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.

Coupling between Replication and Packaging of Flavivirus RNA: Evidence Derived from the Use of DNA-Based Full-Length cDNA Clones of Kunjin Virus

Abstract: In order to study whether flavivirus RNA packaging is dependent on RNA replication, we generated two DNA-based Kunjin virus constructs, pKUN1 and pKUN1dGDD, allowing continuous production of replicating (wild-type) and nonreplicating (with a deletion of the NS5 gene RNA-polymerase motif GDD) full-length Kunjin virus RNAs, respectively, via nuclear transcription by cellular RNA polymerase II. As expected, transfection of pKUN1 plasmid DNA into BHK cells resulted in the recovery of secreted infectious Kunjin virions. Transfection of pKUN1dGDD DNA into BHK cells, however, did not result in the recovery of any secreted virus particles containing encapsidated dGDD RNA, despite an apparent accumulation of this RNA in cells demonstrated by Northern blot analysis and its efficient translation demonstrated by detection of correctly processed labeled structural proteins (at least prM and E) both in cells and in the culture fluid using coimmunoprecipitation analysis with anti-E antibodies. In contrast, when dGDD RNA was produced even in much smaller amounts in pKUN1dGDD DNA-transfected repBHK cells (where it was replicated via complementation), it was packaged into secreted virus particles. Thus, packaging of defective Kunjin virus RNA could occur only when it was replicated. Our results with genome-length Kunjin virus RNA and the results with poliovirus replicon RNA (C. I. Nugent et al., J. Virol. 73:427–435, 1999), both demonstrating the necessity for the RNA to be replicated before it can be packaged, strongly suggest the existence of a common mechanism for minimizing amplification and transmission of defective RNAs among the quasispecies in positive-strand RNA viruses. This mechanism may thus help alleviate the high-copy error rate of RNA-dependent RNA polymerases.

Discovery of an essential nucleotidylating activity associated with a newly delineated conserved domain in the RNA polymerase-containing protein of all nidoviruses

Abstract: RNA viruses encode an RNA-dependent RNA polymerase (RdRp) that catalyzes the synthesis of their RNA(s). In the case of positive-stranded RNA viruses belonging to the order Nidovirales, the RdRp resides in a replicase subunit that is unusually large. Bioinformatics analysis of this non-structural protein has now revealed a nidoviral signature domain (genetic marker) that is N-terminally adjacent to the RdRp and has no apparent homologs elsewhere. Based on its conservation profile, this domain is proposed to have nucleotidylation activity. We used recombinant non-structural protein 9 of the arterivirus equine arteritis virus (EAV) and different biochemical assays, including irreversible labeling with a GTP analog followed by a proteomics analysis, to demonstrate the manganese-dependent covalent binding of guanosine and uridine phosphates to a lysine/histidine residue. Most likely this was the invariant lysine of the newly identified domain, named nidovirus RdRp-associated nucleotidyltransferase (NiRAN), whose substitution with alanine severely diminished the described binding. Furthermore, this mutation crippled EAV and prevented the replication of severe acute respiratory syndrome coronavirus (SARS-CoV) in cell culture, indicating that NiRAN is essential for nidoviruses. Potential functions supported by NiRAN may include nucleic acid ligation, mRNA capping and protein-primed RNA synthesis, possibilities that remain to be explored in future studies.

Targeting RNA with small molecules.

Abstract: Remarkable findings have cemented the central dogma of biology, where the transfer of genetic information from a sequence of nucleotides in DNA to a string of amino acids in proteins is mediated through RNA. Although RNA was recognized as playing a key role in this incredible process, for decades it was considered as a passive carrier of DNA's sequence information. The discovery of ribozymes has changed this view and facilitated a major paradigm shift. 3] RNA is now regarded as a respectable functional biomolecule with impressive catalytic potential. RNA has continued to fascinate and surprise the scientific community with its multifaceted roles in cell biology. The recent high-resolution structures of ribosomes show RNA to be the key component responsible for peptide-bond formation in protein biosynthesis. The discovery of RNA interference, a cellular response to double-stranded RNA that leads to sequence-specific gene silencing, reveals a new and unexpected role for small RNA molecules in gene regulation. Recent findings demonstrating specific interactions between low-molecular-weight metabolites and messenger RNAs (mRNAs) related to their biosynthetic pathways illustrate exciting new regulatory mechanisms at the RNA level. 11] Thus, another paradigm shift is emerging. The capability of RNA to specifically communicate with large and small molecules is central to its diverse biological functions. Revealing the structural and dynamic features of RNA± ligand recognition events will have a direct impact on our ability to ultimately control cell function at the RNA level. It will also open up new opportunities to combat pathogens by specifically targeting their RNA or RNA±protein complexes. With this in mind, we initiated, about a decade ago, a research program aimed at unraveling the fundamentals of RNA± ligand interactions and advancing RNA as a drug target. The purpose of this article is to highlight the inspiration for our program and its evolution. There is remarkable interest in this young and rapidly growing field, and this minireview is not intended to be comprehensive. The interested reader is referred to excellent review articles that summarize advances in this and related fields.

Synthesis of globin ribonucleic acid from duck-reticulocyte chromatin in vitro.

Abstract: The proteins of chromatin serve to restrict the transcription of DNA. The relevance of these findings to the control of gene expression is contingent upon the demonstration that this restriction is specific and mirrors the patterns of RNA synthesis observed in vivo. In this study we demonstrate by RNA-DNA hybridization that the vast majority of the chromatin-directed RNA is synthesized from the unique regions of the reticulocyte genome. Furthermore, by use of the DNA complement of globin mRNA as a probe in annealing reactions, de novo synthesis of globin RNA was detected in RNA transcripts from duck reticulocyte chromatin. No globin sequences were detected in similar preparations of RNA in vitro either from liver chromatin or from DNA freed of protein. These results show that the proteins of chromatin serve to restrict transcription in a very specific manner and provide convincing evidence for the existence of transcriptional control factors in eukaryotes.