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Статті в журналах з теми "RNA Synthesis"

Автор: Grafiati

Опубліковано: 4 червня 2021

Оновлено: 28 січня 2023

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1

Cazenave, C., and O. C. Uhlenbeck. "RNA template-directed RNA synthesis by T7 RNA polymerase." Proceedings of the National Academy of Sciences 91, no. 15 (July 19, 1994): 6972–76. http://dx.doi.org/10.1073/pnas.91.15.6972.

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2

Beerens, Nancy, Barbara Selisko, Stefano Ricagno, Isabelle Imbert, Linda van der Zanden, Eric J. Snijder, and Bruno Canard. "De Novo Initiation of RNA Synthesis by the Arterivirus RNA-Dependent RNA Polymerase." Journal of Virology 81, no. 16 (May 30, 2007): 8384–95. http://dx.doi.org/10.1128/jvi.00564-07.

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ABSTRACT All plus-strand RNA viruses encode an RNA-dependent RNA polymerase (RdRp) that functions as the catalytic subunit of the viral replication/transcription complex, directing viral RNA synthesis in concert with other viral proteins and, sometimes, host proteins. RNA synthesis essentially can be initiated by two different mechanisms, de novo initiation and primer-dependent initiation. Most viral RdRps have been identified solely on the basis of comparative sequence analysis, and for many viruses the mechanism of initiation is unknown. In this study, using the family prototype equine arteritis virus (EAV), we address the mechanism of initiation of RNA synthesis in arteriviruses. The RdRp domains of the members of the arterivirus family, which are part of replicase subunit nsp9, were compared to coronavirus RdRps that belong to the same order of Nidovirales, as well as to other RdRps with known initiation mechanisms and three-dimensional structures. We report here the first successful expression and purification of an arterivirus RdRp that is catalytically active in the absence of other viral or cellular proteins. The EAV nsp9/RdRp initiates RNA synthesis by a de novo mechanism on homopolymeric templates in a template-specific manner. In addition, the requirements for initiation of RNA synthesis from the 3′ end of the viral genome were studied in vivo using a reverse genetics approach. These studies suggest that the 3′-terminal nucleotides of the EAV genome play a critical role in viral RNA synthesis.

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3

Doudna, Jennifer A., and Jack W. Szostak. "RNA-catalysed synthesis of complementary-strand RNA." Nature 339, no. 6225 (June 1989): 519–22. http://dx.doi.org/10.1038/339519a0.

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4

Illangasekare, M., G. Sanchez, T. Nickles, and M. Yarus. "Aminoacyl-RNA synthesis catalyzed by an RNA." Science 267, no. 5198 (February 3, 1995): 643–47. http://dx.doi.org/10.1126/science.7530860.

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5

Doudna, J. A., and J. W. Szostak. "RNA-catalysed synthesis of complementary strand RNA." Trends in Genetics 5 (1989): 323. http://dx.doi.org/10.1016/0168-9525(89)90125-x.

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6

Sivakumaran, K., and C. Cheng Kao. "Initiation of Genomic Plus-Strand RNA Synthesis from DNA and RNA Templates by a Viral RNA-Dependent RNA Polymerase." Journal of Virology 73, no. 8 (August 1, 1999): 6415–23. http://dx.doi.org/10.1128/jvi.73.8.6415-6423.1999.

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ABSTRACT In contrast to the synthesis of minus-strand genomic and plus-strand subgenomic RNAs, the requirements for brome mosaic virus (BMV) genomic plus-strand RNA synthesis in vitro have not been previously reported. Therefore, little is known about the biochemical requirements for directing genomic plus-strand synthesis. Using DNA templates to characterize the requirements for RNA-dependent RNA polymerase template recognition, we found that initiation from the 3′ end of a template requires one nucleotide 3′ of the initiation nucleotide. The addition of a nontemplated nucleotide at the 3′ end of minus-strand BMV RNAs led to initiation of genomic plus-strand RNA in vitro. Genomic plus-strand initiation was specific since cucumber mosaic virus minus-strand RNA templates were unable to direct efficient synthesis under the same conditions. In addition, mutational analysis of the minus-strand template revealed that the −1 nontemplated nucleotide, along with the +1 cytidylate and +2 adenylate, is important for RNA-dependent RNA polymerase interaction. Furthermore, genomic plus-strand RNA synthesis is affected by sequences 5′ of the initiation site.

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7

Röthlisberger, Pascal, Christian Berk, and Jonathan Hall. "RNA Chemistry for RNA Biology." CHIMIA International Journal for Chemistry 73, no. 5 (May 29, 2019): 368–73. http://dx.doi.org/10.2533/chimia.2019.368.

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Анотація:

Advances in the chemical synthesis of RNA have opened new possibilities to address current questions in RNA biology. Access to site-specifically modified oligoribonucleotides is often a pre-requisite for RNA chemical-biology projects. Driven by the enormous research efforts for development of oligonucleotide therapeutics, a wide range of chemical modifications have been developed to modulate the intrinsic properties of nucleic acids in order to fit their use as therapeutics or research tools. The RNA synthesis platform, supported by the NCCR RNA & Disease, aims to provide access to a large variety of chemically modified nucleic acids. In this review, we describe some of the recent projects that involved work of the platform and highlight how RNA chemistry supports new discoveries in RNA biology.

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8

Modahl, Lucy E., Thomas B. Macnaughton, Nongliao Zhu, Deborah L. Johnson, and Michael M. C. Lai. "RNA-Dependent Replication and Transcription of Hepatitis Delta Virus RNA Involve Distinct Cellular RNA Polymerases." Molecular and Cellular Biology 20, no. 16 (August 15, 2000): 6030–39. http://dx.doi.org/10.1128/mcb.20.16.6030-6039.2000.

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ABSTRACT Cellular DNA-dependent RNA polymerase II (pol II) has been postulated to carry out RNA-dependent RNA replication and transcription of hepatitis delta virus (HDV) RNA, generating a full-length (1.7-kb) RNA genome and a subgenomic-length (0.8-kb) mRNA. However, the supporting evidence for this hypothesis was ambiguous because the previous experiments relied on DNA-templated transcription to initiate HDV RNA synthesis. Furthermore, there is no evidence that the same cellular enzyme is involved in the synthesis of both RNA species. In this study, we used a novel HDV RNA-based transfection approach, devoid of any artificial HDV cDNA intermediates, to determine the enzymatic and metabolic requirements for the synthesis of these two RNA species. We showed that HDV subgenomic mRNA transcription was inhibited by a low concentration of α-amanitin (<3 μg/ml) and could be partially restored by an α-amanitin-resistant mutant pol II; however, surprisingly, the synthesis of the full-length (1.7-kb) antigenomic RNA was not affected by α-amanitin to a concentration higher than 25 μg/ml. By several other criteria, such as the differing requirement for the de novo-synthesized hepatitis delta antigen and temperature dependence, we further showed that the metabolic requirements of subgenomic HDV mRNA synthesis are different from those for the synthesis of genomic-length HDV RNA and cellular pol II transcripts. The synthesis of the two HDV RNA species could also be uncoupled under several different conditions. These findings provide strong evidence that pol II, or proteins derived from pol II transcripts, is involved in mRNA transcription from the HDV RNA template. In contrast, the synthesis of the 1.7-kb HDV antigenomic RNA appears not to be dependent on pol II. These results reveal that there are distinct molecular mechanisms for the synthesis of these two RNA species.

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9

Rohayem, Jacques, Katrin Jäger, Ivonne Robel, Ulrike Scheffler, Achim Temme, and Wolfram Rudolph. "Characterization of norovirus 3Dpol RNA-dependent RNA polymerase activity and initiation of RNA synthesis." Journal of General Virology 87, no. 9 (September 1, 2006): 2621–30. http://dx.doi.org/10.1099/vir.0.81802-0.

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Norovirus (NV) 3Dpol is a non-structural protein predicted to play an essential role in the replication of the NV genome. In this study, the characteristics of NV 3Dpol activity and initiation of RNA synthesis have been examined in vitro. Recombinant NV 3Dpol, as well as a 3Dpol active-site mutant were expressed in Escherichia coli and purified. NV 3Dpol was able to synthesize RNA in vitro and displayed flexibility with respect to the use of Mg2+ or Mn2+ as a cofactor. NV 3Dpol yielded two different products when incubated with synthetic RNA in vitro: (i) a double-stranded RNA consisting of two single strands of opposite polarity or (ii) the single-stranded RNA template labelled at its 3′ terminus by terminal transferase activity. Initiation of RNA synthesis occurred de novo rather than by back-priming, as evidenced by the fact that the two strands of the double-stranded RNA product could be separated, and by dissociation in time-course analysis of terminal transferase and RNA synthesis activities. In addition, RNA synthesis was not affected by blocking of the 3′ terminus of the RNA template by a chain terminator, sustaining de novo initiation of RNA synthesis. NV 3Dpol displays in vitro properties characteristic of RNA-dependent RNA polymerases, allowing the implementation of this in vitro enzymic assay for the development and validation of antiviral drugs against NV, a so far non-cultivated virus and an important human pathogen.

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10

Guo, Hui, Mengyue Fan, Zengjin Li, Wei Tang, and Xinrui Duan. "Ratiometric RNA aptamer/fluorophore complex for RNA synthesis detection." Analytical Methods 10, no. 47 (2018): 5629–33. http://dx.doi.org/10.1039/c8ay01880d.

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11

Wang, Lei, Neil A. Smith, Lan Zhang, Elizabeth S. Dennis, Peter M. Waterhouse, Peter J. Unrau, and Ming-Bo Wang. "Synthesis of complementary RNA by RNA-dependent RNA polymerases in plant extracts is independent of an RNA primer." Functional Plant Biology 35, no. 11 (2008): 1091. http://dx.doi.org/10.1071/fp08118.

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RNA-dependent RNA polymerase (RDR) activities were readily detected in extracts from cauliflower and broccoli florets, Arabidopsis thaliana (L.) Heynh callus tissue and broccoli nuclei. The synthesis of complementary RNA (cRNA) was independent of a RNA primer, whether or not the primer contained a 3′ terminal 2′-O-methyl group or was phosphorylated at the 5′ terminus. cRNA synthesis in plant extracts was not affected by loss-of-function mutations in the DICER-LIKE (DCL) proteins DCL2, DCL3, and DCL4, indicating that RDRs function independently of these DCL proteins. A loss-of-function mutation in RDR1, RDR2 or RDR6 did not significantly reduce the amount of cRNA synthesis. This indicates that these RDRs did not account for the bulk RDR activities in plant extracts, and suggest that either the individual RDRs each contribute a fraction of polymerase activity or another RDR(s) is predominant in the plant extract.

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12

Ranjith-Kumar, C. T., Les Gutshall, Min-Ju Kim, Robert T. Sarisky, and C. Cheng Kao. "Requirements for De Novo Initiation of RNA Synthesis by Recombinant Flaviviral RNA-Dependent RNA Polymerases." Journal of Virology 76, no. 24 (December 15, 2002): 12526–36. http://dx.doi.org/10.1128/jvi.76.24.12526-12536.2002.

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ABSTRACT RNA-dependent RNA polymerases (RdRps) that initiate RNA synthesis by a de novo mechanism should specifically recognize the template initiation nucleotide, T1, and the substrate initiation nucleotide, the NTPi. The RdRps from hepatitis C virus (HCV), bovine viral diarrhea virus (BVDV), and GB virus-B all can initiate RNA synthesis by a de novo mechanism. We used RNAs and GTP analogs, respectively, to examine the use of the T1 nucleotide and the initiation nucleotide (NTPi) during de novo initiation of RNA synthesis. The effects of the metal ions Mg2+ and Mn2+ on initiation were also analyzed. All three viral RdRps require correct base pairing between the T1 and NTPi for efficient RNA synthesis. However, each RdRp had some distinct tolerances for modifications in the T1 and NTPi. For example, the HCV RdRp preferred an NTPi lacking one or more phosphates regardless of whether Mn2+ was present or absent, while the BVDV RdRp efficiently used GDP and GMP for initiation of RNA synthesis only in the presence of Mn2+. These and other results indicate that although the three RdRps share a common mechanism of de novo initiation, each has distinct preferences.

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13

Sun, Jin-Hua, Scott Adkins, Greta Faurote, and C. Cheng Kao. "Initiation of (−)-Strand RNA Synthesis Catalyzed by the BMV RNA-Dependent RNA Polymerase: Synthesis of Oligonucleotides." Virology 226, no. 1 (December 1996): 1–12. http://dx.doi.org/10.1006/viro.1996.0622.

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14

Sun, Jin-Hua, Scott Adkins, Greta Faurote, and C. Cheng Kao. "Initiation of (—)-Strand RNA Synthesis Catalyzed by the BMV RNA-Dependent RNA Polymerase: Synthesis of Oligonucleotides." Virology 228, no. 1 (February 1997): 121. http://dx.doi.org/10.1006/viro.1996.8404.

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15

Kao, C. Cheng, Xueyong Yang, Allen Kline, Q. May Wang, Donna Barket, and Beverly A. Heinz. "Template Requirements for RNA Synthesis by a Recombinant Hepatitis C Virus RNA-Dependent RNA Polymerase." Journal of Virology 74, no. 23 (December 1, 2000): 11121–28. http://dx.doi.org/10.1128/jvi.74.23.11121-11128.2000.

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ABSTRACT The RNA-dependent RNA polymerase (RdRp) from hepatitis C virus (HCV), nonstructural protein 5B (NS5B), has recently been shown to direct de novo initiation using a number of complex RNA templates. In this study, we analyzed the features in simple RNA templates that are required to direct de novo initiation of RNA synthesis by HCV NS5B. NS5B was found to protect RNA fragments of 8 to 10 nucleotides (nt) from RNase digestion. However, NS5B could not direct RNA synthesis unless the template contained a stable secondary structure and a single-stranded sequence that contained at least one 3′ cytidylate. The structure of a 25-nt template, named SLD3, was determined by nuclear magnetic resonance spectroscopy to contain an 8-bp stem and a 6-nt single-stranded sequence. Systematic analysis of changes in SLD3 revealed which features in the stem, loop, and 3′ single-stranded sequence were required for efficient RNA synthesis. Also, chimeric molecules composed of DNA and RNA demonstrated that a DNA molecule containing a 3′-terminal ribocytidylate was able to direct RNA synthesis as efficiently as a sequence composed entirely of RNA. These results define the template sequence and structure sufficient to direct the de novo initiation of RNA synthesis by HCV RdRp.

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16

Cedergren, Robert, and Henri Grosjean. "RNA design by in vitro RNA recombination and synthesis." Biochemistry and Cell Biology 65, no. 8 (August 1, 1987): 677–92. http://dx.doi.org/10.1139/o87-090.

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The techniques of in vitro RNA synthesis and recombination are presented. These include the site-specific cleavage of RNA, the manipulation of terminal phosphates, and the ligation of RNA fragments. Areas of promising future research include the establishment of RNA cloning vectors and the use of in vitro transcription of natural or designed RNA genes. The chemical synthesis approach now offers the possibility of making large amounts of biologically active length RNAs and of incorporating modified or reporter nucleotides into RNA sequences for physical studies. The new RNA techniques taken with DNA technology will permit a new approach towards understanding the complexity of RNA metabolism and the relationship of structure to function in RNAs.

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17

Unrau, Peter J., and David P. Bartel. "RNA-catalysed nucleotide synthesis." Nature 395, no. 6699 (September 1998): 260–63. http://dx.doi.org/10.1038/26193.

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18

Coleman, Tricia M., and Faqing Huang. "RNA-Catalyzed Thioester Synthesis." Chemistry & Biology 9, no. 11 (November 2002): 1227–36. http://dx.doi.org/10.1016/s1074-5521(02)00264-8.

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19

Tayon, R. "Completion of RNA synthesis by viral RNA replicases." Nucleic Acids Research 29, no. 17 (September 1, 2001): 3576–82. http://dx.doi.org/10.1093/nar/29.17.3576.

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20

Illangasekare, M., and M. Yarus. "Specific, rapid synthesis of Phe-RNA by RNA." Proceedings of the National Academy of Sciences 96, no. 10 (May 11, 1999): 5470–75. http://dx.doi.org/10.1073/pnas.96.10.5470.

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21

Sun, Xin-Lai, Robert B. Johnson, Michelle A. Hockman, and Q. May Wang. "De Novo RNA Synthesis Catalyzed by HCV RNA-Dependent RNA Polymerase." Biochemical and Biophysical Research Communications 268, no. 3 (February 2000): 798–803. http://dx.doi.org/10.1006/bbrc.2000.2120.

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22

Mairhofer, Elisabeth, Elisabeth Fuchs, and Ronald Micura. "Facile synthesis of a 3-deazaadenosine phosphoramidite for RNA solid-phase synthesis." Beilstein Journal of Organic Chemistry 12 (November 28, 2016): 2556–62. http://dx.doi.org/10.3762/bjoc.12.250.

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Access to 3-deazaadenosine (c3A) building blocks for RNA solid-phase synthesis represents a severe bottleneck in modern RNA research, in particular for atomic mutagenesis experiments to explore mechanistic aspects of ribozyme catalysis. Here, we report the 5-step synthesis of a c3A phosphoramidite from cost-affordable starting materials. The key reaction is a silyl-Hilbert–Johnson nucleosidation using unprotected 6-amino-3-deazapurine and benzoyl-protected 1-O-acetylribose. The novel path is superior to previously described syntheses in terms of efficacy and ease of laboratory handling.

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23

van Dijk, Alberdina A., Eugene V. Makeyev, and Dennis H. Bamford. "Initiation of viral RNA-dependent RNA polymerization." Journal of General Virology 85, no. 5 (May 1, 2004): 1077–93. http://dx.doi.org/10.1099/vir.0.19731-0.

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This review summarizes the combined insights from recent structural and functional studies of viral RNA-dependent RNA polymerases (RdRPs) with the primary focus on the mechanisms of initiation of RNA synthesis. Replication of RNA viruses has traditionally been approached using a combination of biochemical and genetic methods. Recently, high-resolution structures of six viral RdRPs have been determined. For three RdRPs, enzyme complexes with metal ions, single-stranded RNA and/or nucleoside triphosphates have also been solved. These advances have expanded our understanding of the molecular mechanisms of viral RNA synthesis and facilitated further RdRP studies by informed site-directed mutagenesis. What transpires is that the basic polymerase right hand shape provides the correct geometrical arrangement of substrate molecules and metal ions at the active site for the nucleotidyl transfer catalysis, while distinct structural elements have evolved in the different systems to ensure efficient initiation of RNA synthesis. These elements feed the template, NTPs and ions into the catalytic cavity, correctly position the template 3′ terminus, transfer the products out of the catalytic site and orchestrate the transition from initiation to elongation.

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24

Macnaughton, Thomas B., Stephanie T. Shi, Lucy E. Modahl, and Michael M. C. Lai. "Rolling Circle Replication of Hepatitis Delta Virus RNA Is Carried Out by Two Different Cellular RNA Polymerases." Journal of Virology 76, no. 8 (April 15, 2002): 3920–27. http://dx.doi.org/10.1128/jvi.76.8.3920-3927.2002.

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ABSTRACT Hepatitis delta virus (HDV) contains a viroid-like circular RNA that is presumed to replicate via a rolling circle replication mechanism mediated by cellular RNA polymerases. However, the exact mechanism of rolling circle replication for HDV RNA and viroids is not clear. Using our recently described cDNA-free transfection system (L. E. Modahl and M. M. Lai, J. Virol. 72:5449-5456, 1998), we have succeeded in detecting HDV RNA replication by metabolic labeling with [32P]orthophosphate in vivo and obtained direct evidence that HDV RNA replication generates high-molecular-weight multimeric species of HDV RNA, which are processed into monomeric and dimeric forms. Thus, these multimeric RNAs are the true intermediates of HDV RNA replication. We also found that HDV RNA synthesis is highly temperature sensitive, occurring most efficiently at 37 to 40°C and becoming virtually undetectable at temperatures below 30°C. Moreover, genomic HDV RNA synthesis was found to occur at a rate roughly 30-fold higher than that of antigenomic RNA synthesis. Finally, in lysolecithin-permeabilized cells, the synthesis of full-length antigenomic HDV RNA was completely resistant to high concentrations (100 μg/ml) of α-amanitin. In contrast, synthesis of genomic HDV RNA was totally inhibited by α-amanitin at concentrations as low as 2.5 μg/ml. Thus, these results suggest that genomic and antigenomic HDV RNA syntheses are performed by two different host cell enzymes. This observation, combined with our previous finding that hepatitis delta antigen mRNA synthesis is likely performed by RNA polymerase II, suggests that the different HDV RNA species are synthesized by different cellular transcriptional machineries.

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25

ZACHLEDER, V., and I. ŠETLÍK. "Distinct controls of DNA replication and of nuclear division in the cell cycles of the chlorococcal alga Scenedesmus quadricauda." Journal of Cell Science 91, no. 4 (December 1, 1988): 531–39. http://dx.doi.org/10.1242/jcs.91.4.531.

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In the course of the cell cycle of Scenedesmus quadricauda, the syntheses of RNA and total protein occur in steps. Each step represents an approximate doubling of the preceding amount of RNA or protein per cell. The increase in protein content per cell runs parallel to, but with a constant delay behind, the corresponding RNA steps. When protein synthesis is suppressed (e.g. by maintaining the cells in the dark) after an RNA synthesis step has already occurred the cells double their DNA content, but no corresponding nuclear division occurs and uninuclear daughter cells with double the amount of DNA may be formed. Under conditions of phosphorus or nitrogen starvation RNA synthesis is stopped while protein synthesis continues. In this case, the number of DNA replication rounds corresponds to the reduced RNA content while the number of nuclear divisions tends to follow the number of protein synthesis steps until one genome per nucleus is attained. These results indicate that with each doubling of RNA content the cells become committed to DNA replication, while doubling of protein content is required for the commitment to the corresponding nuclear divisions.

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26

Resa-Infante, Patricia, Núria Jorba, Rocio Coloma, and Juan Ortin. "The influenza virus RNA synthesis machine." RNA Biology 8, no. 2 (March 2011): 207–15. http://dx.doi.org/10.4161/rna.8.2.14513.

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27

Fahrenbach, Albert C. "Template-directed nonenzymatic oligonucleotide synthesis: lessons from synthetic chemistry." Pure and Applied Chemistry 87, no. 2 (February 1, 2015): 205–18. http://dx.doi.org/10.1515/pac-2014-1004.

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AbstractThe nonenzymatic synthesis of nucleic acids, in particular, RNA, and the template-directed synthesis of artificial organic molecules, such as macrocycles, catenanes and rotaxanes, have both undergone significant development since the last half of the 20th century. The intersection of these two fields affords insights into how template effects can lead to information copying and storage at the molecular level. Mechanistic examples of model template-directed RNA replication experiments as well as those for totally artificial organic template-directed syntheses will be discussed. The fact that templates typically bind to their reacted products more tightly than their unreacted substrates may be a mechanistic feature necessary to store information in the form of nucleic acids. Understanding the mechanisms of nonenzymatic RNA synthesis is not only essential for testing the RNA world hypothesis in the context of the origin of life on Earth and other planetary bodies, but may one day afford chemists the insights to construct their own artificial molecular replicators.

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28

Walther, Udo Ingbert, Johannes Schulze, and Wolfgang Forth. "Inhibition of protein synthesis by zinc: comparison between protein synthesis and RNA synthesis." Human & Experimental Toxicology 17, no. 12 (December 1998): 661–67. http://dx.doi.org/10.1177/096032719801701203.

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Inhalation of zinc fumes may lead to the acute respiratory distress syndrome. The mechanisms of pulmonary zinc toxicity are not yet understood. Therefore we investigated zinc-dependent depression of protein and RNA synthesis in rat and human lung cell lines. 1 After exposure to 120 or 150 mmol/l zinc, RNA synthesis as assessed by uridine incorporation decreased by 60-70% between 0 and 2 h exposition in rat alveolar type II cells (L2 cells) and human fibroblast-like cells (11Lu and 16Lu cells), and by 90% between 0 and 4 h in carcinoma-derived cells (A549 cells). 2 After 2 h exposure, L2, 11Lu, and 16Lu cells were half-maximally inhibited by 50 mmol/l zinc, whereas A549 cells were more resistant with half-maximal inhibition at 100 mmol/zinc. 3 Protein and RNA synthesis was inhibited in parallel in L2, 11Lu, and A549 cells as indicated by simultaneous determination of uridine and amino acid incorporation. In 16Lu cells, the decline in protein synthesis preceded RNA synthesis inhibition. Pretreatment with RNA synthesis inhibitors (amanitin or actinomycin D) had no effect on time curve and intensity of RNA synthesis inhibition. Taken together, our results indicate that the suppression of RNA and protein synthesis likely are independent phenomena, due to direct zinc effects on these biosynthetic pathways.

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29

Fukuda, Ryuji, and Eriko Hatada. "RNA synthesis of influenza A viruses. Temperature-sensitive mutants defective in RNA synthesis." Uirusu 36, no. 2 (1986): 203–19. http://dx.doi.org/10.2222/jsv.36.203.

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30

Sawicki, S. G., and D. L. Sawicki. "Coronavirus minus-strand RNA synthesis and effect of cycloheximide on coronavirus RNA synthesis." Journal of Virology 57, no. 1 (1986): 328–34. http://dx.doi.org/10.1128/jvi.57.1.328-334.1986.

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31

ILLANGASEKARE, MALI, and MICHAEL YARUS. "A tiny RNA that catalyzes both aminoacyl-RNA and peptidyl-RNA synthesis." RNA 5, no. 11 (November 1999): 1482–89. http://dx.doi.org/10.1017/s1355838299991264.

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32

Heck, Julie A., Xiao Meng, and David N. Frick. "Cyclophilin B stimulates RNA synthesis by the HCV RNA dependent RNA polymerase." Biochemical Pharmacology 77, no. 7 (April 2009): 1173–80. http://dx.doi.org/10.1016/j.bcp.2008.12.019.

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33

Peacock, Thomas P., Carol M. Sheppard, Ecco Staller, and Wendy S. Barclay. "Host Determinants of Influenza RNA Synthesis." Annual Review of Virology 6, no. 1 (September 29, 2019): 215–33. http://dx.doi.org/10.1146/annurev-virology-092917-043339.

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Influenza viruses are a leading cause of seasonal and pandemic respiratory illness. Influenza is a negative-sense single-stranded RNA virus that encodes its own RNA-dependent RNA polymerase (RdRp) for nucleic acid synthesis. The RdRp catalyzes mRNA synthesis, as well as replication of the virus genome (viral RNA) through a complementary RNA intermediate. Virus propagation requires the generation of these RNA species in a controlled manner while competing heavily with the host cell for resources. Influenza virus appropriates host factors to enhance and regulate RdRp activity at every step of RNA synthesis. This review describes such host factors and summarizes our current understanding of the roles they play in viral synthesis of RNA.

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34

Ivanov, S. A., R. Welz, M. B. Gottikh, and S. Müller. "RNA Synthesis by T7 RNA Polymerase Supported Primer Extension." Molecular Biology 38, no. 5 (September 2004): 674–79. http://dx.doi.org/10.1023/b:mbil.0000043937.13698.f0.

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35

Zhu, Bin, Alfredo Hernandez, Min Tan, Jan Wollenhaupt, Stanley Tabor, and Charles C. Richardson. "Synthesis of 2′-Fluoro RNA by Syn5 RNA polymerase." Nucleic Acids Research 43, no. 14 (April 20, 2015): e94-e94. http://dx.doi.org/10.1093/nar/gkv367.

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36

Paul, Aniko V., Jacques H. van Boom, Dmitri Filippov, and Eckard Wimmer. "Protein-primed RNA synthesis by purified poliovirus RNA polymerase." Nature 393, no. 6682 (May 1998): 280–84. http://dx.doi.org/10.1038/30529.

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37

Curran, Joseph, and Daniel Kolakofsky. "Nonsegmented negative-strand RNA virus RNA synthesis in vivo." Virology 371, no. 2 (February 2008): 227–30. http://dx.doi.org/10.1016/j.virol.2007.11.022.

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38

Ortín, Juan, and Jaime Martín-Benito. "The RNA synthesis machinery of negative-stranded RNA viruses." Virology 479-480 (May 2015): 532–44. http://dx.doi.org/10.1016/j.virol.2015.03.018.

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39

Kao, C. Cheng, Paul Singh, and David J. Ecker. "De Novo Initiation of Viral RNA-Dependent RNA Synthesis." Virology 287, no. 2 (September 2001): 251–60. http://dx.doi.org/10.1006/viro.2001.1039.

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40

Murray, Kenneth E., and David J. Barton. "Poliovirus CRE-Dependent VPg Uridylylation Is Required for Positive-Strand RNA Synthesis but Not for Negative-Strand RNA Synthesis." Journal of Virology 77, no. 8 (April 15, 2003): 4739–50. http://dx.doi.org/10.1128/jvi.77.8.4739-4750.2003.

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ABSTRACT The cis-acting replication element (CRE) is a 61-nucleotide stem-loop RNA structure found within the coding sequence of poliovirus protein 2C. Although the CRE is required for viral RNA replication, its precise role(s) in negative- and positive-strand RNA synthesis has not been defined. Adenosine in the loop of the CRE RNA structure functions as the template for the uridylylation of the viral protein VPg. VPgpUpUOH, the predominant product of CRE-dependent VPg uridylylation, is a putative primer for the poliovirus RNA-dependent RNA polymerase. By examining the sequential synthesis of negative- and positive-strand RNAs within preinitiation RNA replication complexes, we found that mutations that disrupt the structure of the CRE prevent VPg uridylylation and positive-strand RNA synthesis. The CRE mutations that inhibited the synthesis of VPgpUpUOH, however, did not inhibit negative-strand RNA synthesis. A Y3F mutation in VPg inhibited both VPgpUpUOH synthesis and negative-strand RNA synthesis, confirming the critical role of the tyrosine hydroxyl of VPg in VPg uridylylation and negative-strand RNA synthesis. trans-replication experiments demonstrated that the CRE and VPgpUpUOH were not required in cis or in trans for poliovirus negative-strand RNA synthesis. Because these results are inconsistent with existing models of poliovirus RNA replication, we propose a new four-step model that explains the roles of VPg, the CRE, and VPgpUpUOH in the asymmetric replication of poliovirus RNA.

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41

Luo, Guangxiang, Robert K. Hamatake, Danielle M. Mathis, Jason Racela, Karen L. Rigat, Julie Lemm, and Richard J. Colonno. "De Novo Initiation of RNA Synthesis by the RNA-Dependent RNA Polymerase (NS5B) of Hepatitis C Virus." Journal of Virology 74, no. 2 (January 15, 2000): 851–63. http://dx.doi.org/10.1128/jvi.74.2.851-863.2000.

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ABSTRACT Hepatitis C virus (HCV) NS5B protein possesses an RNA-dependent RNA polymerase (RdRp) activity, a major function responsible for replication of the viral RNA genome. To further characterize the RdRp activity, NS5B proteins were expressed from recombinant baculoviruses, purified to near homogeneity, and examined for their ability to synthesize RNA in vitro. As a result, a highly active NS5B RdRp (1b-42), which contains an 18-amino acid C-terminal truncation resulting from a newly created stop codon, was identified among a number of independent isolates. The RdRp activity of the truncated NS5B is comparable to the activity of the full-length protein and is 20 times higher in the presence of Mn2+ than in the presence of Mg2+. When a 384-nucleotide RNA was used as the template, two major RNA products were synthesized by 1b-42. One is a complementary RNA identical in size to the input RNA template (monomer), while the other is a hairpin dimer RNA synthesized by a “copy-back” mechanism. Substantial evidence derived from several experiments demonstrated that the RNA monomer was synthesized through de novo initiation by NS5B rather than by a terminal transferase activity. Synthesis of the RNA monomer requires all four ribonucleotides. The RNA monomer product was verified to be the result of de novo RNA synthesis, as two expected RNA products were generated from monomer RNA by RNase H digestion. In addition, modification of the RNA template by the addition of the chain terminator cordycepin at the 3′ end did not affect synthesis of the RNA monomer but eliminated synthesis of the self-priming hairpin dimer RNA. Moreover, synthesis of RNA on poly(C) and poly(U) homopolymer templates by 1b-42 NS5B did not require the oligonucleotide primer at high concentrations (≥50 μM) of GTP and ATP, further supporting a de novo initiation mechanism. These findings suggest that HCV NS5B is able to initiate RNA synthesis de novo.

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42

Cao, Dongdong, Yunrong Gao, and Bo Liang. "Structural Insights into the Respiratory Syncytial Virus RNA Synthesis Complexes." Viruses 13, no. 5 (May 5, 2021): 834. http://dx.doi.org/10.3390/v13050834.

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RNA synthesis in respiratory syncytial virus (RSV), a negative-sense (−) nonsegmented RNA virus, consists of viral gene transcription and genome replication. Gene transcription includes the positive-sense (+) viral mRNA synthesis, 5′-RNA capping and methylation, and 3′ end polyadenylation. Genome replication includes (+) RNA antigenome and (−) RNA genome synthesis. RSV executes the viral RNA synthesis using an RNA synthesis ribonucleoprotein (RNP) complex, comprising four proteins, the nucleoprotein (N), the large protein (L), the phosphoprotein (P), and the M2-1 protein. We provide an overview of the RSV RNA synthesis and the structural insights into the RSV gene transcription and genome replication process. We propose a model of how the essential four proteins coordinate their activities in different RNA synthesis processes.

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43

Kim, Min-Ju, Weidong Zhong, Zhi Hong, and C. Cheng Kao. "Template Nucleotide Moieties Required for De Novo Initiation of RNA Synthesis by a Recombinant Viral RNA-Dependent RNA Polymerase." Journal of Virology 74, no. 22 (November 15, 2000): 10312–22. http://dx.doi.org/10.1128/jvi.74.22.10312-10322.2000.

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ABSTRACT The recombinant RNA-dependent RNA polymerase of the bovine viral diarrhea virus specifically requires a cytidylate at the 3′ end for the de novo initiation of RNA synthesis (C. C. Kao, A. M. Del Vecchio, and W. Zhong, Virology 253:1–7, 1999). Using RNAs containing nucleotide analogs, we found that the N3 and C4-amino group at the initiation cytidine were required for RNA synthesis. However, the ribose C2′-hydroxyl of the initiating cytidylate can accept several modifications and retain the ability to direct synthesis. The only unacceptable modification is a protonated C2′-amino group. Quite strikingly, the recognition of the functional groups for the initiation cytidylate and other template nucleotides are different. For example, a C5-methyl group in cytidine can direct RNA synthesis at all template positions except at the initiation cytidylate and C2′-amino modifications are tolerated better after the +11 position. When a 4-thiouracil (4sU) base analog that allows only imperfect base pairing with the nascent RNA is placed at different positions in the template, the efficiency of synthesis is correlated with the calculated stability of the template-nascent RNA duplex adjacent to the position of the 4sU. These results define the requirements for the specific interactions required for the initiation of RNA synthesis and will be compared to the mechanisms of initiation by other RNA-dependent and DNA-dependent RNA polymerases.

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44

Lai, Vicky C. H., C. Cheng Kao, Eric Ferrari, Justin Park, Annette S. Uss, Jacquelyn Wright-Minogue, Zhi Hong, and Johnson Y. N. Lau. "Mutational Analysis of Bovine Viral Diarrhea Virus RNA-Dependent RNA Polymerase." Journal of Virology 73, no. 12 (December 1, 1999): 10129–36. http://dx.doi.org/10.1128/jvi.73.12.10129-10136.1999.

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ABSTRACT Recombinant bovine viral diarrhea virus (BVDV) nonstructural protein 5B (NS5B) produced in insect cells has been shown to possess an RNA-dependent RNA polymerase (RdRp) activity. Our initial attempt to produce the full-length BVDV NS5B with a C-terminal hexahistidine tag in Escherichia coli failed due to the expression of insoluble products. Prompted by a recent report that removal of the C-terminal hydrophobic domain significantly improved the solubility of hepatitis C virus (HCV) NS5B, we constructed a similar deletion of 24 amino acids at the C terminus of BVDV NS5B. The resulting fusion protein, NS5BΔCT24-His, was purified to homogeneity and demonstrated to direct RNA replication via both primer-dependent (elongative) and primer-independent (de novo) mechanisms. Furthermore, BVDV RdRp was found to utilize a circular single-stranded DNA as a template for RNA synthesis, suggesting that synthesis does not require ends in the template. In addition to the previously described polymerase motifs A, B, C, and D, alignments with other flavivirus sequences revealed two additional motifs, one N-terminal to motif A and one C-terminal to motif D. Extensive alanine substitutions showed that while most mutations had similar effects on both elongative and de novo RNA syntheses, some had selective effects. Finally, deletions of up to 90 amino acids from the N terminus did not significantly affect RdRp activities, whereas deletions of more than 24 amino acids at the C terminus resulted in either insoluble products or soluble proteins (ΔCT179 and ΔCT218) that lacked RdRp activities.

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45

Morasco, B. Joan, Nidhi Sharma, Jessica Parilla, and James B. Flanegan. "Poliovirus cre(2C)-Dependent Synthesis of VPgpUpU Is Required for Positive- but Not Negative-Strand RNA Synthesis." Journal of Virology 77, no. 9 (May 1, 2003): 5136–44. http://dx.doi.org/10.1128/jvi.77.9.5136-5144.2003.

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ABSTRACT The cre(2C) hairpin is a cis-acting replication element in poliovirus RNA and serves as a template for the synthesis of VPgpUpU. We investigated the role of the cre(2C) hairpin on VPgpUpU synthesis and viral RNA replication in preinitiation RNA replication complexes isolated from HeLa S10 translation-RNA replication reactions. cre(2C) hairpin mutations that block VPgpUpU synthesis in reconstituted assays with purified VPg and poliovirus polymerase were also found to completely inhibit VPgpUpU synthesis in preinitiation replication complexes. Surprisingly, blocking VPgpUpU synthesis by mutating the cre(2C) hairpin had no significant effect on negative-strand synthesis but completely inhibited positive-strand synthesis. Negative-strand RNA synthesized in these reactions immunoprecipitated with anti-VPg antibody and demonstrated that it was covalently linked to VPg. This indicated that VPg was used to initiate negative-strand RNA synthesis, although the cre(2C)-dependent synthesis of VPgpUpU was inhibited. Based on these results, we concluded that the cre(2C)-dependent synthesis of VPgpUpU was required for positive- but not negative-strand RNA synthesis. These findings suggest a replication model in which negative-strand synthesis initiates with VPg uridylylated in the 3′ poly(A) tail in virion RNA and positive-strand synthesis initiates with VPgpUpU synthesized on the cre(2C) hairpin. The pool of excess VPgpUpU synthesized on the cre(2C) hairpin should support high levels of positive-strand synthesis and thereby promote the asymmetric replication of poliovirus RNA.

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46

Ranjith-Kumar, C. T., J. Gajewski, L. Gutshall, D. Maley, R. T. Sarisky, and C. Cheng Kao. "Terminal Nucleotidyl Transferase Activity of RecombinantFlaviviridae RNA-Dependent RNA Polymerases: Implication for Viral RNA Synthesis." Journal of Virology 75, no. 18 (September 15, 2001): 8615–23. http://dx.doi.org/10.1128/jvi.75.18.8615-8623.2001.

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ABSTRACT Recombinant hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) was reported to possess terminal transferase (TNTase) activity, the ability to add nontemplated nucleotides to the 3′ end of viral RNAs. However, this TNTase was later purported to be a cellular enzyme copurifying with the HCV RdRp. In this report, we present evidence that TNTase activity is an inherent function of HCV and bovine viral diarrhea virus RdRps highly purified from both prokaryotic and eukaryotic cells. A change of the highly conserved GDD catalytic motif in the HCV RdRp to GAA abolished both RNA synthesis and TNTase activity. Furthermore, the nucleotides added via this TNTase activity are strongly influenced by the sequence near the 3′ terminus of the viral template RNA, perhaps accounting for the previous discrepant observations between RdRp preparations. Last, the RdRp TNTase activity was shown to restore the ability to direct initiation of RNA synthesis in vitro on an initiation-defective RNA substrate, thereby implicating this activity in maintaining the integrity of the viral genome termini.

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47

VANZI, F. "Protein synthesis by single ribosomes." RNA 9, no. 10 (October 1, 2003): 1174–79. http://dx.doi.org/10.1261/rna.5800303.

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48

Kierzek, Ryszard, David W. Kopp, Mary Edmonds, and Marvin H. Caruthers. "Chemical synthesis of branched RNA." Nucleic Acids Research 14, no. 12 (1986): 4751–64. http://dx.doi.org/10.1093/nar/14.12.4751.

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49

Wahl, M. C., B. Ramakrishnan, C. Ban, X. Chen, and M. Sundaralingam. "RNA – Synthesis, Purification and Crystallization." Acta Crystallographica Section D Biological Crystallography 52, no. 4 (July 1, 1996): 668–75. http://dx.doi.org/10.1107/s0907444996002788.

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50

Kompis, Ivan M., Khalid Islam, and Rudolf L. Then. "DNA and RNA Synthesis: Antifolates." Chemical Reviews 105, no. 2 (February 2005): 593–620. http://dx.doi.org/10.1021/cr0301144.

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