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RNA-dependent RNA polymerase

About: RNA-dependent RNA polymerase is a research topic. Over the lifetime, 13904 publications have been published within this topic receiving 767954 citations. The topic is also known as: RdRp & RNA replicase.


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Journal ArticleDOI
TL;DR: A method for localizing and quantitating m6A within a large RNA molecule, the genomic RNA of Rous sarcoma virus, and observing a high frequency of methylation at PuGm6ACU sequences is described.
Abstract: N6-methyladenosine (m6A) residues are present as internal base modifications in most higher eucaryotic mRNAs; however, the biological function of this modification is not known. We describe a method for localizing and quantitating m6A within a large RNA molecule, the genomic RNA of Rous sarcoma virus. Specific fragments of 32P-labeled Rous sarcoma virus RNA were isolated by hybridization with complementary DNA restriction fragments spanning nucleotides 6185 to 8050. RNA was digested with RNase and finger-printed, and individual oligonucleotides were analyzed for the presence of m6A by paper electrophoresis and thin-layer chromatography. With this technique, seven sites of methylation in this region of the Rous sarcoma virus genome were localized at nucleotides 6394, 6447, 6507, 6718, 7414, 7424, and 8014. Further, m6A was observed at two additional sites whose nucleotide assignments remain ambiguous. A clustering of two or more m6A residues was seen at three positions within the RNA analyzed. Modification at certain sites was found to be heterogeneous, in that different molecules of RNA appeared to be methylated differently. Previous studies have determined that methylation occurs only in the sequences Gm6AC and Am6AC. We observed a high frequency of methylation at PuGm6ACU sequences. The possible involvement of m6A in RNA splicing events is discussed.

180 citations

Journal ArticleDOI
TL;DR: Vector particle seed stocks could be amplified after low multiplicity of infection of PCLs, again without generating replication-competent virus, suggesting utility for production of large-scale vector preparations.
Abstract: Alphavirus vectors are being developed for possible human vaccine and gene therapy applications. We have sought to advance this field by devising DNA-based vectors and approaches for the production of recombinant vector particles. In this work, we generated a panel of alphavirus vector packaging cell lines (PCLs). These cell lines were stably transformed with expression cassettes that constitutively produced RNA transcripts encoding the Sindbis virus structural proteins under the regulation of their native subgenomic RNA promoter. As such, translation of the structural proteins was highly inducible and was detected only after synthesis of an authentic subgenomic mRNA by the vector-encoded replicase proteins. Efficient production of biologically active vector particles occurred after introduction of Sindbis virus vectors into the PCLs. In one configuration, the capsid and envelope glycoproteins were separated into distinct cassettes, resulting in vector packaging levels of 107 infectious units/ml, but reducing the generation of contaminating replication-competent virus below the limit of detection. Vector particle seed stocks could be amplified after low multiplicity of infection of PCLs, again without generating replication-competent virus, suggesting utility for production of large-scale vector preparations. Furthermore, both Sindbis virus-based and Semliki Forest virus-based vectors could be packaged with similar efficiency, indicating the possibility of developing a single PCL for use with multiple alphavirus-derived vectors.

180 citations

Journal ArticleDOI
15 Feb 2000-Virology
TL;DR: The utility of this approach is proved by the generation of virus after transfecting the pol I/pol II-promoter-PB1 construct together with vRNA- and protein-expression constructs for the remaining seven segments, and the use of the reverse-genetics system to cell lines for which efficient cotransfection of 17 plasmids cannot be achieved.

180 citations

Journal ArticleDOI
11 Apr 2013-Cell
TL;DR: The exploitation of and targeted interference with regular RNA turnover described here may constitute a general route for small RNAs to rapidly activate both coding and noncoding genes.

180 citations

Journal ArticleDOI
TL;DR: Results provide a molecular basis for inhibition of the SARS-CoV-2 RdRp by these nucleotide analogues and if sufficient efficacy of some of these FDA-approved drugs in inhibiting viral replication in cell culture is established, they may be explored as potential COVID-19 therapeutics.
Abstract: SARS-CoV-2 is responsible for the current COVID-19 pandemic. On the basis of our analysis of hepatitis C virus and coronavirus replication, and the molecular structures and activities of viral inhibitors, we previously demonstrated that three nucleotide analogues (the triphosphates of Sofosbuvir, Alovudine, and AZT) inhibit the SARS-CoV RNA-dependent RNA polymerase (RdRp). We also demonstrated that a library of additional nucleotide analogues terminate RNA synthesis catalyzed by the SARS-CoV-2 RdRp, a well-established drug target for COVID-19. Here, we used polymerase extension experiments to demonstrate that the active triphosphate form of Sofosbuvir (an FDA-approved hepatitis C drug) is incorporated by SARS-CoV-2 RdRp and blocks further incorporation. Using the molecular insight gained from the previous studies, we selected the active triphosphate forms of six other antiviral agents, Alovudine, Tenofovir alafenamide, AZT, Abacavir, Lamivudine, and Emtricitabine, for evaluation as inhibitors of the SARS-CoV-2 RdRp and demonstrated the ability of these viral polymerase inhibitors to be incorporated by SARS-CoV-2 RdRp, where they terminate further polymerase extension with varying efficiency. These results provide a molecular basis for inhibition of the SARS-CoV-2 RdRp by these nucleotide analogues. If sufficient efficacy of some of these FDA-approved drugs in inhibiting viral replication in cell culture is established, they may be explored as potential COVID-19 therapeutics.

180 citations


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Performance
Metrics
No. of papers in the topic in previous years
YearPapers
202358
2022201
2021222
2020200
2019116
2018118