RNA polymerase in normal avian erythrocytes

Significant RNA polymerase activity was demonstrated in isolated nuclei from mature avian erythrocytes. This activity was shown to have characteristics common to mammalian systems, including sensitivity to α-amanatin. A crude fraction of RNA polymerase was solubilized from these nuclei and characterized to provide further support for the existence of the enzyme in these cells.     

RNA-dependent RNA polymerase activity in coronavirus- infected cells

An enzymatic activity which incorporates [3H]UMP into acid-precipitable material in the presence of endogenous template was found in the cytoplasm of porcine cells infected with the transmissible gastroenteritis virus of swine. This activity was not found in uninfected control cells, nor was it found in purified virus. The activity was associated with the mitochondrial fraction of infected cells, suggesting that the enzyme is membrane bound. The activity required the presence of all three ribonucleoside triphosphates in addition to [3H]UTP, and it was not inhibited by actinomycin D. The heated product was digested by RNase but not by DNase. Mg2+ was required for enzymatic activity, and its optimal concentration was approximately 5 mM. The size of the in vitro products was compared by electrophoresis with that of in vivo-synthesized virus-specified RNA to confirm the viral specificity of the polymerase activity. Virus-specified RNA from infected cells consisted of 10 species of single-stranded, polyadenylated RNA with molecular weights of 6.8 X 10(6), 6.2 X 10(6), 3.15 X 10(6), 1.40 X 10(6), 1.05 X 10(6), 0.94 X 10(6), 0.66 X 10(6), 0.39 X 10(6), 0.34 X 10(6), and 0.24 X 10(6). In vitro synthesized RNA consisted of a high-molecular-weight species, of apparently higher molecular weight than genomic RNA, and two single-stranded species that electrophoretically comigrated with the species of 1.40 X 10(6) and 0.66 X 10(6) molecular weight made in vivo.     

The product of ribosome-associated RNA-dependent RNA polymerase in immature chicken erythrocytes

The radioactively labelled product obtained after incubation of chicken erythrocyte ribosomes with [³H]-UTP was shown by sucrose density gradient centrifugation to have a sedimentation coefficient of 4–5S; the ratio of UMP to uridine incorporated was 6.09. The product synthesized and isolated after incubation of intact cells in the presence of [³H]-uridine and actinomycin D was shown to be very similar with respect to sedimentation coefficient and the ratio of UMP/uridine incorporated, providing for the first time evidence of a ribosome-associated terminal ribonucleotidyl-transferase activity in intact cells.     

RNA-dependent RNA polymerase activity associated with endogenous double-stranded RNA in rice

RNA-dependent RNA polymerase (RdRp) activity was detected in the crude microsomal fraction of rice cultured cells that contain a 14 kbp double-stranded RNA (dsRNA). RdRp activity is maximal in the presence of all four nucleotide triphosphates and Mg2+ ion and is resistant to inhibitors of DNA-dependent RNA polymerases (actinomycin D and alpha-amanitin). RdRp activity increases approximately 2.5-fold in the presence of 0.5% deoxycholate. Treatment of purified microsomal fraction with proteinase K plus deoxycholate suggests that the RdRp enzyme complex with its own 14 kb RNA template is located in vesicles. The RdRp enzyme complex was solubilized with Nonidet P-40 and purified by glycerol gradient centrifugation, then exogenous RNA templates were added. Results indicate that exogenous dsRNA reduces RNA synthesis from the endogenous 14 kb RNA template.     

RNA template-mediated inhibition of hepatitis C virus RNA-dependent RNA polymerase activity

The enzymatic activity of hepatitis C virus (HCV) RNA-dependent RNA polymerase NS5B is modulated by the molar ratio of NS5B enzyme and RNA template. Depending on the ratio, either template or enzyme can inhibit activity. Inhibition of NS5B activity by RNA template exhibited characteristics of substrate inhibition, suggesting the template binds to a secondary site on the enzyme forming an inactive complex. Template inhibition was modulated by primer. Increasing concentrations of primer restored NS5B activity and decreased the affinity of template for the secondary site. Conversely, increasing template concentration reduced the affinity of primer binding. The kinetic profiles suggest template inhibition results from the binding of template to a site that interferes with primer binding and the formation of productive replication complexes.     

RNA polymerases during differentiation of avian erythrocytes

DNA-dependent RNA polymerase was analysed during the terminal differentiation stages of avian erythrocytes. It was found that the mature duck erythrocyte, although quiescent in RNA synthesis, contains clearly measurable quantities of RNA polymerase B (or II). Immature polychromatic erythrocytes, derived from anemic ducks and actively synthesizing hemoglobin mRNA, additionally contain significant amounts of RNA polymerase A (or I) and C (or III) previously not detected in these cells. These latter classes of enzymes, although present, are apparently not engaged in RNA synthesis in polychromatic erythrocytes.     

Oligomerization and cooperative RNA synthesis activity of hepatitis C virus RNA-dependent RNA polymerase

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.     

RNA polymerase activity in purified virions of avian reticuloendotheliosis viruses.

An RNA polymerase activity that synthesizes a U-rich RNA hydrogen bonded to a large viral RNA molecule was found in the cores of virions of avian reticuloendotheliosis viruses (REV). The RNA polymerase activity was separable from the DNA polymerase activity of REV virions. The 5'-terminus of the newly synthesized RNA was A. In addition, a tRNA nucleotidyl transferase activity, which added -CpCpA ends to tRNA, appears to be present in the REV virions.     

Direct evidence that ribosome bound RNA-dependent RNA polymerase does not play a role in globin messenger RNA replication.

The radioactively labelled product of RNA-dependent RNA polymerase+ from ribosomes of immature chicken erythrocytes was tested for the presence of newly replicated globin mRNA using unlabelled globin complementary DNA. No radioactively labelled globin mRNA sequences were found in the product, providing direct confirmation that this RNA-dependent RNA polymerase is not involved in globin mRNA amplification.     

Functional oligomerization of poliovirus RNA-dependent RNA polymerase.

Using a hairpin primer/template RNA derived from sequences present at the 3' end of the poliovirus genome, we investigated the RNA-binding and elongation activities of highly purified poliovirus 3D polymerase. We found that surprisingly high polymerase concentrations were required for efficient template utilization. Binding of template RNAs appeared to be the primary determinant of efficient utilization because binding and elongation activities correlated closely. Using a three-filter binding assay, polymerase binding to RNA was found to be highly cooperative with respect to polymerase concentration. At pH 5.5, where binding was most cooperative, a Hill coefficient of 5 was obtained, indicating that several polymerase molecules interact to retain the 110-nt RNA in a filter-bound complex. Chemical crosslinking with glutaraldehyde demonstrated physical polymerase-polymerase interactions, supporting the cooperative binding data. We propose a model in which poliovirus 3D polymerase functions both as a catalytic polymerase and as a cooperative single-stranded RNA-binding protein during RNA-dependent RNA synthesis.     

Hepatitis C virus (HCV) NS5A binds RNA-dependent RNA polymerase (RdRP) NS5B and modulates RNA-dependent RNA polymerase activity.

Hepatitis C virus (HCV) NS5B is RNA-dependent RNA polymerase (RdRP), the essential catalytic enzyme for HCV replication. Recently, NS5A has been reported to be important for the establishment of HCV replication in vitro by the adaptive mutations, although its role in viral replication remains uncertain. Here we report that purified bacterial recombinant NS5A and NS5B directly interact with each other in vitro, detected by glutathione S-transferase (GST) pull-down assay. Furthermore, complex formation of these proteins transiently coexpressed in mammalian cells was detected by coprecipitation. Using terminally and internally truncated NS5A, two discontinuous regions of NS5A (amino acids 105-162 and 277-334) outside of the adaptive mutations were identified to be independently essential for the binding both in vivo and in vitro (Yamashita, T., Kaneko, S., Shirota, Y., Qin, W., Nomura, T., Kobayashi, K., and Mkyrakami, S. (1998) J. Biol. Chem. 273, 15479-15486). We previously examined the effect of His-NS5A on RdRP activity of the soluble recombinant NS5Bt in vitro (see Yamashita et al. above). Wild NS5A weakly stimulated at first (when less than 0.1 molar ratio to NS5B) and then inhibited the NS5Bt RdRP activity in a dose-dependent manner. The internal deletion mutants defective in NS5B binding exhibited no inhibitory effect, indicating that the NS5B binding is necessary for the inhibition. Taken together, our results support the idea that NS5A modulates HCV replication as a component of replication complex.     

Investigation of activity of recombinant mengovirus RNA-dependent RNA polymerase and its mutants

The activities of wild-type mengovirus RNA polymerase (RdRP) and of its three mutants with C-terminal tryp-tophan residue replaced by residues of alanine (W460A), phenylalanine (W460F), or tyrosine (W460Y) were studied. The proteins were expressed in E. coli and purified by affinity chromatography with the IMPACT system. The isolated recombinant proteins were studied using a cell-free replication system on elongation of oligo(U) primer on RNA template corresponding to the 3′-terminal 366-meric fragment of the mengovirus RNA. The activities of the mutant polymerases were comparable to that of the wild-type enzyme.