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1.
López de Quinto S Sáiz M de la Morena D Sobrino F Martínez-Salas E 《Nucleic acids research》2002,30(20):4398-4405
The 3′ end region of foot-and-mouth disease virus (FMDV) consists of two distinct elements, a 90 nt untranslated region (3′-NCR) and a poly(A) tract. Removal of either the poly(A) tract or both the 3′-NCR and the poly(A) tract abrogated infectivity in susceptible cells in the context of a full-length cDNA clone. We have addressed the question of whether the impairment of RNA infectivity is related to defects at the translation level using a double approach. First, compared to the full-length viral RNA, removal of the 3′ sequences reduced the efficiency of translation in vitro. Secondly, a stimulatory effect of the 3′ end sequences on IRES-dependent translation was found in vivo using bicistronic constructs. RNAs carrying the FMDV 3′ end sequences linked to the second cistron showed a significant stimulation of IRES-dependent translation, whereas cap-dependent translation was not affected. Remarkably, IRES-dependent stimulation exerted by the poly(A) tract or the 3′-NCR seems to be the result of two separate events, as the 3′-NCR alone enhanced IRES activity on its own. Under conditions of FMDV Lb protease-induced translation shut-off, the stimulation of IRES activity reached values above 6-fold in living cells. A northern blot analysis indicated that IRES stimulation was not the consequence of a change in the stability of the bicistronic RNA produced in transfected cells. Analysis of the RNA-binding proteins interacting with a mixture of 3′ end and IRES probes showed an additive pattern. Altogether, our results strongly suggest that individual signals in the viral 3′ end ensure stimulation of FMDV translation. 相似文献
2.
Joep Joosten Gijs J Overheul Ronald P Van
Rij Pascal Miesen 《Nucleic acids research》2021,49(15):8886
In the germline of animals, PIWI interacting (pi)RNAs protect the genome against the detrimental effects of transposon mobilization. In Drosophila, piRNA-mediated cleavage of transposon RNA triggers the production of responder piRNAs via ping-pong amplification. Responder piRNA 3′ end formation by the nuclease Zucchini is coupled to the production of downstream trailer piRNAs, expanding the repertoire of transposon piRNA sequences. In Aedes aegypti mosquitoes, piRNAs are generated from viral RNA, yet, it is unknown how viral piRNA 3′ ends are formed and whether viral RNA cleavage gives rise to trailer piRNA production. Here we report that in Ae. aegypti, virus- and transposon-derived piRNAs have sharp 3′ ends, and are biased for downstream uridine residues, features reminiscent of Zucchini cleavage of precursor piRNAs in Drosophila. We designed a reporter system to study viral piRNA 3′ end formation and found that targeting viral RNA by abundant endogenous piRNAs triggers the production of responder and trailer piRNAs. Using this reporter, we identified the Ae. aegypti orthologs of Zucchini and Nibbler, two nucleases involved in piRNA 3′ end formation. Our results furthermore suggest that autonomous piRNA production from viral RNA can be triggered and expanded by an initial cleavage event guided by genome-encoded piRNAs. 相似文献
3.
Polyadenylation of genomic RNA and initiation of antigenomic RNA in a positive-strand RNA virus are controlled by the same cis-element 总被引:1,自引:0,他引:1
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van Ooij MJ Polacek C Glaudemans DH Kuijpers J van Kuppeveld FJ Andino R Agol VI Melchers WJ 《Nucleic acids research》2006,34(10):2953-2965
Genomes and antigenomes of many positive-strand RNA viruses contain 3′-poly(A) and 5′-poly(U) tracts, respectively, serving as mutual templates. Mechanism(s) controlling the length of these homopolymeric stretches are not well understood. Here, we show that in coxsackievirus B3 (CVB3) and three other enteroviruses the poly(A) tract is ~80–90 and the poly(U) tract is ~20 nt-long. Mutagenesis analysis indicate that the length of the CVB3 3′-poly(A) is determined by the oriR, a cis-element in the 3′-noncoding region of viral RNA. In contrast, while mutations of the oriR inhibit initiation of (−) RNA synthesis, they do not affect the 5′-poly(U) length. Poly(A)-lacking genomes are able to acquire genetically unstable AU-rich poly(A)-terminated 3′-tails, which may be generated by a mechanism distinct from the cognate viral RNA polyadenylation. The aberrant tails ensure only inefficient replication. The possibility of RNA replication independent of oriR and poly(A) demonstrate that highly debilitated viruses are able to survive by utilizing ‘emergence’, perhaps atavistic, mechanisms. 相似文献
4.
Rotavirus RNA Replication Requires a Single-Stranded 3′ End for Efficient Minus-Strand Synthesis
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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. 相似文献
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6.
Iwakawa HO Mizumoto H Nagano H Imoto Y Takigawa K Sarawaneeyaruk S Kaido M Mise K Okuno T 《Journal of virology》2008,82(20):10162-10174
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. 相似文献
7.
Solution structure of an arabinonucleic acid (ANA)/RNA duplex in a chimeric hairpin: comparison with 2′-fluoro-ANA/RNA and DNA/RNA hybrids
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Alexei Yu. Denisov Anne M. Noronha Christopher J. Wilds Jean-Franois Trempe Richard T. Pon Kalle Gehring Masad J. Damha 《Nucleic acids research》2001,29(21):4284-4293
Hybrids of RNA and arabinonucleic acid (ANA) as well as the 2′-fluoro-ANA analog (2′F-ANA) were recently shown to be substrates of the enzyme RNase H. Although RNase H binds to double-stranded RNA, no cleavage occurs with such duplexes. Therefore, knowledge of the structure of ANA/RNA hybrids may prove helpful in the design of future antisense oligonucleotide analogs. In this study, we have determined the NMR solution structures of ANA/RNA and DNA/RNA hairpin duplexes and compared them to the recently published structure of a 2′F-ANA/RNA hairpin duplex. We demonstrate here that the sugars of RNA nucleotides of the ANA/RNA hairpin stem adopt the C3′-endo (north, A-form) conformation, whereas those of the ANA strand adopt a ‘rigid’ O4′-endo (east) sugar pucker. The DNA strand of the DNA/RNA hairpin stem is flexible, but the average DNA/RNA hairpin structural parameters are close to the ANA/RNA and 2′F-ANA/RNA hairpin parameters. The minor groove width of ANA/RNA, 2′F-ANA/RNA and DNA/RNA helices is 9.0 ± 0.5 Å, a value that is intermediate between that of A- and B-form duplexes. These results rationalize the ability of ANA/RNA and 2′F-ANA/RNA hybrids to elicit RNase H activity. 相似文献
8.
Jin Wang Hongping Dong Yok Hian Chionh Megan E. McBee Sasilada Sirirungruang Richard P. Cunningham Pei-Yong Shi Peter C. Dedon 《Nucleic acids research》2016,44(18):8962-8975
The misincorporation of 2′-deoxyribonucleotides (dNs) into RNA has important implications for the function of non-coding RNAs, the translational fidelity of coding RNAs and the mutagenic evolution of viral RNA genomes. However, quantitative appreciation for the degree to which dN misincorporation occurs is limited by the lack of analytical tools. Here, we report a method to hydrolyze RNA to release 2′-deoxyribonucleotide-ribonucleotide pairs (dNrN) that are then quantified by chromatography-coupled mass spectrometry (LC-MS). Using this platform, we found misincorporated dNs occurring at 1 per 103 to 105 ribonucleotide (nt) in mRNA, rRNAs and tRNA in human cells, Escherichia coli, Saccharomyces cerevisiae and, most abundantly, in the RNA genome of dengue virus. The frequency of dNs varied widely among organisms and sequence contexts, and partly reflected the in vitro discrimination efficiencies of different RNA polymerases against 2′-deoxyribonucleoside 5′-triphosphates (dNTPs). Further, we demonstrate a strong link between dN frequencies in RNA and the balance of dNTPs and ribonucleoside 5′-triphosphates (rNTPs) in the cellular pool, with significant stress-induced variation of dN incorporation. Potential implications of dNs in RNA are discussed, including the possibilities of dN incorporation in RNA as a contributing factor in viral evolution and human disease, and as a host immune defense mechanism against viral infections. 相似文献
9.
A 68-Nucleotide Sequence within the 3′ Noncoding Region of Simian Hemorrhagic Fever Virus Negative-Strand RNA Binds to Four MA104 Cell Proteins
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The 3′ noncoding region (NCR) of the negative-strand RNA [3′(−)NCR RNA] of the arterivirus simian hemorrhagic fever virus (SHFV) is 209 nucleotides (nt) in length. Since this 3′ region, designated 3′(−)209, is the site of initiation of full-length positive-strand RNA and is the template for the synthesis of the 5′ leader sequence, which is found on both full-length and subgenomic mRNAs, it is likely to contain cis-acting signals for RNA synthesis and to interact with cellular and viral proteins to form replication complexes. Gel mobility shift assays showed that cellular proteins in MA104 S100 cytoplasmic extracts formed two complexes with the SHFV 3′(−)209 RNA, and results from competition gel mobility shift assays demonstrated that these interactions were specific. Four proteins with molecular masses of 103, 86, 55, and 36 kDa were detected in UV-induced cross-linking assays, and three of these proteins (103, 55, and 36 kDa) were also detected by Northwestern blotting assays. Identical gel mobility shift and UV-induced cross-linking patterns were obtained with uninfected and SHFV-infected extracts, indicating that the four proteins detected are cellular, not viral, proteins. The binding sites for the four cellular proteins were mapped to the region between nt 117 and 184 (68-nt sequence) from the 3′ end of the SHFV negative-strand RNA. This 68-nt sequence was predicted to form two stem-loops, SL4 and SL5. The 3′(−)NCR RNA of another arterivirus, lactate dehydrogenase-elevating virus C (LDV-C), competed with the SHFV 3′(−)209 RNA in competition gel mobility shift assays. UV-induced cross-linking assays showed that four MA104 cellular proteins with the same molecular masses as those that bind to the SHFV 3′(−)209 RNA also bind to the LDV-C 3′(−)NCR RNA and equine arteritis virus 3′(−)NCR RNA. However, each of these viral RNAs also bound to an additional MA104 protein. The binding sites for the MA104 cellular proteins were shown to be located in similar positions in the LDV-C 3′(−)NCR and SHFV 3′(−)209 RNAs. These data suggest that the binding sites for a set of the cellular proteins are conserved in all arterivirus RNAs and that these cell proteins may be utilized as components of viral replication complexes. 相似文献
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Sakamoto S Tamura T Furukawa T Komatsu Y Ohtsuka E Kitamura M Inoue H 《Nucleic acids research》2003,31(5):1416-1425
Based on our recent studies of RNA cleavage by oligonucleotide–terpyridine·Cu(II) complex 5′- and/or 3′-conjugates, we designed 2′-O-methyloligonucleotides with two terpyridine-attached nucleosides at contiguous internal sites. To connect the 2′-terpyridine-modified uridine residue at the 5′-side to the 5′-O-terpyridyl nucleoside residue at the 3′-side, a dimethoxytrityl derivative of 5-hydroxypropyl-5′-O-terpyridyl-2′-deoxyuridine-3′-phosphoramidite was newly synthesized. Using this unit, we constructed two terpyridine conjugates, with either an unusual phophodiester bond or the bond extended by a propanediol(s)-containing linker. Cleavage reactions of the target RNA oligomer, under the conditions of conjugate excess in the presence of Cu(II), indicated that the conjugates precisely cleaved the RNA at the predetermined site and that one propanediol-containing linker was the most appropriate for inducing high cleavage activity. Furthermore, a comparison of the activity of the propanediol agent with those of the control conjugates with one complex confirmed that the two complexes are required for efficient RNA cleavage. The reaction of the novel cleaver revealed a bell-shaped pH–rate profile with a maximum at pH ~7.5, which is a result of the cooperative action of the complexes. In addition, we demonstrated that the agent catalytically cleaves an excess of the RNA, with the kinetic parameter kcat/Km = 0.118 nM–1 h–1. 相似文献
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Molinaro RJ Jha BK Malathi K Varambally S Chinnaiyan AM Silverman RH 《Nucleic acids research》2006,34(22):6684-6695
The antiviral and antitumor functions of RNase L are enabled by binding to the allosteric effectors 5′-phosphorylated, 2′,5′-linked oligoadenylates (2-5A). 2-5A is produced by interferon-inducible 2′,5′-oligoadenylate synthetases (OAS) upon activation by viral double-stranded RNA (dsRNA). Because mutations in RNase L have been implicated as risk factors for prostate cancer, we sought to determine if OAS activators are present in prostate cancer cells. We show that prostate cancer cell lines (PC3, LNCaP and DU145), but not normal prostate epithelial cells (PrEC), contain RNA fractions capable of binding to and activating OAS. To identify the RNA activators, we developed a cDNA cloning strategy based on stringent affinity of RNAs for OAS. We thus identified mRNAs for Raf kinase inhibitor protein (RKIP) and poly(rC)-binding protein 2 (PCBP2) that bind and potently activate OAS. In addition, human endogenous retrovirus (hERV) envelope RNAs were present in PC3 cells that bind and activate OAS. Analysis of several gene expression profiling studies indicated that PCBP2 RNA was consistently elevated in metastatic prostate cancer. Results suggest that OAS activation may occur in prostate cancer cells in vivo stimulated by cellular mRNAs for RKIP and PCBP2. 相似文献
14.
Replication of plus-strand RNA viruses depends on recruited host factors that aid several critical steps during replication. Several of the co-opted host factors bind to the viral RNA, which plays multiple roles, including mRNA function, as an assembly platform for the viral replicase (VRC), template for RNA synthesis, and encapsidation during infection. It is likely that remodeling of the viral RNAs and RNA-protein complexes during the switch from one step to another requires RNA helicases. In this paper, we have discovered a second group of cellular RNA helicases, including the eIF4AIII-like yeast Fal1p and the DDX5-like Dbp3p and the orthologous plant AtRH2 and AtRH5 DEAD box helicases, which are co-opted by tombusviruses. Unlike the previously characterized DDX3-like AtRH20/Ded1p helicases that bind to the 3′ terminal promoter region in the viral minus-strand (−)RNA, the other class of eIF4AIII-like RNA helicases bind to a different cis-acting element, namely the 5′ proximal RIII(−) replication enhancer (REN) element in the TBSV (−)RNA. We show that the binding of AtRH2 and AtRH5 helicases to the TBSV (−)RNA could unwind the dsRNA structure within the RIII(−) REN. This unique characteristic allows the eIF4AIII-like helicases to perform novel pro-viral functions involving the RIII(−) REN in stimulation of plus-strand (+)RNA synthesis. We also show that AtRH2 and AtRH5 helicases are components of the tombusvirus VRCs based on co-purification experiments. We propose that eIF4AIII-like helicases destabilize dsRNA replication intermediate within the RIII(−) REN that promotes bringing the 5′ and 3′ terminal (−)RNA sequences in close vicinity via long-range RNA-RNA base pairing. This newly formed RNA structure promoted by eIF4AIII helicase together with AtRH20 helicase might facilitate the recycling of the viral replicases for multiple rounds of (+)-strand synthesis, thus resulting in asymmetrical viral replication. 相似文献
15.
Kathleen Mahias Neveen Ahmed-El-Sayed Cyril Masante Juliette Bitard Cathy Staedel Fabien Darfeuille Michel Ventura Thérèse Astier-Gin 《Nucleic acids research》2010,38(12):4079-4091
The replication of the genomic RNA of the hepatitis C virus (HCV) of positive polarity involves the synthesis of a replication intermediate of negative polarity by the viral RNA-dependent RNA polymerase (NS5B). In vitro and likely in vivo, the NS5B initiates RNA synthesis without primers. This de novo mechanism needs specific interactions between the polymerase and viral RNA elements. Cis-acting elements involved in the initiation of (–) RNA synthesis have been identified in the 3′ non-coding region and in the NS5B coding region of the HCV RNA. However, the detailed contribution of sequences and/or structures of (–) RNA involved in the initiation of (+) RNA synthesis has been less studied. In this report, we identified an RNA element localized between nucleotides 177 and 222 from the 3′-end of the (–) RNA that is necessary for efficient initiation of RNA synthesis by the recombinant NS5B. By site-directed mutagenesis experiments, we demonstrate that the structure rather than the primary sequence of this domain is important for RNA synthesis. We also demonstrate that the intact structure of this RNA element is also needed for efficient RNA synthesis when the viral NS5B functions in association with other viral and cellular proteins in cultured hepatic cells. 相似文献
16.
Targeted inhibition of the hepatitis C internal ribosomal entry site genomic RNA with oligonucleotide conjugates
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Hepatitis C is a major public health concern, with an estimated 170 million people infected worldwide and an urgent need for new drug development. An attractive therapeutic approach is to prevent the ‘cap-independent’ translation initiation of the viral proteins by interfering with both the structure and function of the hepatitis C viral internal ribosomal entry site (HCV IRES). Towards this goal, we report the design, synthesis and purification of novel bi-functional molecules containing DNA or RNA antisenses attached to functional groups performing RNA hydrolysis. These 5′ or 3′-coupled conjugates bind the HCV IRES with affinity and specificity and elicit targeted hydrolysis of the viral genomic RNA after short (1 h) incubation at low (500 nM) concentration at 37°C in vitro. Additional secondary cleavage sites are induced and their mapping within the RNA structure indicates that functional domains IIIb-e are excised from the IRES that, based on cryo-EM studies, becomes incapable of binding the small ribosomal subunit and initiation factor 3 (eIF3). All these molecules inhibit, in a dose-dependent manner, the ‘IRES-dependent’ translation in vitro. The 5′-coupled imidazole conjugate reduces viral protein synthesis by half at a 300 nM concentration (IC50), corresponding to a 4-fold increase of activity when compared to the naked oligonucleotide. These new conjugates are now being tested for activity on infected hepatic cell lines. 相似文献
17.
William G. Davis Mausumi Basu Elizabeth J. Elrod Markus W. Germann Margo A. Brinton 《Journal of virology》2013,87(13):7622-7636
The 3′-terminal nucleotides (nt) of West Nile virus (WNV) genomic RNA form a penultimate 16-nt small stem-loop (SSL) and an 80-nt terminal stem-loop (SL). These RNA structures are conserved in divergent flavivirus genomes. A previous in vitro study using truncated WNV 3′ RNA structures predicted a putative tertiary interaction between the 5′ side of the 3′-terminal SL and the loop of the SSL. Although substitution or deletion of the 3′ G (nt 87) within the SSL loop, which forms the only G-C pair in the predicted tertiary interaction, in a WNV infectious clone was lethal, a finding consistent with the involvement in a functionally relevant pseudoknot interaction, extensive mutagenesis of nucleotides in the terminal SL did not identify a cis-acting pairing partner for this SSL 3′ G. However, both the sequence and the structural context of two adjacent base pairs flanked by symmetrical internal loops in the 3′-terminal SL were shown to be required for efficient viral RNA replication. Nuclear magnetic resonance analysis confirmed the predicted SSL and SL structures but not the tertiary interaction. The SSL was previously reported to contain one of three eEF1A binding sites, and G87 in the SSL loop was shown to be involved in eEF1A binding. The nucleotides at the bottom part of the 3′-terminal SL switch between 3′ RNA-RNA and 3′-5′ RNA-RNA interactions. The data suggest that interaction of the 3′ SL RNA with eEF1A at three sites and a unique metastable structural feature may participate in regulating structural changes in the 3′-terminal SL. 相似文献
18.
Uniformly 32P-labeled, double-stranded genome RNA isolated from purified reovirus contains two types of 5′-terminal sequences. One strand contains a phosphatase-resistant 5′-terminal structure, XpppG*pCpU, which is also present in the viral mRNA. The 5′ blocking group, X, is removed by β-elimination indicating that it is a nucleoside containing free 2′,3′-hydroxyls. G*pC is an alkaline-resistant, 2′-O-methylated sequence. The other strand contains a phosphatase-sensitive 5′ sequence, ppGpPupPyp. The results are discussed in relation to blocked 5′-terminal structures in other viral and cellular RNAs. 相似文献
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The RNA genome of the hepatitis C virus (HCV) contains multiple conserved structural cis domains that direct protein synthesis, replication, and infectivity. The untranslatable regions (UTRs) play essential roles in the HCV cycle. Uncapped viral RNAs are translated via an internal ribosome entry site (IRES) located at the 5′ UTR, which acts as a scaffold for recruiting multiple protein factors. Replication of the viral genome is initiated at the 3′ UTR. Bioinformatics methods have identified other structural RNA elements thought to be involved in the HCV cycle. The 5BSL3.2 motif, which is embedded in a cruciform structure at the 3′ end of the NS5B coding sequence, contributes to the three-dimensional folding of the entire 3′ end of the genome. It is essential in the initiation of replication. This paper reports the identification of a novel, strand-specific, long-range RNA–RNA interaction between the 5′ and 3′ ends of the genome, which involves 5BSL3.2 and IRES motifs. Mutants harboring substitutions in the apical loop of domain IIId or in the internal loop of 5BSL3.2 disrupt the complex, indicating these regions are essential in initiating the kissing interaction. No complex was formed when the UTRs of the related foot and mouth disease virus were used in binding assays, suggesting this interaction is specific for HCV sequences. The present data firmly suggest the existence of a higher-order structure that may mediate a protein-independent circularization of the HCV genome. The 5′–3′ end bridge may have a role in viral translation modulation and in the switch from protein synthesis to RNA replication. 相似文献