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At least 19 recordsLinked to original sources
PubMed2003

Quantitative analysis of influenza virus RNP interaction with RNA cap structures and comparison to human cap binding protein eIF4E.

Influenza virus polymerase uses capped RNA primers for transcription initiation in infected cells. This unique mechanism involves the specific binding of the polymerase to capped mRNA precursors in the nucleus of infected cells. These host RNAs are then cleaved by a polymerase associated endonuclease at a position 10-15 nucleotides downstream of the cap structure. The resulting capped RNA oligonucleotides function as primers for transcription initiation. The viral cap binding site has previously been mapped to the PB2 subunit of the trimeric influenza polymerase complex. We have established a quantitative assay system for the analysis of cap interaction with PB2 as part of the native, viral ribonucleoprotein complex (RNP) using a specific UV cross-linking approach. Cap binding was not affected by the RNase pretreatment of the capped RNA substrate and cap binding was not inhibited by excess uncapped RNA, indicating that under the assay conditions, the majority of the binding energy was contributed by the interaction with the cap structure. Binding to 7-methyl-GTP was found to involve synergistic interaction with 7-methyl guanosine and triphosphate binding subsites. A similar mode of interaction with 7-methyl-GTP was found for human cap binding protein eIF4E. However, the potency of 7-methyl-GTP for cap binding inhibition was 200-fold stronger with eIF4E and had a higher contribution from the triphosphate moiety as compared to influenza RNP. Due to this difference in cap subsite interaction, it was possible to identify novel cap analogues, which selectively interact with influenza virus, but not human cap binding protein.

Base Sequence↗
PubMed1982

RNA capping by HeLa cell RNA guanylyltransferase. Characterization of a covalent protein-guanylate intermediate.

RNA capping by partially purified HeLa cell GTP:RNA guanylyltransferase has been shown to occur in the following sequence of two partial reactions involving a covalent protein-guanylate intermediate: (i) E(P68) + GTP in equilibrium E(P68-GMP) + PPi (ii) E(P68-GMP) + ppRNA in equilibrium GpppRNA + E(P68) Initially, the enzyme reacts with GTP in the absence of an RNA cap acceptor to form a covalent protein-guanylate complex. This complex consists of a GMP residue linked via a phosphoamide bond to a Mr = 68,000 protein. The enzyme then transfers the guanylate residue from the Mr = 68,000 polypeptide to the 5' end of diphosphate-terminated poly(a) to yield the capped derivative GpppA(pA)n. Both partial reactions have been shown to be reversible. In the reverse of Reaction i, E(P68--GMP) reacts with PPi to regenerate GTP. In the reverse of Reaction ii, the enzyme catalyzes the transfer of the 5'-GMP from capped RNA to the Mr = 68,000 protein to form protein-guanylate complex. A divalent cation is required for both partial reactions. The Mr = 68,000 protein is presumed to be a subunit of the HeLa guanylyltransferase. This interpretation is consistent with the sedimentation coefficient of 4.2 S of the native enzyme. Preliminary studies of RNA guanylyltransferase from mouse myeloma tumors suggest a similar mechanism of transguanylylation involving a Mr = 68,000 protein-guanylate complex. These data, in conjunction with previous studies of vaccinia virus guanylyltransferase (Shuman, S., and Hurwitz, J. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 187-191) suggests that covalent GMP-enzyme intermediates may be a general feature of the RNA capping reaction.

Animals↗
PubMed1997

Differential effect of modified capped RNA substrates on influenza virus transcription.

The RNA-dependent RNA polymerase of influenza virus transcribes messenger RNA through a unique cap scavenging mechanism. Viral enzyme binds to the cap structure of host mRNA, cleaves the molecule 9-15 bases downstream of the cap, and uses the short capped oligonucleotide as a primer for mRNA synthesis. Previously, we have shown that the viral polymerase can efficiently bind capped RNAs shorter than 9 nucleotides in length, but the viral enzyme can not utilize these RNAs as primers. For this reason, these short capped oligonucleotides are potent inhibitors of influenza virus transcription. In these studies, it is now shown that short capped oligomers inhibit capped-RNA dependent transcription at the initial step of cap binding. In contrast, low concentrations of these short capped RNAs can actually stimulate viral transcription primed with high concentrations of the dinucleotide ApG. Another capped RNA derivative containing phosphorothioate oligonucleotides was also investigated as a potential polymerase inhibitor. This longer capped RNA was able to bind to the polymerase, but could not be cleaved to primer length by the enzyme associated endonuclease. Thus, the capped phosphorothioate RNA inhibited cap-primed transcription at the step of cap binding. However, in contrast to the short capped oligonucleotide, it also inhibited ApG primed viral transcription.

Oligoribonucleotides↗
PubMed1998

Recognition of capped RNA substrates by VP39, the vaccinia virus-encoded mRNA cap-specific 2'-O-methyltransferase.

We have investigated the interaction of VP39, the vaccinia-encoded mRNA cap-specific 2'-O-methyltransferase, with its capped RNA substrate. Two sites on the protein surface, responsible for binding the terminal cap nucleotide (m7G) and cap-proximal RNA, were characterized, and a third (downstream RNA binding) site was identified. Regarding the crystallographically defined m7G binding pocket, VP39 showed significant activity with adenine-capped RNA. Although VP39 mutants lacking specific m7G-contact side chains within the pocket showed reduced catalytic activity, none was transformed into a cap-independent RNA methyltransferase. Moreover, each retained a preference for m7G and A over unmethylated G as the terminal cap nucleotide, indicating a redundancy of m7G-contact residues able to confer cap-type specificity. Despite containing the 2'-O-methylation site, m7GpppG (cap dinucleotide) could not be methylated by VP39, but m7GpppGUbiotinp could. This indicated the minimum-length 2'-O-methyltransferase substrate to be either m7GpppGp, m7GpppGpN, or m7GpppGpNp. RNA-protein contacts immediately downstream of the m7GpppG moiety were found to be pH-sensitive. This was detectable only in the context of a weakened interaction of near-minimum-length substrates with VP39's m7G binding pocket (through the use of either adenine-capped substrate or a VP39 pocket mutant), as a dramatic elevation of KM at pH values above 7.5. KM values for substrates with RNA chain lengths of 2-6 nt were between 160 and 230 nM, but dropped to 9-15 nM upon increasing chain lengths to 20-50 nt. This suggested the binding of regions of the RNA substrate >6 nt from the 5' terminus to a previously unknown site on the VP39 surface.

Amino Acid Substitution↗
PubMed1979

Transmethylation and transguanylylation in 5'-RNA capping system isolated from rat liver nuclei.

Rat liver nuclei were isolated and sonicated for extraction in order to study the capping of RNA. The guanosine 7-methyltransferase was purified from the extract by hydroxylapatite column chromatography with stepwise addition of phosphate buffer. It was assayed by using as methyl acceptor synthetic G(5')ppp(5')G and S-adenosylmethionine as donor. The enzyme appeared in a sharp peak at 160 mM. The same peak fraction was subsequently found to contain the enzyme that guanylylates short synthetic polynucleotides and low molecular weight yeast RNA as acceptors. The two enzymatic activities were separated on Sephadex G-150 chromatography, yielding guanylyltransferase and guanosine 7-methyltransferase with molecular weights of approximately 65,000 and 130,000 respectively. Guanylyltransferase was further purified by CM-Sephadex chromatography, whereby G-7-methyltransferase was completely removed. Dithiothreitol was essential for guanylylation, and 2 mM Mn2+ (optimum) was twice as active as 8 mM Mg2+ (optimum). The alpha-32P of [32P]GTP, but not its beta- or gamma-32P was incorporated into the cap structure. By using unlabeled GTP with [beta-32P]ppGpCpC-poly(A2,U2,G) as acceptor, [beta'-32P]-GpppG... was formed. Our purified transguanylylation enzyme was found to catalyze a [32P]pyrophosphate exchange with GTP, which may be useful as a rapid assay for transguanylylation.

Animals↗
PubMed2004

The broad spectrum antiviral nucleoside ribavirin as a substrate for a viral RNA capping enzyme.

The broad spectrum antiviral nucleoside ribavirin displays activity against a variety of RNA and DNA viruses. A number of possible mechanisms have been proposed during the past 30 years to account for the antiviral activity of ribavirin, including the possibility that ribavirin might have a negative effect on the synthesis of the RNA cap structure of viral RNA transcripts. In the present study, we investigated the possibility that ribavirin can directly serve as a substrate for the vaccinia virus RNA capping enzyme. We demonstrate that ribavirin triphosphate can be used as a substrate by the capping enzyme and can form a covalent ribavirin monophosphate-enzyme intermediate reminiscent of the classical GMP-enzyme intermediate. Furthermore, our data indicate that ribavirin monophosphate can be transferred to the diphosphate end of an RNA transcript to form the unusual RpppN structure. Finally, we provide evidence that RNA transcripts that possess ribavirin as the blocking nucleoside are more stable than unblocked transcripts. However, in vitro translation assays indicate that RNA transcripts blocked with ribavirin are not translated efficiently. Our study provides the first biochemical evidences that ribavirin can directly interact with a viral capping enzyme. The ability of a purified RNA capping enzyme to utilize ribavirin as a substrate has not been previously documented and has implications for our understanding of the catalytic mechanisms of RNA capping enzymes. The biological implications of these findings for the proposed ribavirin-mediated inhibition of capping are discussed.

Antiviral Agents↗
PubMed1999

Conversion of a DNA ligase into an RNA capping enzyme.

In eukaryotes, newly synthesised mRNA is 'capped' by the addition of GMP to the 5" end by RNA capping enzymes. Recent structural studies have shown that RNA capping enzymes and DNA ligases have similar protein folds, suggesting a conserved catalytic mechanism. To explore these similarities we have produced a chimeric enzyme comprising the N-terminal domain 1 of a DNA ligase fused to the C-terminal domain 2 of a mRNA capping enzyme. This report shows that this hybrid enzyme retains adenylation activity, characteristic of DNA ligases but, remarkably, the chimera has ATP-dependent mRNA capping activity. This is the first observation of ATP-dependent RNA capping. These results suggest that nucleotidyltransferases may have evolved from a common ancestral gene.

Amino Acid Sequence↗
PubMed2002

An RNA cap (nucleoside-2'-O-)-methyltransferase in the flavivirus RNA polymerase NS5: crystal structure and functional characterization.

Viruses represent an attractive system with which to study the molecular basis of mRNA capping and its relation to the RNA transcription machinery. The RNA-dependent RNA polymerase NS5 of flaviviruses presents a characteristic motif of S-adenosyl-L-methionine-dependent methyltransferases at its N-terminus, and polymerase motifs at its C-terminus. The crystal structure of an N-terminal fragment of Dengue virus type 2 NS5 is reported at 2.4 A resolution. We show that this NS5 domain includes a typical methyltransferase core and exhibits a (nucleoside-2'-O-)-methyltransferase activity on capped RNA. The structure of a ternary complex comprising S-adenosyl-L-homocysteine and a guanosine triphosphate (GTP) analogue shows that 54 amino acids N-terminal to the core provide a novel GTP-binding site that selects guanine using a previously unreported mechanism. Binding studies using GTP- and RNA cap-analogues, as well as the spatial arrangement of the methyltransferase active site relative to the GTP-binding site, suggest that the latter is a specific cap-binding site. As RNA capping is an essential viral function, these results provide a structural basis for the rational design of drugs against the emerging flaviviruses.

Amino Acid Sequence↗
PubMed1981

Selected host cell capped RNA fragments prime influenza viral RNA transcription in vivo.

Influenza viral RNA transcription in vitro is primed by capped RNA fragments cleaved from capped RNAs by a viral endonuclease. The present study was undertaken to determine whether the specificities of the viral endonuclease and transcriptase observed in in vitro studies are also observed in the infected cell. The NS (nonstructural) gene of influenza WSN virus was cloned in pBR322 by using a double-stranded DNA containing a cDNA copy of both virion RNA (vRNA) and in vivo viral mRNA. We determined the 5' terminal sequence of the particular NS viral mRNA molecule which was cloned and also the 5' terminal sequences of the entire population of in vivo NS viral mRNAs synthesized in two different cell lines. For the latter determination we used a restriction fragment from the cloned DNA for the reverse transcriptase-catalyzed extension of total in vivo viral mRNA. The results indicate that in vivo and in vitro viral RNA transcription are similar in two important respects: (i) transcription initiates not with an A residue directed by the 3' terminal U of the vRNA, but with a G residue directed by the 3' penultimate C of the vRNA; and (ii) capped RNA fragments containing a 3' terminal A residue are preferentially used as primers, therapy generating an AG sequence in the viral mRNA complementary to the 3' terminal UC of the vRNA. Actually, for in vivo transcription, a subset of A-terminated capped fragments, namely those containing a 3' penultimate C residue, are the preferred primers. The latter specificity had not been observed in previous in vitro studies.

Base Sequence↗
PubMed2000

Helicase and capping enzyme active site mutations in brome mosaic virus protein 1a cause defects in template recruitment, negative-strand RNA synthesis, and viral RNA capping.

Brome mosaic virus (BMV) encodes two RNA replication proteins: 1a, which contains RNA capping and helicase-like domains, and 2a, which is related to polymerases. BMV 1a and 2a can direct virus-specific RNA replication in the yeast Saccharomyces cerevisiae, which reproduces the known features of BMV replication in plant cells. We constructed single amino acid point mutations at the predicted capping and helicase active sites of 1a and analyzed their effects on BMV RNA3 replication in yeast. The helicase mutants showed no function in any assays used: they were strongly defective in template recruitment for RNA replication, as measured by 1a-induced stabilization of RNA3, and they synthesized no detectable negative-strand or subgenomic RNA. Capping domain mutants divided into two groups. The first exhibited increased template recruitment but nevertheless allowed only low levels of negative-strand and subgenomic mRNA synthesis. The second was strongly defective in template recruitment, made very low levels of negative strands, and made no detectable subgenomes. To distinguish between RNA synthesis and capping defects, we deleted chromosomal gene XRN1, encoding the major exonuclease that degrades uncapped mRNAs. XRN1 deletion suppressed the second but not the first group of capping mutants, allowing synthesis and accumulation of large amounts of uncapped subgenomic mRNAs, thus providing direct evidence for the importance of the viral RNA capping function. The helicase and capping enzyme mutants showed no complementation. Instead, at high levels of expression, a helicase mutant dominantly interfered with the function of the wild-type protein. These results are discussed in relation to the interconnected functions required for different steps of positive-strand RNA virus replication.

Bromovirus↗
PubMed2006

A general RNA-capping ribozyme retains stereochemistry during cap exchange.

Numerous natural and artificial ribozymes have been shown to facilitate reactions that invert stereochemistry. Here, we demonstrate that an RNA-capping ribozyme retains stereochemistry at a phosphorus reaction center. The ribozyme synthesizes a broad range of 5'-5' RNA caps by exchanging phosphate groups around the alpha-phosphate found at the 5' terminus of the ribozyme. A ribozyme prepared with an Rp adenosine(5')alpha-thiotetraphosphate cap was found to exchange this cap for an Rp 4-thiouridine(5')alpha-thiotetraphosphate cap when incubated with 4-thiouridine triphosphate. The same Rp capped construct, when incubated with [gamma-(32)P]-ATP, exchanged the unlabeled ATP for a radiolabeled one while maintaining the same stereoconfiguration. In contrast, ribozymes prepared with an Sp cap failed to react even in the presence of thiophilic metal ions such as manganese. The kinetics of capping was also unusual as compared to inverting ribozymes. When the ribozyme was prepared with a triphosphate, capping was found to follow Michaelis-Menten-type kinetics even though the rate of pyrophosphate release was completely independent of nucleotide substrate concentration. Interestingly, the rate of capping and hydrolysis, when summed, was found to be indistinguishable from the rate of pyrophosphate release, indicating that an early rate-limiting step precedes both capping and hydrolysis. Together the retention of stereochemistry and kinetics imply that capping utilizes two inverting chemical steps that are separated by the transient formation of a rate-limiting covalent intermediate. As all protein enzymes that mediate similar capping reactions utilize a covalent intermediate, chemical necessity may have strongly guided the evolution of both protein and RNA-capping catalysts.

Adenosine Triphosphate↗
PubMed1983

RNA polymerase of influenza virus. IV. Catalytic properties of the capped RNA endonuclease associated with the RNA polymerase.

Catalytic properties of the capped RNA-specific endonuclease associated with the influenza virus RNA polymerase were analyzed with use of synthetic hetero- and homopolymers containing 32P-labeled CAP structures at their 5' termini. The endonuclease displays its intrinsic activity provided that substrate RNA contains both the CAP-1 structure (m7GpppGm) and either A or U residues at 9 to 11 nucleotides distant from the CAP structure. Independent recognition of multiple RNA signals by the endonuclease was further supported by the findings that dinucleotide ApG, free CAP structures and RNA without the CAP structure inhibited the endonuclease activity to different extents. In the presence of four species of ribonucleoside 5'-triphosphates, the endonucleolytically cleaved fragments with the CAP-1 structure were incorporated into polynucleotides, supporting the concept that they are used as the primers for the transcription. The initial nucleotide linked to the primers was a G residue, the nucleotide complementary to the second base of the 3' termini of the vRNA segments.

Animals↗
PubMed2005

Capped RNA transcripts of full-length cDNA clones of swine hepatitis E virus are replication competent when transfected into Huh7 cells and infectious when intrahepatically inoculated into pigs.

Swine hepatitis E virus (swine HEV), the first animal strain of HEV to be isolated, is a zoonotic agent. We report here the construction and in vitro and in vivo characterizations of infectious cDNA clones of swine HEV. Eight overlapping fragments spanning the entire genome were amplified by reverse transcription-PCR and assembled into a full-length cDNA clone, clone C, which contained 14 mutations compared to the consensus sequence of swine HEV. RNA transcripts from clone C were not infectious, as determined by intrahepatic inoculation into pigs and by in vitro transfection of Huh7 cells. Multiple site-based site-directed mutagenesis was performed to generate three new cDNA clones (pSHEV-1, pSHEV-2, and pSHEV-3) which differed from each other. The transfection of capped RNA transcripts into human liver Huh7 cells resulted in the synthesis of both ORF2 capsid and ORF3 proteins, indicating that the cDNA clones were replication competent. Each of the three clones resulted in active swine HEV infections after the intrahepatic inoculation of pigs with capped RNA transcripts. The patterns of seroconversion, viremia, and fecal virus shedding for pigs inoculated with RNA transcripts from clones pSHEV-2 and pSHEV-3 were similar to each other and to those for pigs inoculated with wild-type swine HEV, suggesting that the nucleotide differences between these two cDNA clones were not critical for replication. Pigs inoculated with RNA transcripts from clone pSHEV-1, which contained three nonsilent mutations in the ORF2 capsid gene, had a delayed appearance of seroconversion and fecal virus shedding and had undetectable viremia. The availability of these infectious cDNA clones affords us an opportunity to understand the mechanisms of cross-species infection by constructing chimeric human and swine HEVs.

Animals↗
PubMed1994

Purification of human U6 small nuclear RNA capping enzyme. Evidence for a common capping enzyme for gamma-monomethyl-capped small RNAs.

To understand the mechanism of gamma-monomethyl (meppp) cap formation, we attempted to identify and purify the U6 small nuclear RNA capping enzyme. Although more than one protein was cross-linked to U6, 7SK, B2, or plant U3 RNA, only one protein of approximately 130 kDa was common to all four meppp-capped RNAs; 5 S RNA, which is an uncapped RNA, was not cross-linked to this protein. In addition to specific cross-linking with meppp-capped RNAs, an approximately 130-kDa protein was also cross-linked to 3H-labeled AdoMet. We purified the capping enzyme from a HeLa cell S-100 extract by several successive chromatographic steps, and an approximately 130-kDa protein was purified along with the capping activity. The capping activity and the approximately 130-kDa protein also cosedimented on a glycerol gradient. The purified enzyme catalyzed meppp cap formation of U6, 7SK, B2, and plant U3 RNA, and this enzyme is probably responsible for the capping of multiple RNAs in vivo. The capping activity is distinct from U6 snRNA N6-adenosine methyltransferase, and this is the first methyltransferase to be purified that methylates gamma-phosphate residues in RNAs.

Catalysis↗
PubMed2004

Functional interactions of RNA-capping enzyme with factors that positively and negatively regulate promoter escape by RNA polymerase II.

Capping of the 5' ends of nascent RNA polymerase II transcripts is the first pre-mRNA processing event in all eukaryotic cells. Capping enzyme (CE) is recruited to transcription complexes soon after initiation by the phosphorylation of Ser-5 of the carboxyl-terminal domain of the largest subunit of RNA polymerase II. Here, we analyze the role of CE in promoter clearance and its functional interactions with different factors that are involved in promoter clearance. FCP1-mediated dephosphorylation of the carboxyl-terminal domain results in a drastic decrease in cotranscriptional capping efficiency but is reversed by the presence of DRB sensitivity-inducing factor (DSIF). These results suggest involvement of DSIF in CE recruitment. Importantly, CE relieves transcriptional repression by the negative elongation factor, indicating a critical role of CE in the elongation checkpoint control mechanism during promoter clearance. This functional interaction between CE and the negative elongation factor documents a dynamic role of CE in promoter clearance beyond its catalytic activities.

Nuclear Proteins↗
PubMed1992

Spatial localization of distinct rheumatic disease-associated epitopes and the RNA "cap" of the U1 snRNP particle.

The spatial organization of two rheumatic disease-associated epitopes and the RNA "cap" structure of the U1 small nuclear ribonucleoprotein (snRNP2) was analyzed both in situ and in vitro by two independent interference immuno-assays. Sm and RNP autoantibodies, associated with systemic lupus erythematosus and mixed connective tissue disease, respectively, were used to probe the epitope locations. The Sm epitope on the U1 snRNP structure was localized proximal to the RNP. Experiments with an anti-m7G (mRNA "cap") monoclonal antibody revealed that an in situ association of the Sm and RNP epitopes with the mRNA "cap" structure may exist. Our findings, together with previous observations by others, suggest a model for the spatial arrangement of these rheumatic disease-associated protein epitopes, and the U1 RNA within the U1 snRNP particle.

Antibodies, Monoclonal↗