In vitro production of synthetic viral RNAs and their delivery into mammalian cells and the application of viral RNAs in the study of innate interferon responses

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http://hdl.handle.net/10138/323338

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Jiang , M , Österlund , P , Poranen , M & Julkunen , I 2020 , ' In vitro production of synthetic viral RNAs and their delivery into mammalian cells and the application of viral RNAs in the study of innate interferon responses ' , Methods , vol. 183 , pp. 21-29 . https://doi.org/10.1016/j.ymeth.2019.10.013

Title: In vitro production of synthetic viral RNAs and their delivery into mammalian cells and the application of viral RNAs in the study of innate interferon responses
Author: Jiang, Miao; Österlund, Pamela; Poranen, Minna; Julkunen, Ilkka
Contributor: University of Helsinki, Molecular and Integrative Biosciences Research Programme
University of Helsinki, Molecular and Translational Virology
Date: 2020-11-01
Language: eng
Number of pages: 9
Belongs to series: Methods
ISSN: 1046-2023
URI: http://hdl.handle.net/10138/323338
Abstract: Mammalian cells express different types of RNA molecules that can be classified as protein coding RNAs (mRNA) and non-coding RNAs (ncRNAs) the latter of which have housekeeping and regulatory functions in cells. Cellular RNAs are not recognized by cellular pattern recognition receptors (PRRs) and innate immunity is not activated. RNA viruses encode and express RNA molecules that usually differ from cell-specific RNAs and they include for instance 5'capped and 5-mono- and triphosphorylated RNAs, small viral RNAs and viral RNA-protein complexes called vRNPs. These molecules are recognized by certain members of Toll-like receptor (TLR) and RIG-I-like receptor (RLR) families leading to activation of innate immune responses and the production of antiviral cytokines, such as type I and type III interferons (IFNs). Virus-specific ssRNA and dsRNA molecules that mimic the viral genomic RNAs or their replication intermediates can efficiently be produced by bacteriophage T7 DNA-dependent RNA polymerase and bacteriophage phi6 RNA-dependent RNA polymerase, respectively. These molecules can then be delivered into mammalian cells and the mechanisms of activation of innate immune responses can be studied. In addition, synthetic viral dsRNAs can be processed to small interfering RNAs (siRNAs) by a Dicer enzyme to produce a swarm of antiviral siRNAs. Here we describe the biology of RNAs, their in vitro production and delivery into mammalian cells as well as how these molecules can be used to inhibit virus replication and to study the mechanisms of activation of the innate immune system.
Subject: 1182 Biochemistry, cell and molecular biology
1183 Plant biology, microbiology, virology
RNA
Virus
Dendritic cells (DCs)
Interferon (IFN) response
In vitro RNA production
RNA delivery
DsiRNA
Innate immunity
DOUBLE-STRANDED-RNA
SUBGENOMIC FLAVIVIRUS RNA
DENDRITIC CELLS
RIG-I
CHEMICAL-SYNTHESIS
SIRNA DELIVERY
SMALL NUCLEAR
RECOGNITION
VIRUS
POOLS
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