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1.
We reported previously that polycytidylate [poly(C)]-dependent RNA polymerase activity was a property of small spherical or triangular reovirus-specific particles which sedimented at 13 to 19S and were composed solely of the reovirus protein, sigma NS. Depending on the fraction of cellular extracts from which they were obtained, these particles exhibited marked differences in stability. Most 13 to 19S particles from a particular fraction repeatedly disaggregated into smaller 4 to 5S subunits with no enzymatic activity. Disruption of many particles could be prevented and polymerase activity retained after these particles had bound different single-stranded (ss) RNAs. Our previous results indicated that there was heterogeneity among the 13 to 19S particles in that possession of poly(C)-dependent RNA polymerase activity was a property of only some. Support for this heterogeneity was derived from the demonstration in this report that there were at least three types of binding sites present within particles in any purified preparation: (i) those binding only poly(C); (ii) those binding only reovirus ss RNAs; and (iii) those binding one or the other, but not both at the same time. It is suggested that only those particles able to bind either poly(C) or reovirus ss RNAs had poly(C)-dependent RNA polymerase activity, as reovirus ss RNAs markedly inhibited the polymerase activity. All three size classes of reovirus ss RNAs were equally effective in binding, but once bound, they were not copied. It is possible that heterogeneity in binding capacity of different particles comprised of only one protein, sigma NS, could result from the ability of subunits containing this protein to assemble into slightly different 13 to 19S particles with specificity of binding or polymerase activity conferred by the configuration of the assembled particles. The high capacity of sigma NS to bind many different nucleic acids with some specificity suggests that these particles may act during infection as condensing agents to bring together 10 reovirus ss RNA templates in preparation for double-stranded RNA synthesis.  相似文献   

2.
Characterization of purified DNA-dependent RNA polymerase (EC 2.7.7.6) of Caulobacter crescentus, strain CB15 has led to the conclusion that this enzyme catalyzes poly(A) synthesis in the absence of template. Poly(A) synthetase activity co-purifies with both holoenzyme and core polymerase on DNA-cellulose columns, and core polymerase purified to 98% homogeneity by glycerol gradient centrifugation is still capable of catalyzing poly(A) polymerization. Both RNA synthesis and poly(A) polymerization activities are sensitive to rifampicin. In addition, RNA polymerase purified from partially rifampicin-sensitive mutants exhibits the same partial sensitivity in vitro to the drug in the synthesis of RNA and poly(A). The enzyme used in these studies was prepared by a simple method which allows a high yield of pure RNA polymerase from large batches of exponential cells. The procedure includes high speed centrifugation of cell extracts, DEAE-cellulose column, DNA-affinity chromatography, and low salt glycerol gradient centrifugation. Holoenzyme can be resolved into core and sigma subunit by either DNA-cellulose chromatography or glycerol gradient centrifugation, and the latter step allows recovery of pure sigma factor.  相似文献   

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This report describes the complete nucleotide sequence of human reovirus (Dearing strain) genome segment S3. Previous studies indicated that this RNA encodes the major non-structural viral polypeptide sigma NS, a protein that binds ssRNAs (Huisman & Joklik, Virology 70, 411-424, 1976) and has a poly(C)-dependent poly(G) polymerase activity (Gomatos et al., J. Virol. 39, 115-124, 1981). The genome segment consists of 1,198 nucleotides and possesses an open reading frame that extends 366 codons from the first AUG triplet (residues 28-30). There is no significant sequence homology between the plus strand of genome segment S3 and that of genome segment S2 determined previously (Cashdollar et al., PNAS 79, 7644-7648, 1982). However, S3 RNA has significant dyad symmetry and regions that can potentially hybridize (delta G = -26 KCal/mole) with S2 RNA. From the predicted amino acid sequence a possible secondary structure for sigma NS protein was determined. Structural features of reovirus RNA and sigma NS are discussed in relation to their role(s) in viral genome assembly.  相似文献   

5.
Hepatitis C virus (HCV) non-structural protein 5B (NS5B) is an RNA replicase. We expressed full-length NS5B (591 amino acid residues) in Escherichia coli as a fusion protein with maltose binding protein (MBP-NS5B). MBP-NS5B was recovered in the soluble fraction after centrifugation at 40,000 x g and affinity-purified with amylose resin. The purified MBP-NS5B had a high-level of poly (A), oligo (U)-dependent UMP incorporation with a Km of 2 microM for UTP. Surprisingly, the enzymatically active MBP-NS5B was sedimented by ultracentrifugation at 160,000 x g. The pellet contained 16S and 23S ribosomal RNAs, suggesting that ribosomes were associated with MBP-NS5B. Ribosomes and MBP-NS5B were subsequently co-purified on amylose resin. Deletion study revealed that either the N-terminal (amino acid residues 1-107) or the C-terminal (amino acid residues 498-591) region of NS5B were sufficient for this association with ribosomes. We further found that NS5B also bound with human ribosomes. Our results implicate a novel mechanism of coupling between replication and translation of the viral genome in the life cycle of HCV.  相似文献   

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A poliovirus-specific polyuridylic acid [poly(U)] polymerase that copies a polyadenylic acid template complexed to an oligouridylic acid primer was isolated from the membrane fraction of infected HeLa cells and was found to sediment at 4 to 5S on a linear 5 to 20% glycerol gradient. When the poly(U) polymerase was isolated from cells labeled with [(35)S]methionine and was analyzed by glycerol gradient centrifugation and polyacrylamide gel electrophoresis, the position of only one viral protein was found to correlate with the location of enzyme activity. This protein had an apparent molecular weight of 62,500 based on its electrophoretic mobility relative to that of several molecular weight standards and was designated p63. When the poly(U) polymerase was isolated from the soluble fraction of a cytoplasmic extract, the activity was found to sediment at about 7S. In this case, however, both p63 and NCVP2 (77,000-dalton precursor of p63) cosedimented with the 7S activity peak. When the 7S polymerase activity was purified by phosphocellulose chromatography, both p63 and NCVP2 were found to co-chromatograph with poly(U) polymerase activity. The poliovirus replicase complexed with its endogenous RNA template was isolated from infected cells labeled with [(35)S]methionine and was centrifuged through a linear 15 to 30% glycerol gradient. The major viral polypeptide component in a 26S peak of replicase activity was p63, but small amounts of other poliovirus proteins were also present. When the replicase-template complex was treated with RNase T1 before centrifugation, a single peak of activity was found that sedimented at 20S and contained only labeled p63. Thus, p63 was found to be the only viral polypeptide in the replicase bound to its endogenous RNA template, and appears to be active as a poly(U) polymerase either as a monomer protein or as a 7S complex.  相似文献   

9.
A template independent poly (A)·poly (U) synthesizing activity has been isolated from Bacillus subtilis. This activity is eluted from a DNA-cellulose column along with DNA-dependent RNA polymerase. The column fractions which exhibit this activity contain RNA polymerase holoenzyme plus a polypeptide which is slightly larger than sigma factor; pure RNA polymerase holoenzyme did not synthesize poly (A)·poly (U). The activity was dependent on the presence of ATP, UTP, and Mn++ (Mg++ could not substitute), and was inhibited by rifampicin, streptolydigin, and Cibacron Blue. The incorporation of nucleotides was not linear with time, but appeared after a lag period. The results suggest that a modified form of DNA-dependent RNA polymerase analogous to Escherichia coli holoenzyme II is catalyzing the synthesis of poly (A)·poly (U).  相似文献   

10.
Dengue fever, a neglected emerging disease for which no vaccine or antiviral agents exist at present, is caused by dengue virus, a member of the Flavivirus genus, which includes several important human pathogens, such as yellow fever and West Nile viruses. The NS5 protein from dengue virus is bifunctional and contains 900 amino acids. The S-adenosyl methionine transferase activity resides within its N-terminal domain, and residues 270 to 900 form the RNA-dependent RNA polymerase (RdRp) catalytic domain. Viral replication begins with the synthesis of minus-strand RNA from the dengue virus positive-strand RNA genome, which is subsequently used as a template for synthesizing additional plus-strand RNA genomes. This essential function for the production of new viral particles is catalyzed by the NS5 RdRp. Here we present a high-throughput in vitro assay partly recapitulating this activity and the crystallographic structure of an enzymatically active fragment of the dengue virus RdRp refined at 1.85-A resolution. The NS5 nuclear localization sequences, previously thought to fold into a separate domain, form an integral part of the polymerase subdomains. The structure also reveals the presence of two zinc ion binding motifs. In the absence of a template strand, a chain-terminating nucleoside analogue binds to the priming loop site. These results should inform and accelerate the structure-based design of antiviral compounds against dengue virus.  相似文献   

11.
DNA polymerase was purified from Drosophila melanogaster embryos by a combination of phosphocellulose adsorption, Sepharose 6B gel filtration, and DEAE-cellulose chromatography. Three enzyme forms, designated enzymes I, II, and III, were separated by differential elution from DEAE-cellulose and were further purified by glycerol gradient centrifugation. Purification was monitored with two synthetic primer-templates, poly(dA) . (dT)-16 and poly(rA) . (dT)-16. At the final step of purification, enzymes I, II, and III were purified approximately 1700-fold, 2000-fold and 1000-fold, respectively, on the basis of their activities with poly(dA) . (dT)-16. The DNA polymerase eluted heterogeneously as anomalously high-molecular-weight molecules from Sepharose 6B gel filtration columns. On DEAE-cellulose chromatography enzymes I and II eluted as distinct peaks and enzyme III eluted heterogeneously. On glycerol velocity gradients enzyme I sedimented at 5.5-7.3 S, enzyme II sedimented at 7.3-8.3 S, and enzyme III sedimented at 7.3-9.0 S. All enzymes were active with both synthetic primer-templates, except the 9.0 S component of enzyme III, which was inactive with poly(rA) . (dT)-16. Non-denaturing polyacrylamide gel electrophoresis did not separate poly(dA) . (dT)-16 activity from poly(rA) . (dT)-16 activity. The DNA polymerase preferred poly(dA) . (dT)-16 (with Mg2+) as a primer-template, although it was also active with poly(rA) . (dT)-16 (with Mn2+), and it preferred activated calf thymus DNA to native or heat-denatured calf thymus DNA. All three primer-template activities were inhibited by N-ethylmaleimide. Enzyme activity with activated DNA and poly(dA) . (dT)-16 was inhibited by K+ and activity with poly(rA) . (dT)-16 was stimulated by K+ and by spermidine. The optimum pH for enzyme activity with the synthetic primer-templates was 8.5. The DNA polymerases did not exhibit deoxyribonuclease or ATPase activities. The results of this study suggest that the forms of DNA polymerase from Drosophila embryos have physical properties similar to those of DNA polymerase-alpha and enzymatic properties similar to those of all three vertebrate DNA polymerases.  相似文献   

12.
The NS5B protein of the hepatitis C virus (HCV) is an RNA-dependent RNA polymerase (RdRp) (S.-E. Behrens, L. Tomei, and R. De Francesco, EMBO J. 15:12-22, 1996) that is assumed to be required for replication of the viral genome. To further study the biochemical and structural properties of this enzyme, an NS5B-hexahistidine fusion protein was expressed with recombinant baculoviruses in insect cells and purified to near homogeneity. The enzyme was found to have a primer-dependent RdRp activity that was able to copy a complete in vitro-transcribed HCV genome in the absence of additional viral or cellular factors. Filter binding assays and competition experiments showed that the purified enzyme binds RNA with no clear preference for HCV 3'-end sequences. Binding to homopolymeric RNAs was also examined, and the following order of specificity was observed: poly(U) > poly(G) > poly(A) > poly(C). An inverse order was found for the RdRp activity, which used poly(C) most efficiently as a template but was inactive on poly(U) and poly(G), suggesting that a high binding affinity between polymerase and template interferes with processivity. By using a mutational analysis, four amino acid sequence motifs crucial for RdRp activity were identified. While most substitutions of conserved residues within these motifs severely reduced the enzymatic activities, a single substitution in motif D which enhanced the RdRp activity by about 50% was found. Deletion studies indicate that amino acid residues at the very termini, in particular the amino terminus, are important for RdRp activity but not for RNA binding. Finally, we found a terminal transferase activity associated with the purified enzyme. However, this activity was also detected with NS5B proteins with an inactive RdRp, with an NS4B protein purified in the same way, and with wild-type baculovirus, suggesting that it is not an inherent activity of NS5B.  相似文献   

13.
The time course of vaccinia deoxyribonucleic acid (DNA)-dependent ribonucleic acid (RNA) polymerase synthesis and its intracellular localization were studied with virus-infected HeLa cells. Viral RNA polymerase activity could be meassured shortly after viral infection in the cytoplasmic fraction of infected cells in vitro. However, unless the cells were broken in the presence of the nonionic detergent Triton-X-100, no significant synthesis of new RNA polymerase was detected during the viral growth cycle. When cells were broken in the presence of this detergent, extensive increases in viral RNA polymerase activity were observed late in the infection cycle. The onset of new RNA polymerase synthesis was dependent on prior viral DNA replication. Fluorodeoxyuridine (5 x 10(-5)m) prevented the onset of viral polymerase synthesis. Streptovitacin A, a specific and complete inhibitor of protein synthesis in HeLa cells, prevented the synthesis of RNA polymerase. Thus, the synthesis of RNA polymerase is a "late" function of the virus. The newly synthesized RNA polymerase activity was primarily bound to particles which sedimented during high-speed centrifugation. These particles have been characterized by sucrose gradient centrifugation. A major class of active RNA polymerase particles were considerably "lighter" than whole virus in sucrose gradients. These particles were entirely resistant to the action of added pancreatic deoxyribonuclease, and they were not stimulated by added calf thymus primer DNA. It is concluded that these particles are not active in RNA synthesis in vivo, and that activation occurs as a result of detergent treatment in vitro.  相似文献   

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The visna viral RNA-dependent DNA polymerase has been resolved into two forms by affinity chromatography. Glycerine gradient centrifugation of the two forms showed that one form sedimented at 6.9 S corresponding to an apparent molecular weight of 135 000 and the other at 6.3 S corresponding to 118 000. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of the two forms indicated that the 6.9 S enzyme is composed of 2 molecules of 68 000 mol. wt. chain and the 6.3 S is a single chain enzyme. The latter form has been identified as a glycoprotein. The 6.9 S form can be completely inactivated in 20 min at 45 degrees C, prefers poly(rC) over poly(rA) as template and has high efficiency in utilizing visna 70 S RNA as template. The 6.3 S form is stable at 45 degrees C, active with 70 S viral RNA as template, prefers poly(rA) over poly(rC), and requires higher concentration of Mn2+ (0.4 mM) for maximum activity than the 6.9 S form does (0.1 mM) with synthetic homopolymers as templates. However, both 6.9 S and 6.3 S forms prefer Mg2+ over Mn2+ regardless of the nature of the templates.  相似文献   

17.
The RNA-dependent RNA polymerase of influenza virus A/PR/8 was isolated from virus particles by stepwise centrifugation in cesium salts. First, RNP (viral RNA-NP-P proteins) complexes were isolated by glycerol gradient centrifugation of detergent-treated viruses and subsequently NP was dissociated from RNP by cesium chloride gradient centrifugation. The P-RNA (P proteins-viral RNA) complexes were further dissociated into P proteins and viral RNA by cesium trifluoroacetate (CsTFA) gradient centrifugation. The nature of P proteins was further analyzed by glycerol gradient centrifugation and immunoblotting using monospecific antibodies against each P protein. The three P proteins, PB1, PB2, and PA, sedimented altogether as fast as the marker protein with the molecular weight of about 250,000 Da. Upon addition of the template vRNA, the RNA-free P protein complex exhibited the activities of capped RNA cleavage and limited RNA synthesis. When a cell line stably expressing cDNAs for three P proteins and NP protein was examined, the three P proteins were found to be co-precipitated by antibodies against the individual P proteins. These results indicate that the influenza virus RNA-dependent RNA polymerase is a heterocomplex composed of one each of the three P proteins and that the RNA-free RNA polymerase can be isolated in an active form from virus particles. Furthermore, the three P proteins form a complex in the absence of vRNA.  相似文献   

18.
The NS5B RNA-dependent RNA polymerase encoded by hepatitis C virus (HCV) plays a key role in viral replication. Reported here is evidence that HCV NS5B polymerase acts as a functional oligomer. Oligomerization of HCV NS5B protein was demonstrated by gel filtration, chemical cross-linking, temperature sensitivity, and yeast cell two-hybrid analysis. Mutagenesis studies showed that the C-terminal hydrophobic region of the protein was not essential for its oligomerization. Importantly, HCV NS5B polymerase exhibited cooperative RNA synthesis activity with a dissociation constant, K(d), of approximately 22 nM, suggesting a role for the polymerase-polymerase interaction in the regulation of HCV replicase activity. Further functional evidence includes the inhibition of the wild-type NS5B polymerase activity by a catalytically inactive form of NS5B. Finally, the X-ray crystal structure of HCV NS5B polymerase was solved at 2.9 A. Two extensive interfaces have been identified from the packing of the NS5B molecules in the crystal lattice, suggesting a higher-order structure that is consistent with the biochemical data.  相似文献   

19.
It has been shown that DNA primase activity is tightly associated with 10S DNA polymerase alpha from calf thymus (Yoshida, S. et al. (1983) Biochim. Biophys. Acta 741, 348-357). In the present study, the primase activity was separated from DNA polymerase alpha by treating purified 10S DNA polymerase alpha with 3.4 M urea followed by a fast column chromatography (Pharmacia FPLC, Mono Q column equilibrated with 2 M urea). Ten to 20 % of the primase activity was separated from 10S DNA polymerase alpha by this procedure but 80-90% remained in the complex. The separated primase activity sedimented at 5.6S through a gradient of glycerol. The separated primase was strongly inhibited by araATP (Ki = 10 microM) and was also sensitive to salts such as KCl (50% inhibition at 30 mM). The primase used poly(dT) or poly(dC) as templates efficiently, but showed little activity with poly(dA) or poly(dI). These properties agree well with those of the primase activity in the DNA polymerase alpha-primase complex (10S DNA polymerase alpha). These results indicate that the calf thymus primase may be a part of the 10S DNA polymerase alpha and its enzymological characters are preserved after separation from the complex.  相似文献   

20.
Viruses of the family Flaviviridae are important human and animal pathogens. Among them, the Flaviviruses dengue (DENV) and West Nile (WNV) cause regular outbreaks with fatal outcomes. The RNA-dependent RNA polymerase (RdRp) activity of the non-structural protein 5 (NS5) is a key activity for viral RNA replication. In this study, crystal structures of enzymatically active and inactive WNV RdRp domains were determined at 3.0- and 2.35-A resolution, respectively. The determined structures were shown to be mostly similar to the RdRps of the Flaviviridae members hepatitis C and bovine viral diarrhea virus, although with unique elements characteristic for the WNV RdRp. Using a reverse genetic system, residues involved in putative interactions between the RNA-cap methyltransferase (MTase) and the RdRp domain of Flavivirus NS5 were identified. This allowed us to propose a model for the structure of the full-length WNV NS5 by in silico docking of the WNV MTase domain (modeled from our previously determined structure of the DENV MTase domain) onto the RdRp domain. The Flavivirus RdRp domain structure determined here should facilitate both the design of anti-Flavivirus drugs and structure-function studies of the Flavivirus replication complex in which the multifunctional NS5 protein plays a central role.  相似文献   

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