RTPase Activity Research Articles

The large protein \u2018L\u2019 of Peste-des-petits-ruminants virus exhibits RNA triphosphatase activity, the first enzyme in mRNA capping pathway

Peste-des-petits-ruminants is a highly contagious and fatal disease of goats and sheep caused by non-segmented, negative strand RNA virus belonging to the Morbillivirus genus—Peste-des-petits-ruminants virus (PPRV) which is evolutionarily closely related to Rinderpest virus (RPV). The large protein ‘L’ of the members of this genus is a multifunctional catalytic protein, which transcribes and replicates the viral genomic RNA as well as possesses mRNA capping, methylation and polyadenylation activities; however, the detailed mechanism of mRNA capping by PPRV L protein has not been studied. We have found earlier that the L protein of RPV has RNA triphosphatase (RTPase), guanylyltransferase (GTase) and methyltransferase activities, and unlike vesicular stomatitis virus (VSV), follows the conventional pathway of mRNA capping. In the present work, using a 5′-end labelled viral RNA as substrate, we demonstrate that PPRV L protein has RTPase activity when present in the ribonucleoprotein complex of purified virus as well as recombinant L–P complex expressed in insect cells. Further, a minimal domain in the C-terminal region (aa1640–1840) of the L protein has been expressed in E. coli and shown to exhibit RTPase activity. The RTPase activity of PPRV L protein is metal-dependent and functions with a divalent cation, either magnesium or manganese. In addition, RTPase associated nucleotide triphosphatase activity (NTPase) of PPRV L protein is also demonstrated. This work provides the first detailed study of RTPase activity and identifies the RTPase domain of PPRV L protein.

The RNA triphosphatase domain of L protein of Rinderpest virus exhibits pyrophosphatase and tripolyphosphatase activities.

L protein of the Rinderpest virus, an archetypal paramyxovirus possesses RNA-dependent RNA polymerase activity which transcribes the genome into mRNAs as well as replicates the RNA genome. The protein also possesses RNA triphosphatase (RTPase), guanylyltransferase (GTase) and methyltransferase enzyme activities responsible for capping the mRNAs in a conventional pathway similar to that of the host pathway. Subsequent to the earlier characterization of the GTase activity of L protein and identification of the RTPase domain of the L protein, we report here, additional enzymatic activities associated with the RTPase domain. We have characterized the pyrophosphatase and tripolyphosphatase activities of the L-RTPase domain which are metal-dependent and proceed much faster than the RTPase activity. Interestingly, the mutant proteins E1645A and E1647A abrogated the pyrophosphatase and tripolyphosphatase significantly, indicating a strong overlap of the active sites of these activities with that of RTPase. We discuss the likely role of GTase-associated L protein pyrophosphatase in the polymerase function. We also discuss a possible biological role for the tripolyphosphatase activity hitherto considered insignificant for the viruses possessing such activity.

A carboxy terminal domain of the L protein of rinderpest virus possesses RNA triphosphatase activity – The first enzyme in the viral mRNA capping pathway

The large protein L of negative-sense RNA viruses is a multifunctional protein involved in transcription and replication of genomic RNA. It also possesses enzymatic activities involved in capping and methylation of viral mRNAs. The pathway for mRNA capping followed by the L protein of the viruses in the Morbillivirus genus has not been established, although it has been speculated that these viruses may follow the unconventional capping pathway as has been shown for some viruses of Rhabdoviridae family.We had earlier shown that the large protein L of Rinderpest virus expressed as recombinant L–P complex in insect cells as well as the ribonucleoprotein complex from purified virus possesses RNA triphosphatase (RTPase) and guanylyltransferase activities, in addition to RNA dependent RNA polymerase activity. In the present work, we demonstrate that RTPase as well as nucleoside triphosphatase (NTPase) activities are exhibited by a subdomain of the L protein in the C terminal region (a.a. 1640–1840). The RTPase activity depends absolutely on a divalent cation, either magnesium or manganese. Both the RTPase and NTPase activities of the protein show dual metal specificity. Two mutant proteins having alanine mutations in the glutamic acid residues in motif-A of the RTPase domain did not show RTPase activity, while exhibiting reduced NTPase activity suggesting overlapping active sites for the two enzymatic functions. The RTPase and NTPase activities of the L subdomain resemble those of the Vaccinia capping enzyme D1 and the baculovirus LEF4 proteins.

Crystallographic Analysis of Rotavirus NSP2-RNA Complex Reveals Specific Recognition of 5' GG Sequence for RTPase Activity.

Rotavirus nonstructural protein NSP2, a functional octamer, is critical for the formation of viroplasms, which are exclusive sites for replication and packaging of the segmented double-stranded RNA (dsRNA) rotavirus genome. As a component of replication intermediates, NSP2 is also implicated in various replication-related activities. In addition to sequence-independent single-stranded RNA-binding and helix-destabilizing activities, NSP2 exhibits monomer-associated nucleoside and 5' RNA triphosphatase (NTPase/RTPase) activities that are mediated by a conserved H225 residue within a narrow enzymatic cleft. Lack of a 5' γ-phosphate is a common feature of the negative-strand RNA [(-)RNA] of the packaged dsRNA segments in rotavirus. Strikingly, all (-)RNAs (of group A rotaviruses) have a 5' GG dinucleotide sequence. As the only rotavirus protein with 5' RTPase activity, NSP2 is implicated in the removal of the γ-phosphate from the rotavirus (-)RNA. To understand how NSP2, despite its sequence-independent RNA-binding property, recognizes (-)RNA to hydrolyze the γ-phosphate within the catalytic cleft, we determined a crystal structure of NSP2 in complex with the 5' consensus sequence of minus-strand rotavirus RNA. Our studies show that the 5' GG of the bound oligoribonucleotide interacts extensively with highly conserved residues in the NSP2 enzymatic cleft. Although these residues provide GG-specific interactions, surface plasmon resonance studies suggest that the C-terminal helix and other basic residues outside the enzymatic cleft account for sequence-independent RNA binding of NSP2. A novel observation from our studies, which may have implications in viroplasm formation, is that the C-terminal helix of NSP2 exhibits two distinct conformations and engages in domain-swapping interactions, which result in the formation of NSP2 octamer chains.

NTPase and 5′-RNA Triphosphatase Activities of Chikungunya Virus nsP2 Protein

Chikungunya virus (CHIKV) is an insect borne virus (genus: Alphavirus) which causes acute febrile illness in humans followed by a prolonged arthralgic disease that affects the joints of the extremities. Re-emergence of the virus in the form of outbreaks in last 6–7 years has posed a serious public health problem. CHIKV has a positive sense single stranded RNA genome of about 12,000 nt. Open reading frame 1 of the viral genome encodes a polyprotein precursor, nsP1234, which is processed further into different non structural proteins (nsP1, nsP2, nsP3 and nsP4). Sequence based analyses have shown helicase domain at the N-terminus and protease domain at C-terminus of nsP2. A detailed biochemical analysis of NTPase/RNA helicase and 5′-RNA phosphatase activities of recombinant CHIKV-nsP2T protein (containing conserved NTPase/helicase motifs in the N-terminus and partial papain like protease domain at the C-terminus) was carried out. The protein could hydrolyze all NTPs except dTTP and showed better efficiency for ATP, dATP, GTP and dGTP hydrolysis. ATP was the most preferred substrate by the enzyme. CHIKV-nsP2T also showed 5′-triphosphatase (RTPase) activity that specifically removes the γ-phosphate from the 5′ end of RNA. Both NTPase and RTPase activities of the protein were completely dependent on Mg2+ ions. RTPase activity was inhibited by ATP showing sharing of the binding motif by NTP and RNA. Both enzymatic activities were drastically reduced by mutations in the NTP binding motif (GKT) and co-factor, Mg2+ ion binding motif (DEXX) suggesting that they have a common catalytic site.

1235 A phase-I study of the combination of intravenous reovirus (REOLYSIN ®) and gemcitabine in patients with advanced cancer

A low-copy component of mammalian reovirus particles is μ2, an 83-kDa protein encoded by the M1 viral genome segment and packaged within the viral core. Previous studies have identified μ2 as a nucleoside triphosphate phosphohydrolase (NTPase) as well as an RNA 5′-triphosphate phosphohydrolase (RTPase), putatively involved in reovirus RNA synthesis and/or 5′-capping. Other studies have identified μ2 as a microtubule-binding protein, which also associates with the viral factory matrix protein μNS and thereby anchors the factories to cellular microtubules during infections by most reovirus strains. To extend studies of μ2 functions during infection, we tested a small interfering RNA (siRNA) directed against the M1 plus-strand RNAs of reovirus strains Type 1 Lang (T1L) and Type 3 Dearing (T3D). The siRNA strongly suppressed μ2 expression by either strain and reduced infectious yields in a strain-dependent manner. This first strain difference was genetically mapped to the M1 genome segment and tentatively assigned to a single μ2 sequence polymorphism, Pro/Ser208, which also determines a T1L–T3D strain difference in microtubule association. The siRNA-based defect in μ2 expression was rescued by plasmids, containing silent mutations in the siRNA-targeted sequence, which encoded either T1L or T3D μ2, but the growth defect was rescued only by T1L μ2. This second strain difference was also mapped to Pro/Ser208, in that swapping this one residue between T1L and T3D μ2 reversed the rescue phenotypes. Thus, the T1L–T3D strain difference in μ2–microtubule association was correlated not only with the extent of reduction in infectious yields by the siRNA but also with the extent of rescue by plasmid-derived μ2. In addition, the rescue capacity of T1L μ2 was abrogated by nocodazole treatment, providing independent evidence for the importance of μ2–microtubule association in plasmid-based rescue. In two separate cases, the results revealed functional differences between virus- and plasmid-derived μ2. Ala substitutions within the NTP-binding motif of T1L μ2 also abrogated its rescue capacity, suggesting that the NTPase or RTPase activity of μ2 is additionally required for effective viral growth.

Analysis of the nucleoside triphosphatase, RNA triphosphatase, and unwinding activities of the helicase domain of dengue virus NS3 protein

The helicase domain of dengue virus NS3 protein (DENV NS3H) contains RNA-stimulated nucleoside triphosphatase (NTPase), ATPase/helicase, and RNA 5′-triphosphatase (RTPase) activities that are essential for viral RNA replication and capping. Here, we show that DENV NS3H unwinds 3′-tailed duplex with an RNA but not a DNA loading strand, and the helicase activity is poorly processive. The substrate of the divalent cation-dependent RTPase activity is not restricted to viral RNA 5′-terminus, a protruding 5′-terminus made the RNA 5′-triphosphate readily accessible to DENV NS3H. DENV NS3H preferentially binds RNA to DNA, and the functional interaction with RNA is sensitive to ionic strength.

Avian reovirus core protein μA expressed in Escherichia coli possesses both NTPase and RTPase activities

Analysis of the amino acid sequence of core protein muA of avian reovirus has indicated that it may share similar functions to protein mu2 of mammalian reovirus. Since mu2 displayed both nucleotide triphosphatase (NTPase) and RNA triphosphatase (RTPase) activities, the purified recombinant muA ( muA) was designed and used to test these activities. muA was thus expressed in bacteria with a 4.5 kDa fusion peptide and six His tags at its N terminus. Results indicated that muA possessed NTPase activity that enabled the protein to hydrolyse the beta-gamma phosphoanhydride bond of all four NTPs, since NDPs were the only radiolabelled products observed. The substrate preference was ATP>CTP>GTP>UTP, based on the estimated k(cat) values. Alanine substitutions for lysines 408 and 412 (K408A/K412A) in a putative nucleotide-binding site of muA abolished NTPase activity, further suggesting that NTPase activity is attributable to protein muA. The activity of muA is dependent on the divalent cations Mg(2+) or Mn(2+), but not Ca(2+) or Zn(2+). Optimal NTPase activity of muA was achieved between pH 5.5 and 6.0. In addition, muA enzymic activity increased with temperature up to 40 degrees C and was almost totally inhibited at temperatures higher than 55 degrees C. Tests of phosphate release from RNA substrates with muA or K408A/K412A muA indicated that muA, but not K408A/K412A muA, displayed RTPase activity. The results suggested that both NTPase and RTPase activities of muA might be carried out at the same active site, and that protein muA could play important roles during viral RNA synthesis.

Silencing and complementation of reovirus core protein μ2: Functional correlations with μ2–microtubule association and differences between virus- and plasmid-derived μ2

A low-copy component of mammalian reovirus particles is μ2, an 83-kDa protein encoded by the M1 viral genome segment and packaged within the viral core. Previous studies have identified μ2 as a nucleoside triphosphate phosphohydrolase (NTPase) as well as an RNA 5′-triphosphate phosphohydrolase (RTPase), putatively involved in reovirus RNA synthesis and/or 5′-capping. Other studies have identified μ2 as a microtubule-binding protein, which also associates with the viral factory matrix protein μNS and thereby anchors the factories to cellular microtubules during infections by most reovirus strains. To extend studies of μ2 functions during infection, we tested a small interfering RNA (siRNA) directed against the M1 plus-strand RNAs of reovirus strains Type 1 Lang (T1L) and Type 3 Dearing (T3D). The siRNA strongly suppressed μ2 expression by either strain and reduced infectious yields in a strain-dependent manner. This first strain difference was genetically mapped to the M1 genome segment and tentatively assigned to a single μ2 sequence polymorphism, Pro/Ser208, which also determines a T1L–T3D strain difference in microtubule association. The siRNA-based defect in μ2 expression was rescued by plasmids, containing silent mutations in the siRNA-targeted sequence, which encoded either T1L or T3D μ2, but the growth defect was rescued only by T1L μ2. This second strain difference was also mapped to Pro/Ser208, in that swapping this one residue between T1L and T3D μ2 reversed the rescue phenotypes. Thus, the T1L–T3D strain difference in μ2–microtubule association was correlated not only with the extent of reduction in infectious yields by the siRNA but also with the extent of rescue by plasmid-derived μ2. In addition, the rescue capacity of T1L μ2 was abrogated by nocodazole treatment, providing independent evidence for the importance of μ2–microtubule association in plasmid-based rescue. In two separate cases, the results revealed functional differences between virus- and plasmid-derived μ2. Ala substitutions within the NTP-binding motif of T1L μ2 also abrogated its rescue capacity, suggesting that the NTPase or RTPase activity of μ2 is additionally required for effective viral growth.

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.

Histidine Triad-like Motif of the Rotavirus NSP2 Octamer Mediates both RTPase and NTPase Activities

Rotavirus NSP2 is an abundant non-structural RNA-binding protein essential for forming the viral factories that support replication of the double-stranded RNA genome. NSP2 exists as stable doughnut-shaped octamers within the infected cell, representing the tail-to-tail interaction of two tetramers. Extending diagonally across the surface of each octamer are four highly basic grooves that function as binding sites for single-stranded RNA. Between the N and C-terminal domains of each monomer is a deep electropositive cleft containing a catalytic site that hydrolyzes the γ-β phosphoanhydride bond of any NTP. The catalytic site has similarity to those of the histidine triad (HIT) family of nucleotide-binding proteins. Due to the close proximity of the grooves and clefts, we investigated the possibility that the RNA-binding activity of the groove promoted the insertion of the 5′-triphosphate moiety of the RNA into the cleft, and the subsequent hydrolysis of its γ-β phosphoanhydride bond. Our results show that NSP2 hydrolyzes the γP from RNAs and NTPs through Mg 2+-dependent activities that proceed with similar reaction velocities, that require the catalytic His225 residue, and that produce a phosphorylated intermediate. Competition assays indicate that although both substrates enter the active site, RNA is the preferred substrate due to its higher affinity for the octamer. The RNA triphosphatase (RTPase) activity of NSP2 may account for the absence of the 5′-terminal γP on the (−) strands of the double-stranded RNA genome segments. This is the first report of a HIT-like protein with a multifunctional catalytic site, capable of accommodating both NTPs and RNAs during γP hydrolysis.

The RNA helicase, nucleotide 5′-triphosphatase, and RNA 5′-triphosphatase activities of Dengue virus protein NS3 are Mg 2+-dependent and require a functional Walker B motif in the helicase catalytic core

The nonstructural protein 3 (NS3) of Dengue virus (DV) is a multifunctional enzyme carrying activities involved in viral RNA replication and capping: helicase, nucleoside 5′-triphosphatase (NTPase), and RNA 5′-triphosphatase (RTPase). Here, a 54-kDa C-terminal domain of NS3 (ΔNS3) bearing all three activities was expressed as a recombinant protein. Structure-based sequence analysis in comparison with Hepatitis C virus (HCV) helicase indicates the presence of a HCV-helicase-like catalytic core domain in the N-terminal part of ΔNS3, whereas the C-terminal part seems to be different. In this report, we show that the RTPase activity of ΔNS3 is Mg 2+-dependent as are both helicase and NTPase activities. Mutational analysis shows that the RTPase activity requires an intact NTPase/helicase Walker B motif in the helicase core, consistent with the fact that such motifs are involved in the coordination of Mg 2+. The R513A substitution in the C-terminal domain of ΔNS3 abrogates helicase activity and strongly diminishes RTPase activity, indicating that both activities are functionally coupled. DV RTPase seems to belong to a new class of Mg 2+-dependent RTPases, which use the active center of the helicase/NTPase catalytic core in conjunction with elements in the C-terminal domain.

The Caenorhabditis elegansmRNA 5′-Capping Enzyme: IN VITRO AND IN VIVO CHARACTERIZATION

Eukaryotic mRNA capping enzymes are bifunctional, carrying both RNA triphosphatase (RTPase) and guanylyltransferase (GTase) activities. The Caenorhabditis elegans CEL-1 capping enzyme consists of an N-terminal region with RTPase activity and a C-terminal region that resembles known GTases, However, CEL-1 has not previously been shown to have GTase activity. Cloning of the cel-1 cDNA shows that the full-length protein has 623 amino acids, including an additional 38 residues at the C termini and 12 residues at the N termini not originally predicted from the genomic sequence. Full-length CEL-1 has RTPase and GTase activities, and the cDNA can functionally replace the capping enzyme genes in Saccharomyces cerevisiae. The CEL-1 RTPase domain is related by sequence to protein-tyrosine phosphatases; therefore, mutagenesis of residues predicted to be important for RTPase activity was carried out. CEL-1 uses a mechanism similar to protein-tyrosine phosphatases, except that there was not an absolute requirement for a conserved acidic residue that acts as a proton donor. CEL-1 shows a strong preference for RNA substrates of at least three nucleotides in length. RNA-mediated interference in C. elegans embryos shows that lack of CEL-1 causes development to arrest with a phenotype similar to that seen when RNA polymerase II elongation activity is disrupted. Therefore, capping is essential for gene expression in metazoans.

A Plasmodium falciparum protein related to fungal RNA 5′-triphosphatases

Messenger RNA of eukaryotic cells and most of their viruses carry a ‘cap’ structure at their 5 end. RNA 5 -triphosphatase (RTPase) activity is required for the first step of cap biosynthesis. This activity removes the -phosphate from the 5 end of a nascent mRNA to leave a diphosphate end. mRNA guanylyltransferase (GTase) subsequently adds GMP in a 5 –5 orientation to form the structure, GpppN1-. These two activities are tightly associated and co-purify as ‘mRNA capping enzyme’ [1]. One or more methyltransferase completes the capping modifications. At least two groups of cellular RTPases have been identified. Metazoan capping enzymes consist of a single polypeptide with two separable domains; a GTase domain and a RTPase domain that resembles the protein tyrosine phosphatases (PTPs) [2–4]. This type of RTPase domain appears to use a mechanism similar to that of PTPs, as a conserved nucleophilic cysteine is required for activity [2,5–8]. The capping enzymes of the yeasts Saccharomyces cere isiae and Candida albicans define a second class of RTPases. In these organisms, GTase and RTPase are carried by distinct proteins that form a heterodimer complex. The amino acid sequence of the catalytic region of yeast RTPase subunit has no obvious similarity to PTPs [9,10]. Unlike metazoan RTPases, the yeast proteins require divalent cations for activity [11,12]. It has been suggested that fungal RTPases comprise a new family of metal-dependent nucleotide phosphohydrolase (NTPase)/RTPase. This family may also include capping RTPases encoded by some DNA viruses. Although there is no extensive sequence similarity between the viral and yeast RTPases, several short sequence elements found in all of these proteins have been shown to be essential for the activity [11–14]. To identify other possible members of the yeast RTPase family, we used the BLAST algorithm [15] to search GenBank for proteins with similarity to Cet1, the S. cere isiae capping enzyme RTPase subunit [9]. Known relatives of Cet1 include CaCet1, the C. albicans RTPase subunit [10] and Ctl1 (also known as Cth1), a second RTPase from S. cere isiae whose function is unknown but nonessential for viability [16,17]. In addition to these fungal proteins, an open reading frame from human parasite Plasmodium falciparum (PFC0985c, accession number: CAB39040) was found to have significant similarity to Cet1 (P=3e-5). No related proteins were found in the complete Drosophila, C. elegans, or human genomes. PFC0985c is a hypothetical protein with 591 amino acids whose predicted size is 69 kDa. This protein has 109 asparagine residues and 89 acidic residues that are largely clustered in the N-terminal (amino acids 100– 250) and the C-terminal (aa 405–591) regions (Fig. Abbre iations: CIP, calf intestine alkaline phopshatase; GTase, mRNA guanylytransferase; IPTG, isopropyl -D-thiogalactopyranoside; NTPase, nucleotide phosphohydrolase; PCR, polymerase chain reaction; PEI, polyethyleneimine; PTP, protein tyrosine phosphatase; RTPase, RNA 5 -triphosphatase; TLC, thin-layer chromatography. * Corresponding author. Tel.: +1-617-4320696; fax: +1-6177380516. E-mail address: steveb@hms.harvard.edu (S. Buratowski). 1 Present address: Shionogi BioResearch Corporation, 45 Hartwell Avenue, Lexington, MA 02421, USA.