replication of plus-strand RNA viruses. In this report, a heat shock protein 90 homologue of Nicotiana benthamiana, NbHsp90, was identified in association with partially purified replicase complexes from BaMV-infected tissue, and shown to specifically interact with the 3′ untranslated region (3′ UTR) of BaMV genomic RNA, but not with the 3′ UTR of BaMV-associated satellite RNA (satBaMV RNA) or that of genomic RNA of other viruses, such as Potato virus X (PVX) or Cucumber mosaic virus (CMV). Mutational analyses revealed that the interaction occurs between the middle domain of NbHsp90 and domain E of the BaMV 3′ UTR. The knockdown or inhibition of NbHsp90 suppressed BaMV infectivity, but not that of satBaMV RNA, PVX, or CMV in N. benthamiana. Time-course analysis further revealed that the inhibitory effect of 17-AAG is significant only during the immediate early stages of BaMV replication. Moreover, yeast two-hybrid and GST pull-down assays demonstrated the existence of an interaction between NbHsp90 and the BaMV RNA-dependent RNA polymerase. These results reveal a novel role for NbHsp90 in the selective enhancement of BaMV replication, most likely through direct interaction with the 3′ UTR of BaMV RNA during the initiation of BaMV RNA replication.     

A complex RNA motif defined by three discontinuous 5-nucleotide-long strands is essential for Flavivirus RNA replication

Tertiary or higher-order RNA motifs that regulate replication of positive-strand RNA viruses are as yet poorly understood. Using Japanese encephalitis virus (JEV), we now show that a key element in JEV RNA replication is a complex RNA motif that includes a string of three discontinuous complementary sequences (TDCS). The TDCS consists of three 5-nt-long strands, the left (L) strand upstream of the translation initiator AUG adjacent to the 5′-end of the genome, and the middle (M) and right (R) strands corresponding to the base of the Flavivirus-conserved 3′ stem–loop structure near the 3′-end of the RNA. The three strands are arranged in an antiparallel configuration, with two sets of base-pairing interactions creating L-M and M-R duplexes. Disrupting either or both of these duplex regions of TDCS completely abolished RNA replication, whereas reconstructing both duplex regions, albeit with mutated sequences, fully restored RNA replication. Modeling of replication-competent genomes recovered from a large pool of pseudorevertants originating from six replication-incompetent TDCS mutants suggests that both duplex base-pairing potentials of TDCS are required for RNA replication. In all cases, acquisition of novel sequences within the 3′M-R duplex facilitated a long-range RNA–RNA interaction of its 3′M strand with either the authentic 5′L strand or its alternative (invariably located upstream of the 5′ initiator), thereby restoring replicability. We also found that a TDCS homolog is conserved in other flaviviruses. These data suggest that two duplex base-pairings defined by the TDCS play an essential regulatory role in a key step(s) of Flavivirus RNA replication.     

In Vivo Addition of Poly(A) Tail and AU-Rich Sequences to the 3′ Terminus of the Sindbis Virus RNA Genome: a Novel 3′-End Repair Pathway

Alphaviruses are mosquito-transmitted RNA viruses that cause important diseases in both humans and livestock. Sindbis virus (SIN), the type species of the alphavirus genus, carries a 11.7-kb positive-sense RNA genome which is capped at its 5′ end and polyadenylated at its 3′ end. The 3′ nontranslated region (3′NTR) of the SIN genome carries many AU-rich motifs, including a 19-nucleotide (nt) conserved element (3′CSE) and a poly(A) tail. This 3′CSE and the adjoining poly(A) tail are believed to regulate the synthesis of negative-sense RNA and genome replication in vivo. We have recently demonstrated that the SIN genome lacking the poly(A) tail was infectious and that de novo polyadenylation could occur in vivo (K. R. Hill, M. Hajjou, J. Hu, and R. Raju, J. Virol. 71:2693–2704, 1997). Here, we demonstrate that the 3′-terminal 29-nt region of the SIN genome carries a signal for possible cytoplasmic polyadenylation. To further investigate the polyadenylation signals within the 3′NTR, we generated a battery of mutant genomes with mutations in the 3′NTR and tested their ability to generate infectious virus and undergo 3′ polyadenylation in vivo. Engineered SIN genomes with terminal deletions within the 19-nt 3′CSE were infectious and regained their poly(A) tail. Also, a SIN genome carrying the poly(A) tail but lacking a part or the entire 19-nt 3′CSE was also infectious. Sequence analysis of viruses generated from these engineered SIN genomes demonstrated the addition of a variety of AU-rich sequence motifs just adjacent to the poly(A) tail. The addition of AU-rich motifs to the mutant SIN genomes appears to require the presence of a significant portion of the 3′NTR. These results indicate the ability of alphavirus RNAs to undergo 3′ repair and the existence of a pathway for the addition of AU-rich sequences and a poly(A) tail to their 3′ end in the infected host cell. Most importantly, these results indicate the ability of alphavirus replication machinery to use a multitude of AU-rich RNA sequences abutted by a poly(A) motif as promoters for negative-sense RNA synthesis and genome replication in vivo. The possible roles of cytoplasmic polyadenylation machinery, terminal transferase-like enzymes, and the viral polymerase in the terminal repair processes are discussed.     

Amiloride derivatives inhibit coxsackievirus B3 RNA replication

Amiloride derivatives are known blockers of the cellular Na⁺/H⁺ exchanger and the epithelial Na⁺ channel. More recent studies demonstrate that they also inhibit ion channels formed by a number of viral proteins. We previously reported that 5-(N-ethyl-N-isopropyl)amiloride (EIPA) modestly inhibits intracellular replication and, to a larger extent, release of human rhinovirus 2 (HRV2) (E. V. Gazina, D. N. Harrison, M. Jefferies, H. Tan, D. Williams, D. A. Anderson and S. Petrou, Antiviral Res. 67:98-106, 2005). Here, we demonstrate that amiloride and EIPA strongly inhibit coxsackievirus B3 (CVB3) RNA replication and do not inhibit CVB3 release, in contrast to our previous findings on HRV2. Passaging of plasmid-derived CVB3 in the presence of amiloride generated mutant viruses with amino acid substitutions in position 299 or 372 of the CVB3 polymerase. Introduction of either of these mutations into the CVB3 plasmid produced resistance to amiloride and EIPA, suggesting that they act as inhibitors of CVB3 polymerase, a novel mechanism of antiviral activity for these compounds.     

RNA Polymerase Activity and Specific RNA Structure Are Required for Efficient HCV Replication in Cultured Cells

We have previously reported that the NS3 helicase (N3H) and NS5B-to-3′X (N5BX) regions are important for the efficient replication of hepatitis C virus (HCV) strain JFH-1 and viral production in HuH-7 cells. In the current study, we investigated the relationships between HCV genome replication, virus production, and the structure of N5BX. We found that the Q377R, A450S, S455N, R517K, and Y561F mutations in the NS5B region resulted in up-regulation of J6CF NS5B polymerase activity in vitro. However, the activation effects of these mutations on viral RNA replication and virus production with JFH-1 N3H appeared to differ. In the presence of the N3H region and 3′ untranslated region (UTR) of JFH-1, A450S, R517K, and Y561F together were sufficient to confer HCV genome replication activity and virus production ability to J6CF in cultured cells. Y561F was also involved in the kissing-loop interaction between SL3.2 in the NS5B region and SL2 in the 3′X region. We next analyzed the 3′ structure of HCV genome RNA. The shorter polyU/UC tracts of JFH-1 resulted in more efficient RNA replication than J6CF. Furthermore, 9458G in the JFH-1 variable region (VR) was responsible for RNA replication activity because of its RNA structures. In conclusion, N3H, high polymerase activity, enhanced kissing-loop interactions, and optimal viral RNA structure in the 3′UTR were required for J6CF replication in cultured cells.     

La autoantigen is required for the internal ribosome entry site-mediated translation of Coxsackievirus B3 RNA

Translation initiation in Coxsackievirus B3 (CVB3) occurs via ribosome binding to an internal ribosome entry site (IRES) located in the 5′-untranslated region (UTR) of the viral RNA. This unique mechanism of translation initiation requires various trans-acting factors from the host. We show that human La autoantigen (La) binds to the CVB3 5′-UTR and also demonstrate the dose-dependent effect of exogenously added La protein in stimulating CVB3 IRES-mediated translation. The requirement of La for CVB3 IRES mediated translation has been further demonstrated by inhibition of translation as a result of sequestering La and its restoration by exogenous addition of recombinant La protein. The abundance of La protein in various mouse tissue extracts has been probed using anti-La antibody. Pancreatic tissue, a target organ for CVB3 infection, was found to have a large abundance of La protein which was demonstrated to interact with the CVB3 5′-UTR. Furthermore, exogenous addition of pancreas extract to in vitro translation reactions resulted in a dose dependent stimulation of CVB3 IRES-mediated translation. These observations indicate the role of La in CVB3 IRES-mediated translation, and suggest its possible involvement in the efficient translation of the viral RNA in the pancreas.     

Small RNA Modules Confer Different Stabilities and Interact Differently with Multiple Targets

Bacterial Hfq-associated small regulatory RNAs (sRNAs) parallel animal microRNAs in their ability to control multiple target mRNAs. The small non-coding MicA RNA represses the expression of several genes, including major outer membrane proteins such as ompA, tsx and ecnB. In this study, we have characterised the RNA determinants involved in the stability of MicA and analysed how they influence the expression of its targets. Site-directed mutagenesis was used to construct MicA mutated forms. The 5′linear domain, the structured region with two stem-loops, the A/U-rich sequence or the 3′ poly(U) tail were altered without affecting the overall secondary structure of MicA. The stability and the target regulation abilities of the wild-type and the different mutated forms of MicA were then compared. The 5′ domain impacted MicA stability through an RNase III-mediated pathway. The two stem-loops showed different roles and disruption of stem-loop 2 was the one that mostly affected MicA stability and abundance. Moreover, STEM2 was found to be more important for the in vivo repression of both ompA and ecnB mRNAs while STEM1 was critical for regulation of tsx mRNA levels. The A/U-rich linear sequence is not the only Hfq-binding site present in MicA and the 3′ poly(U) sequence was critical for sRNA stability. PNPase was shown to be an important exoribonuclease involved in sRNA degradation. In addition to the 5′ domain of MicA, the stem-loops and the 3′ poly(U) tail are also shown to affect target-binding. Disruption of the 3′U-rich sequence greatly affects all targets analysed. In conclusion, our results have shown that it is important to understand the “sRNA anatomy” in order to modulate its stability. Furthermore, we have demonstrated that MicA RNA can use different modules to regulate its targets. This knowledge can allow for the engineering of non-coding RNAs that interact differently with multiple targets.     

Characterization of the E.coli poly(A) polymerase: nucleotide specificity, RNA-binding affinities and RNA structure dependence

Polyadenylation of RNA molecules in bacteria and chloroplasts has been implicated as part of the RNA degradation pathway. The polyadenylation reaction is performed in Escherichia coli mainly by the enzyme poly(A) polymerase I (PAP I). In order to understand the molecular mechanism of RNA polyadenylation in bacteria, we characterized the biochemical properties of this reaction in vitro using the purified enzyme. Unlike the PAP from yeast nucleus, which is specific for ATP, E.coli PAP I can use all four nucleotide triphosphates as substrates for addition of long ribohomopolymers to RNA. PAP I displays a high binding activity to poly(U), poly(C) and poly(A) ribohomopolymers, but not to poly(G). The 3′-ends of most of the mRNA molecules in bacteria are characterized by a stem–loop structure. We show here that in vitro PAP I activity is inhibited by a stem–loop structure. A tail of two to six nucleotides located 3′ to the stem–loop structure is sufficient to overcome this inhibition. These results suggest that the stem–loop structure located in most of the mRNA 3′-ends may function as an inhibitor of polyadenylation and degradation of the corresponding RNA molecule. However, RNA 3′-ends produced by endonucleolytic cleavage by RNase E in single-strand regions of mRNA molecules may serve as efficient substrates for polyadenylation that direct these molecules for rapid exonucleolytic degradation.     

A viral noncoding RNA generated by cis-element-mediated protection against 5'->3' RNA decay represses both cap-independent and cap-dependent translation

Positive-strand RNA viruses use diverse mechanisms to regulate viral and host gene expression for ensuring their efficient proliferation or persistence in the host. We found that a small viral noncoding RNA (0.4 kb), named SR1f, accumulated in Red clover necrotic mosaic virus (RCNMV)-infected plants and protoplasts and was packaged into virions. The genome of RCNMV consists of two positive-strand RNAs, RNA1 and RNA2. SR1f was generated from the 3′ untranslated region (UTR) of RNA1, which contains RNA elements essential for both cap-independent translation and negative-strand RNA synthesis. A 58-nucleotide sequence in the 3′ UTR of RNA1 (Seq1f58) was necessary and sufficient for the generation of SR1f. SR1f was neither a subgenomic RNA nor a defective RNA replicon but a stable degradation product generated by Seq1f58-mediated protection against 5′→3′ decay. SR1f efficiently suppressed both cap-independent and cap-dependent translation both in vitro and in vivo. SR1f trans inhibited negative-strand RNA synthesis of RCNMV genomic RNAs via repression of replicase protein production but not via competition of replicase proteins in vitro. RCNMV seems to use cellular enzymes to generate SR1f that might play a regulatory role in RCNMV infection. Our results also suggest that Seq1f58 is an RNA element that protects the 3′-side RNA sequences against 5′→3′ decay in plant cells as reported for the poly(G) tract and stable stem-loop structure in Saccharomyces cerevisiae.     

Identification of an Internal RNA Element Essential for Replication and Translational Enhancement of Tobacco Necrosis Virus A C

Different regulatory elements function are involved in plant virus gene expression and replication by long-distance RNA-RNA interactions. A cap-independent functional element of the Barley yellow dwarf virus (BYDV) – like translational enhancer (BTE) is present in Tobacco necrosis virus A (TNV-A), a Necrovirus member in the Tombusviridae family. In this paper, an RNA stretch flanking the 5′ proximal end of the TNV-A^C coat protein (CP) gene was shown to be essential for viral replication in Chenopodium amaranticolor plants and tobacco cells. This internal sequence functioned in transient expression of β-glucuronidase (GUS) when present at either the 5′ or 3′ sides of the GUS open reading frame. Serial deletion analyses revealed that nine nucleotides from nt 2609 to 2617 (−3 to +6 of the CP initiation site) within TNV-A^C RNA are indispensable for viral replication in whole plants and tobacco cells. Fusion of this RNA element in mRNAs translated in tobacco cells resulted in a remarkable enhancement of luciferase expression from in vitro synthesised chimaeric RNAs or DNA expression vectors. Interestingly, the element also exhibited increased translational activity when fused downstream of the reporter genes, although the efficiency was lower than with upstream fusions. These results provide evidence that an internal RNA element in the genomic (g) RNA of TNV-A^C, ranging approximately from nt 2543 to 2617, plays a bifunctional role in viral replication and translation enhancement during infection, and that this element may use novel strategies differing from those previously reported for other viruses.     

Poly(A) Polymerase from Vaccinia Virus-Infected Cells II. Product and Primer Characterization

The product of the in vitro reaction of a vaccinia virus-induced poly(A) polymerase (see preceding paper) with ATP is shown to be poly(A) by nuclease resistance and by annealing with poly(U). Polyacrylamide gel electrophoresis indicates that the in vitro synthesized poly(A) is associated with large RNA which is sensitive to RNase. RNA which co-purifies with the virus-induced enzyme is similar to vaccinia virus-specific RNA with respect to size and poly(A) content. Double labeling studies indicate that the RNA which co-purifies with the enzyme becomes associated with the poly(A) synthesized in vitro. The poly(A) formed in vitro is located on the 3′-OH terminus of this RNA. During in vitro poly(A) synthesis ³²P from α-[³²P]ATP is transferred to nucleosides other than 2′,3′-AMP, primarily to CMP. Inclusion of poly(U) in the in vitro reactions results in an increase in the transfer of ³²P to UMP.     

Inhibition of Hepatitis C Virus (HCV) Replication by Specific RNA Aptamers against HCV NS5B RNA Replicase

This study identified specific and avid RNA aptamers consisting of 2′-hydroxyl- or 2′-fluoropyrimidines against hepatitis C virus (HCV) NS5B replicase, an enzyme that is essential for HCV replication. These aptamers acted as potent decoys to competitively impede replicase-catalyzed RNA synthesis activity. Cytoplasmic expression of the 2′-hydroxyl aptamer efficiently inhibited HCV replicon replication in human liver cells through specific interaction with, and sequestration of, the target protein without either off-target effects or escape mutant generation. A selected 2′-fluoro aptamer could be truncated to a chemically manufacturable length of 29 nucleotides (nt), with increase in the affinity to HCV NS5B. Noticeably, transfection of the truncated aptamer efficiently suppressed HCV replication in cells without escape mutant appearance. The aptamer was further modified through conjugation of a cholesterol or galactose-polyethylene glycol ligand for in vivo availability and liver-specific delivery. The conjugated aptamer efficiently entered cells and inhibited genotype 1b subgenomic and genotype 2a full-length HCV JFH-1 RNA replication without toxicity and innate immunity induction. Importantly, a therapeutically feasible amount of the conjugated aptamer was delivered in vivo to liver tissue in mice. Therefore, cytoplasmic expression of 2′-hydroxyl aptamer or direct administration of chemically synthesized and ligand-conjugated 2′-fluoro aptamer against HCV NS5B could be a potent anti-HCV approach.     

Characterization of a Nodavirus Replicase Revealed a de Novo Initiation Mechanism of RNA Synthesis and Terminal Nucleotidyltransferase Activity

Nodaviruses are a family of positive-stranded RNA viruses with a bipartite genome of RNAs. In nodaviruses, genomic RNA1 encodes protein A, which is recognized as an RNA-dependent RNA polymerase (RdRP) and functions as the sole viral replicase protein responsible for its RNA replication. Although nodaviral RNA replication has been studied in considerable detail, and nodaviruses are well recognized models for investigating viral RNA replication, the mechanism(s) governing the initiation of nodaviral RNA synthesis have not been determined. In this study, we characterized the RdRP activity of Wuhan nodavirus (WhNV) protein A in detail and determined that this nodaviral protein A initiates RNA synthesis via a de novo mechanism, and this RNA synthesis initiation could be independent of other viral or cellular factors. Moreover, we uncovered that WhNV protein A contains a terminal nucleotidyltransferase (TNTase) activity, which is the first time such an activity has been identified in nodaviruses. We subsequently found that the TNTase activity could function in vitro to repair the 3′ initiation site, which may be digested by cellular exonucleases, to ensure the efficiency and accuracy of viral RNA synthesis initiation. Furthermore, we determined the cis-acting elements for RdRP or TNTase activity at the 3′-end of positive or negative strand RNA1. Taken together, our data establish the de novo synthesis initiation mechanism and the TNTase activity of WhNV protein A, and this work represents an important advance toward understanding the mechanism(s) of nodaviral RNA replication.     

Effect of rye embryo ribosome nuclease on double-stranded RNA

In extracts obtained by treating rye embryo ribosomes with 0.5 M NH₄Cl, nuclease activity was noted towards double-stranded RNA from virus of Penicillium chrysogenum and towards synthetic poly (A)-poly (U) and poly (I)-poly (C) complexes.     

Rotavirus RNA Replication Requires a Single-Stranded 3′ End for Efficient Minus-Strand Synthesis

The segmented double-stranded (ds) RNA genome of the rotaviruses is replicated asymmetrically, with viral mRNA serving as the template for the synthesis of minus-strand RNA. Previous studies with cell-free replication systems have shown that the highly conserved termini of rotavirus gene 8 and 9 mRNAs contain cis-acting signals that promote the synthesis of dsRNA. Based on the location of the cis-acting signals and computer modeling of their secondary structure, the ends of the gene 8 or 9 mRNAs are proposed to interact in cis to form a modified panhandle structure that promotes the synthesis of dsRNA. In this structure, the last 11 to 12 nucleotides of the RNA, including the cis-acting signal that is essential for RNA replication, extend as a single-stranded tail from the panhandled region, and the 5′ untranslated region folds to form a stem-loop motif. To understand the importance of the predicted secondary structure in minus-strand synthesis, mutations were introduced into viral RNAs which affected the 3′ tail and the 5′ stem-loop. Analysis of the RNAs with a cell-free replication system showed that, in contrast to mutations which altered the structure of the 5′ stem-loop, mutations which caused complete or near-complete complementarity between the 5′ end and the 3′ tail significantly inhibited (≥10-fold) minus-strand synthesis. Likewise, incubation of wild-type RNAs with oligonucleotides which were complementary to the 3′ tail inhibited replication. Despite their replication-defective phenotype, mutant RNAs with complementary 5′ and 3′ termini were shown to competitively interfere with the replication of wild-type mRNA and to bind the viral RNA polymerase VP1 as efficiently as wild-type RNA. These results indicate that the single-strand nature of the 3′ end of rotavirus mRNA is essential for efficient dsRNA synthesis and that the specific binding of the RNA polymerase to the mRNA template is required but not sufficient for the synthesis of minus-strand RNA.