Tobacco Acid Pyrophosphatase Research Articles

RppH can faithfully replace TAP to allow cloning of 5′-triphosphate carrying small RNAs

RNA interference was first described in the nematode Caenorhabditis elegans. Ever since, several new endogenous small RNA pathways have been described and characterized to different degrees. The very prominent secondary small interfering RNAs, also called 22G-RNAs, bear a 5′ triphosphate group after loading into an Argonaute protein. This creates a technical issue, since 5′PPP groups decrease cloning efficiency for small RNA sequencing. To increase cloning efficiency of these small RNA species, a common practice in the field is the treatment of RNA samples, prior to library preparation, with Tobacco Acid pyrophosphatase (TAP). Recently, TAP production and supply was discontinued, so an alternative must be devised. We turned to RNA 5′ pyrophosphohydrolase (RppH), a commercially available pyrophosphatase isolated from E. coli. Here we directly compare TAP and RppH in their use for small RNA library preparation. We show that RppH-treated samples faithfully recapitulate TAP-treated samples. Specifically, there is enrichment for 22G-RNAs and mapped small RNA reads show no small RNA transcriptome-wide differences between RppH and TAP treatment. We propose that RppH can be used as a small RNA pyrophosphatase to enrich for triphosphorylated small RNA species and show that RppH- and TAP-derived datasets can be used in direct comparison.•We show that treatment of small RNA samples with RppH prior to sequencing library preparation increases the cloning efficiency of 5′ triphosphorylated small RNAs;•RppH treatment is a valid alternative to TAP treatment.

GTSF-1 is required for formation of a functional RNA-dependent RNA Polymerase complex in Caenorhabditis elegans.

Argonaute proteins and their associated small RNAs (sRNAs) are evolutionarily conserved regulators of gene expression. Gametocyte-specific factor 1 (Gtsf1) proteins, characterized by two tandem CHHC zinc fingers and an unstructured C-terminal tail, are conserved in animals and have been shown to interact with Piwi clade Argonautes, thereby assisting their activity. We identified the Caenorhabditis elegans Gtsf1 homolog, named it gtsf-1 and characterized it in the context of the sRNA pathways of C.elegans We report that GTSF-1 is not required for Piwi-mediated gene silencing. Instead, gtsf-1 mutants show a striking depletion of 26G-RNAs, a class of endogenous sRNAs, fully phenocopying rrf-3 mutants. We show, both invivo and invitro, that GTSF-1 interacts with RRF-3 via its CHHC zinc fingers. Furthermore, we demonstrate that GTSF-1 is required for the assembly of a larger RRF-3 and DCR-1-containing complex (ERIC), thereby allowing for 26G-RNA generation. We propose that GTSF-1 homologs may act to drive the assembly of larger complexes that act in sRNA production and/or in imposing sRNA-mediated silencing activities.

Application of a Schizosaccharomyces pombe Edc1-fused Dcp1-Dcp2 decapping enzyme for transcription start site mapping.

Changes in the 5′ leader of an mRNA can have profound effects on its translational efficiency with little effect on abundance. Sequencing-based methods to accurately map the 5′ leader by identifying the first transcribed nucleotide rely on enzymatic removal of the 5′ eukaryotic cap structure by tobacco acid pyrophosphatase (TAP). However, commercial TAP production has been problematic and has now been discontinued. RppH, a bacterial enzyme that can also cleave the 5′ cap, and Cap-Clip, a plant-derived enzyme, have been marketed as TAP replacements. We have engineered a Schizosaccharomyces pombe Edc1-fused Dcp1–Dcp2 decapping enzyme that functions as a superior TAP replacement. It can be purified from E. coli overexpression in high yields using standard biochemical methods. This constitutively active enzyme is four orders of magnitude more catalytically efficient than RppH at 5′ cap removal, compares favorably to Cap-Clip, and the 5′ monophosphorylated RNA product is suitable for standard RNA cloning methods. This engineered enzyme is a better replacement for TAP treatment than the current marketed use of RppH and can be produced cost-effectively in a general laboratory setting, unlike Cap-Clip.

Cross talk between ABC transporter mRNAs via a target mRNA-derived sponge of the GcvB small RNA.

There is an expanding list of examples by which one mRNA can posttranscriptionally influence the expression of others. This can involve RNA sponges that sequester regulatory RNAs of mRNAs in the same regulon, but the underlying molecular mechanism of such mRNA cross talk remains little understood. Here, we report sponge-mediated mRNA cross talk in the posttranscriptional network of GcvB, a conserved Hfq-dependent small RNA with one of the largest regulons known in bacteria. We show that mRNA decay from the gltIJKL locus encoding an amino acid ABC transporter generates a stable fragment (SroC) that base-pairs with GcvB. This interaction triggers the degradation of GcvB by RNase E, alleviating the GcvB-mediated mRNA repression of other amino acid-related transport and metabolic genes. Intriguingly, since the gltIJKL mRNA itself is a target of GcvB, the SroC sponge seems to enable both an internal feed-forward loop to activate its parental mRNA in cis and activation of many trans-encoded mRNAs in the same pathway. Disabling this mRNA cross talk affects bacterial growth when peptides are the sole carbon and nitrogen sources.

Riboswitch discovery by combining RNA-seq and genome-wide identification of transcriptional start sites.

Deep-sequencing technologies applied to RNA have tremendous potential to identify novel transcripts with single-nucleotide resolution. By combining whole-transcript cDNA sequencing (RNA-seq) and genome-wide identification of transcription start sites (dRNA-seq), it is possible to characterize long 5'-untranslated regions potentially endowed with regulatory capacities and to detect premature termination of transcription. This can be used to identify new potential riboswitches. In this chapter, we provide a detailed protocol of the dRNA-seq method based on differential pretreatment of RNAs with tobacco acid pyrophosphatase to differentiate between 5'-ends of primary and processed RNAs. We also give a briefer protocol of the preparation of RNA-seq libraries and of how to go through data bioinformatics analysis and data visualization using genome browsers. This approach is powerful to identify novel riboswitches and to demonstrate the functionality of riboswitches predicted in silico.

Characterization and comparison of the full 3′ and 5′ untranslated genomic regions of diverse isolates of infectious salmon anaemia virus by using a rapid and universal method

The 3′ and 5′ untranslated regions (UTRs) of the gene segments of orthomyxoviruses interact closely with the polymerase complex and are important for viral replication and transcription regulation. Despite this, the 3′ and 5′ RNA UTRs of the infectious salmon anaemia virus (ISAV) genome have only been partially characterized and little is known about the level of conservation between different virus subtypes. This report details for the first time, the adaptation of a rapid method for the simultaneous characterization of the 3′ and 5′ UTRs of each viral segment of ISAV. This was achieved through self circularization of segments using T4 RNA ligase, followed by PCR and sequencing. Dephosphorylation of 5′ ends using tobacco acid pyrophosphatase (TAP) proved to be a specific requirement for ligation of ISAV ends which was not essential for characterization of influenza virus in a similar manner. The development of universal primers facilitated the characterization of 4 genetically distinct ISAV isolates from Canada, Norway and Scotland. Comparison of the UTR regions revealed a similarity in organization and presence of conserved terminal sequences as reported for other orthomyxoviruses. Interestingly, the 3′ ends of ISAV segments including segments 1, 5 and 6, were shorter and 5′ UTRs generally longer than in their influenza counterparts.

Critical Role of 7,8-Didemethyl-8-hydroxy-5-deazariboflavin for Photoreactivation in Chlamydomonas reinhardtii

DNA photolyases use two noncovalently bound chromophores to catalyze photoreactivation, the blue light-dependent repair of DNA that has been damaged by ultraviolet light. FAD is the catalytic chromophore for all photolyases and is essential for photoreactivation. The identity of the second chromophore is often 7,8-didemethyl-8-hydroxy-5-deazariboflavin (FO). Under standard light conditions, the second chromophore is considered nonessential for photoreactivation because DNA photolyase bound to only FAD is sufficient to catalyze the repair of UV-damaged DNA. phr1 is a photoreactivation-deficient strain of Chlamydomonas. In this work, the PHR1 gene of Chlamydomonas was cloned through molecular mapping and shown to encode a protein similar to known FO synthases. Additional results revealed that the phr1 strain was deficient in an FO-like molecule and that this deficiency, as well as the phr1 photoreactivation deficiency, could be rescued by transformation with DNA constructs containing the PHR1 gene. Furthermore, expression of a PHR1 cDNA in Escherichia coli produced a protein that generated a molecule with characteristics similar to FO. Together, these results indicate that the Chlamydomonas PHR1 gene encodes an FO synthase and that optimal photoreactivation in Chlamydomonas requires FO, a molecule known to serve as a second chromophore for DNA photolyases.

Transcriptional Regulation of the Novel Toll-like Receptor Tlr13

Little has been known about Tlr13 (Toll-like receptor 13), a novel member of the Toll-like receptor family. To elucidate the molecular basis of murine Tlr13 gene expression, the activity of the Tlr13 gene promoter was characterized. Reporter gene analysis and electrophoretic mobility shift assays demonstrated that Tlr13 gene transcription was regulated through three cis-acting elements that interacted with the Ets2, Sp1, and PU.1 transcription factors. Furthermore, our work suggests that these transcription factors may cooperate, culminating in maximal transcription of the Tlr13 gene. In contrast, NF-kappaB appeared to act as an inhibitor of Tlr13 transcription. Overexpression of Ets2 caused a strong increase in the transcriptional activity of the Tlr13 promoter; however, overexpression of NF-kappaB p65 dramatically inhibited it. Additionally, interferon-beta is capable of acting Tlr13 transcription, but the activated signaling of lipopolysaccharide/TLR4 and peptidoglycan/TLR2 strongly inhibited the Tlr13 gene promoter. Thus, these findings reveal the mechanism of Tlr13 gene regulation, thereby providing insight into the function of Tlr13 in the immune response to pathogen.

Mutagenesis of the Dengue Virus Type 2 NS5 Methyltransferase Domain

The Flavivirus NS5 protein possesses both (guanine-N7)-methyltransferase and nucleoside-2'-O methyltransferase activities required for sequential methylation of the cap structure present at the 5' end of the Flavivirus RNA genome. Seventeen mutations were introduced into the dengue virus type 2 NS5 methyltransferase domain, targeting amino acids either predicted to be directly involved in S-adenosyl-l-methionine binding or important for NS5 conformation and/or charged interactions. The effects of the mutations on (i) (guanine-N7)-methyltransferase and nucleoside-2'-O methyltransferase activities using biochemical assays based on a bacterially expressed NS5 methyltransferase domain and (ii) viral replication using a dengue virus type 2 infectious cDNA clone were examined. Clustered mutations targeting the S-adenosyl-l-methionine binding pocket or an active site residue abolished both methyltransferase activities and viral replication, demonstrating that both methyltransferase activities utilize a single S-adenosyl-l-methionine binding pocket. Substitutions to single amino acids binding S-adenosyl-l-methionine decreased both methyltransferase activities by varying amounts. However, viruses that replicated at wild type levels could be recovered with mutations that reduced both activities by >75%, suggesting that only a threshold level of methyltransferase activity was required for virus replication in vivo. Mutation of residues outside of regions directly involved in S-adenosyl-l-methionine binding or catalysis also affected methyltransferase activity and virus replication. The recovery of viruses containing compensatory second site mutations in the NS5 and NS3 proteins identified regions of the methyltransferase domain important for overall stability of the protein or likely to play a role in virus replication distinct from that of cap methylation.

Cap accessibility correlates with the initiation efficiency of alfalfa mosaic virus RNAs.

The rate of cap removal from the alfalfa mosaic virus (A1MV) RNAs with tobacco acid pyrophosphatase (TAP) depends on the RNA species. At 37 degrees C and in the absence of divalent cation, RNA 3 reacts more slowly than the other three, which are decapped at similar rates. In the presence of magnesium, at 25 degrees C, TAP also discriminates against RNA 1. Thus the order of reactivity with TAP largely mimics the hierarchy of initiation efficiencies of the A1MV RNAs (Godefroy-Colburn et al., preceding paper in this journal). Our interpretation of these findings is that cap accessibility is what limits the rate of reaction with initiation factors as well as with TAP. In this hypothesis, translational discrimination between naturally capped messages would be related to the rate of 'breathing' of their 5' ends.

Complete nucleotide sequence of a South African isolate of grapevine leafroll-associated virus 3 reveals a 5′UTR of 737 nucleotides

Grapevine leafroll-associated virus 3 (GLRaV-3) is the type species of the genus Ampelovirus (family Closteroviridae) [1]. It is an economically important virus that is known to only infect Vitis spp. and that has a negative impact on the wine and table grape industries worldwide. To date, there has been only one report that claims the complete nucleotide sequence of GLRaV-3, by Ling et al. in 2004 (isolate NY-1, AF037268) [2]. In that report the 50 untranslated region (UTR) was found to be 158 nt in length. Here we report that the sequence of GLRaV-3, isolate GP18, has a 50UTR of 737 nt. This extended UTR was found in all other South African isolates of GLRaV-3 that were analysed. Grapevine material (Cabernet Sauvignon) was harvested in the Somerset West wine-producing region in South Africa from a monitored vineyard. RNA of isolate GP18 was extracted from a vine that displayed symptoms for the first time (Pietersen, pers comm). GP18 double-stranded RNA (dsRNA) was extracted from phloem tissue of wooded canes using an adapted cellulose (CF11) column method first described by Hu et al. [3]. RT-PCR was performed with primer sets designed to cover a large portion of the genome (nucleotides 1,835–17,905 of AF037268) in 10 overlapping clones (Fig. 1). Amplicons were cloned and sequenced and a consensus sequence was generated using BioEdit [4]. Primers were not included in the consensus sequence assembly of these overlapping sequences. Poly(A) tailing was performed on dsRNA in an attempt to reach the 50and 30terminal ends (Fig. 1) [5]. The 30-terminus of GP18 was found to be similar to that of the NY-1 isolate. However, we were unable to reach the reported 50-end of the NY-1 isolate and consistently found amplicons that started at +50 nt. After further experimentation with PCR conditions, a range of amplicons were generated that extended beyond the NY-1 sequence’s 50-end. A possible explanation for this inconsistency, using poly(A) tailing, might be the high uracil content in the 50-region upstream of the +50 site in the NY-1 strain that leads to stretches of adenines on the negative strand, which could act as priming sites for the oligo(dT) primer and lead to fragments shorter than the true genomic size. To determine the 50 end of the GP18 genome, total RNA was extracted from grapevine phloem tissue and subjected to RNA ligase-mediated rapid amplification of cDNA Ends (RLM-RACE) using the FirstChoice RLM-RACE kit (Ambion, USA) as per the manufacturer’s instructions. The amplicon generated for the 50-end of the GP18 genome was larger than expected (Fig. 1). It was cloned and four clones sequenced. The 50UTR was found to extend beyond the sequence reported for the NY-1 isolate [2]. The adapter ligation reaction was repeated on the same total RNA treated with calf intestine alkaline phosphatase (CIP) and tobacco acid pyrophosphatase (TAP), and a further five clones were sequenced. All nine clones showed the first 365 nucleotides of the NY-1 sequence and an additional 579 nucleotides upstream of the 50-end. The efficacy of RLM-RACE to determine the 50-termini of multiple ssRNA viruses from total RNA in a single reaction was also investigated. RLM-RACE was performed on total RNA extracted from three grapevine plants (K1, K5, K6) that contained a mixture of viruses (GLRaV-2, -3 and grapevine rupestris stem pitting-associated virus H. J. Maree M.-J. Freeborough J. T. Burger (&) Department of Genetics, Stellenbosch University, Private Bag X1, Matieland 7602, South Africa e-mail: jtb@sun.ac.za 1 The GenBank accession number for the sequence reported in this paper is EU259806.

The TbMTr1 Spliced Leader RNA Cap 1 2 ′-O-Ribose Methyltransferase from Trypanosoma brucei Acts with Substrate Specificity

In metazoa cap 1 (m(7)GpppNmp-RNA) is linked to higher levels of translation; however, the enzyme responsible remains unidentified. We have validated the first eukaryotic encoded cap 1 2'-O-ribose methyltransferase, TbMTr1, a member of a conserved family that modifies the first transcribed nucleotide of spliced leader and U1 small nuclear RNAs in the kinetoplastid protozoan Trypanosoma brucei. In addition to cap 0 (m(7)GpppNp-RNA), mRNA in these parasites has ribose methylations on the first four nucleotides with base methylations on the first and fourth (m(7)Gpppm(6,6)AmpAmpCmpm(3)Ump-SL RNA) conveyed via trans-splicing of a universal spliced leader. The function of this cap 4 is unclear. Spliced leader is the majority RNA polymerase II transcript; the RNA polymerase III-transcribed U1 small nuclear RNA has the same first four nucleotides as spliced leader, but it receives an m(2,2,7)G cap with hypermethylation at position one only (m(2,2,7)Gpppm(6,6)AmpApCpUp-U1 snRNA). Here we examine the biochemical properties of recombinant TbMTr1. Active over a pH range of 6.0 to 9.5, TbMTr1 is sensitive to Mg(2+). Positions Lys(95)-Asp(204)-Lys(259)-Glu(285) constitute the conserved catalytic core. A guanosine cap on RNA independent of its N(7) methylation status is required for substrate recognition, but an m(2,2,7G)-cap is not recognized. TbMTr1 favors the spliced leader 5' sequence, as reflected by a preference for A at position 1 and modulation of activity for substrates with base changes at positions 2 and 3. With similarities to human cap 1 methyltransferase activity, TbMTr1 is an excellent model for higher eukaryotic cap 1 methyltransferases and the consequences of cap 1 modification.

Unconventional Mechanism of mRNA Capping by the RNA-Dependent RNA Polymerase of Vesicular Stomatitis Virus

All known eukaryotic and some viral mRNA capping enzymes (CEs) transfer a GMP moiety of GTP to the 5'-diphosphate end of the acceptor RNA via a covalent enzyme-GMP intermediate to generate the cap structure. In striking contrast, the putative CE of vesicular stomatitis virus (VSV), a prototype of nonsegmented negative-strand (NNS) RNA viruses including rabies, measles, and Ebola, incorporates the GDP moiety of GTP into the cap structure of transcribing mRNAs. Here, we report that the RNA-dependent RNA polymerase L protein of VSV catalyzes the capping reaction by an RNA:GDP polyribonucleotidyltransferase activity, in which a 5'-monophosphorylated viral mRNA-start sequence is transferred to GDP generated from GTP via a covalent enzyme-RNA intermediate. Thus, the L proteins of VSV and, by extension, other NNS RNA viruses represent a new class of viral CEs, which have evolved independently from known eukaryotic CEs.

Cross-species and Cross-compartmental Aminoacylation of Isoaccepting tRNAs by a Class II tRNA Synthetase

Cross-species and Cross-compartmental Aminoacylation of Isoaccepting tRNAs by a Class II tRNA Synthetase

Identification, Evolution, and Regulation of Expression of Guinea Pig Trappin with an Unusually Long Transglutaminase Substrate Domain

Trappins are found in human, bovine, hippopotamus, and members of the pig family, but not in rat and mouse. To clarify the evolution of the trappin genes and the functional significance of their products, we isolated the trappin gene in guinea pig, a species belonging to a rodent family distinct from rat and mouse. Guinea pig trappin was confirmed to encode the same domain structure as trappin, consisting of a signal sequence, an extra large transglutaminase substrate domain, and a whey acidic protein motif. Northern blot analysis and in situ hybridization histochemistry as well as immunohistochemistry demonstrated that guinea pig trappin is expressed solely in the secretory epithelium of the seminal vesicle and that its expression is androgen-dependent. We confirmed that guinea pig trappin is cross-linked by prostate transglutaminase and that the whey acidic protein motif derived from guinea pig trappin has an inhibitory activity against leukocyte elastase. Genome sequence analysis showed that guinea pig trappin belongs to the family of REST (rapidly evolving seminal vesicle transcribed) genes.

The nucleotide sequence and genome organization of Japanese iris necrotic ring virus , a new species in the genus Carmovirus

The genome of Japanese iris necrotic ring virus (JINRV) consists of a positive-sense ssRNA of 4014 nucleotides with six major open reading frames (ORFs). A 5'-non-coding region of 31 nucleotides precedes the first initiation codon. Like Carnation mottle virus (CarMV), the 5'-proximal three ORFs encode a 26 kDa protein (p26) and two readthrough proteins, i.e. an 85 kDa putative RNA replicase (p85) and a 99 kDa protein (p99). The central ORF encodes a small 8 kDa protein (p8). The 3'-proximal ORF encodes a 38 kDa capsid protein (p38). Another ORF encoding a 12 kDa protein (p12) overlaps the p99 ORF.JINRV RNA treated with bacterial alkaline phosphatase and tobacco acid pyrophosphatase could not be ligated to an oligoribonucleotide using T4 RNA ligase, indicating that the 5' end of the viral RNA is uncapped. The 3' end is not polyadenylated. Comparison of the genomic organization and the predicted amino acid sequences with those of other viruses confirmed that JINRV should be classified as a member of the genus Carmovirus, family Tombusviridae.

Termini of All mRNA Species of Marburg Virus: Sequence and Secondary Structure

The 3′ and 5′ ends of Marburg virus (MBG)-specific mRNA species have been determined using reverse transcription-PCR, rapid amplification of cDNA ends, or the reverse ligation-mediated PCR procedure after removal of cap structures with tobacco acid pyrophosphatase. The polyadenylation sites of all MBG-specific mRNAs were strictly conserved and corresponded to the predicted transcriptional stop signals of genomic RNA. Determination of the 5′ ends of the mRNA species showed that mRNA synthesis started precisely at the first nucleotide of a highly conserved transcriptional start site. The 5′ ends of the mRNA species can build a stable secondary structure with the conserved nucleotides always located in the stem region of a hairpin. Nucleotide substitutions in the conserved 5′ regions are accompanied by compensatory mutations of the complementary nucleotide thus leading to a conservation of the secondary structures. Compensatory mutations were also found when 5′ ends of mRNA of MBG strain Musoke were compared with MBG strain Popp or the closely related Ebola virus, indicating that the secondary structures will be conserved even if the sequence is altered.

Oligo-capping: a simple method to replace the cap structure of eukaryotic mRNAs with oligoribonucleotides

We have devised a method to replace the cap structure of a mRNA with an oligoribonucleotide (r-oligo) to label the 5′ end of eukaryotic mRNAs. The method consists of removing the cap with tobacco acid pyrophosphatase (TAP) and ligating r-oligos to decapped mRNAs with T4 RNA ligase. This reaction was made cap-specific by removing 5′-phosphates of non-capped RNAs with alkaline phosphatase prior to TAP treatment. Unlike the conventional methods that label the 5′ end of cDNAs, this method specifically labels the capped end of the mRNAs with a synthetic r-oligo prior to first-strand cDNA synthesis. The 5′ end of the mRNA was identified quite simply by reverse transcription-polymerase chain reaction (RT-PCR).

Human beta-globin mRNAs that harbor a nonsense codon are degraded in murine erythroid tissues to intermediates lacking regions of exon I or exons I and II that have a cap-like structure at the 5' termini.

Previous studies have demonstrated that nonsense codons within beta zero-thalassemic or in vitro-mutagenized human beta-globin transgenes result in the production of mRNAs that are degraded abnormally rapidly in the cytoplasm of murine erythroid cells. As a consequence, three RNA degradative intermediates are formed that lack sequences from either exon I or exons I and II. We show here that the intermediates, like the full-length mRNA from which they derive and the endogenous murine beta maj-globin mRNA, bind to the anticap monoclonal antibody H-20 in a way that is competed by the cap analogue m7G and eliminated by prior exposure to tobacco acid pyrophosphatase. Furthermore, the intermediates, like the two full-length mRNAs, are resistant to a 5'----3' exonuclease activity isolated from HeLa cell nuclei that degrades uncapped but not capped ribopolymers. Based on these observations, the intermediates appear to possess a structure that is indistinguishable from the cap at the 5' end of mRNA, i.e. a methylated nucleoside that is linked to the RNA by a 5'-5' phosphodiester bond. Detection of the intermediates during murine development was concomitant with detection of full-length thalassemic mRNA. Intermediate production appears to be influenced by RNA structure as indicated by the products that derive from a beta zero-thalassemic beta-globin transgene harboring a structural alteration (a 4 bp deletion) that was larger than any of those previously studied.

Nucleotide sequencing of 5′ and 3′ termini of bovine viral diarrhea virus by RNA ligation and PCR

Genomic RNA was extracted from cytopathic (CP) bovine viral diarrhea virus (BVDV) strain NADL, CP strain 72, and noncytopathic (NCP) strain SD-1 purified by ultracentrifugation. Assuming the presence of a cap structure, de-blocking of the 5′ capped end of the genomic RNA was done by treatment with tobacco acid pyrophosphatase (TAP). Following decapping, the RNA molecules were ligated using T4 RNA ligase and the ligated tandem RNA templates were then amplified by primer-directed amplification (PCR). cDNA synthesis was done using reverse transcriptase with random primers and cDNA amplification was done using a negative sense primer 231–248 and a positive sense primer 12434–12451. The nucleotide sequence of the amplified product was determined by double-stranded sequencing using the Sanger di-deoxy chain termination method and an additional ‘CCCCC’ nucleotide sequence was identified at the ligation site. Following dATP tailing of cDNA and amplification across the 5′ terminus and nucleotide sequencing, no additional nucleotides were identified on the 5′ terminus. The 5′ terminus as published by Collett et al., 1988b was confirmed as previously reported. Therefore, the 3′ terminus includes an additional ‘CCCCC’ nucleotide sequence to that previously reported. Identical results were obtained when the BVDV genomic RNA was not decapped prior to RNA ligation and amplification.