Topic
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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TL;DR: A hierarchy among vRNA segments for virion incorporation is suggested and may imply intersegment association of vRNAs during virus assembly.
Abstract: The genome of influenza A viruses comprises eight negative-strand RNA segments. Although all eight segments must be present in cells for efficient viral replication, the mechanism(s) by which these viral RNA (vRNA) segments are incorporated into virions is not fully understood. We recently found that sequences at both ends of the coding regions of the HA, NA, and NS vRNA segments of A/WSN/33 play important roles in the incorporation of these vRNAs into virions. In order to similarly identify the regions of the PB2, PB1, and PA vRNAs of this strain that are critical for their incorporation, we generated a series of mutant vRNAs that possessed the green fluorescent protein gene flanked by portions of the coding and noncoding regions of the respective segments. For all three polymerase segments, deletions at the ends of their coding regions decreased their virion incorporation efficiencies. More importantly, these regions not only affected the incorporation of the segment in which they reside, but were also important for the incorporation of other segments. This effect was most prominent with the PB2 vRNA. These findings suggest a hierarchy among vRNA segments for virion incorporation and may imply intersegment association of vRNAs during virus assembly.
191 citations
TL;DR: Sequence‐specific hybridization probes of high specific activity are prepared by cloning the probe sequence downstream of a bacteriophage promoter, and the plasmid DNA is transcribed with bacteriophile RNA polymerase, which efficiently transcribes the cloned sequence into a discrete RNA species of known specific activity and high abundance.
Abstract: Sequence-specific hybridization probes of high specific activity are prepared by cloning the probe sequence downstream of a bacteriophage promoter. The plasmid is cleaved with a restriction enzyme, and the plasmid DNA is transcribed with bacteriophage RNA polymerase, which efficiently transcribes the cloned sequence into a discrete RNA species of known specific activity and high abundance. The RNA is purified by removal of the DNA template, protein, and the unincorporated label. Alternatively, the probe is purified by gel electrophoresis, as described in a support protocol. The probe RNA is hybridized to sample RNAs and the hybridization reactions are treated with ribonuclease to remove free probe, leaving intact fragments of probe annealed to homologous sequences in the sample RNA. These fragments are analyzed by electrophoresis on a sequencing gel and the presence of the target mRNA is revealed by the appearance of an appropriately sized fragment of the probe.
191 citations
TL;DR: The structures of the NS5B polymerase/non-nucleoside inhibitor complexes bind at a common binding site, which is nearly 35 Å away from the polymerase active site and is located in the thumb domain, and the enzyme inhibitor complexes are stabilized by hydrogen bonding and van der Waals interactions.
191 citations
TL;DR: Current understanding of SARS-CoV enzymes involved in RNA biochemistry is summarized, such as the in vitro characterization of a highly active and processive RNA polymerase complex which can associate with methyltransferase and 3′–5′ exoribonuclease activities involved inRNA capping, and RNA proofreading, respectively.
191 citations
TL;DR: Recent progress on the identification and mechanism of the key components including viral sensors, viral triggers, effectors, and amplifiers, of the small RNA‐directed viral immunity are reviewed.
Abstract: Suppression of viral infection by RNA in a nucleotide sequence homology-dependent manner was first reported in plants in early 1990 s. Studies in the past 15 years have established a completely new RNA-based immune system against viruses that is mechanistically related to RNA silencing or RNA interference (RNAi). This viral immunity begins with recognition of viral double-stranded or structured RNA by the Dicer nuclease family of host immune receptors. In fungi, plants and invertebrates, the viral RNA trigger is processed into small interfering RNAs (siRNAs) to direct specific silencing of the homologous viral genomic and/or messenger RNAs by an RNaseH-like Argonaute protein. Deep sequencing of virus-derived siRNAs indicates that the immunity against viruses with a positive-strand RNA genome is induced by Dicer recognition of dsRNA formed during the initiation of viral progeny (+)RNA synthesis. The RNA-based immune pathway in these organisms overlaps the canonical dsRNA-siRNA pathway of RNAi and may require amplification of viral siRNAs by host RNA-dependent RNA polymerase in plants and nematodes. Production of virus-derived small RNAs is undetectable in mammalian cells infected with RNA viruses. However, infection of mammals with several nucleus-replicating DNA viruses induces production of virus-derived microRNAs capable of silencing host and viral mRNAs as found for viral siRNAs. Remarkably, recent studies indicate that prokaryotes also produce virus-derived small RNAs known as CRISPR RNAs to guide antiviral defense in a manner that has yet to be defined. In this article, we review the recent progress on the identification and mechanism of the key components including viral sensors, viral triggers, effectors, and amplifiers, of the small RNA-directed viral immunity. We also highlight some of the many unresolved questions.
191 citations