Versatile Trans-Replication Systems for Chikungunya Virus Allow Functional Analysis and Tagging of Every Replicase Protein

Show full item record



Permalink

http://hdl.handle.net/10138/161549

Citation

Utt , A , Quirin , T , Saul , S , Hellstrom , K , Ahola , T & Merits , A 2016 , ' Versatile Trans-Replication Systems for Chikungunya Virus Allow Functional Analysis and Tagging of Every Replicase Protein ' , PLoS One , vol. 11 , no. 3 , 0151616 . https://doi.org/10.1371/journal.pone.0151616

Title: Versatile Trans-Replication Systems for Chikungunya Virus Allow Functional Analysis and Tagging of Every Replicase Protein
Author: Utt, Age; Quirin, Tania; Saul, Sirle; Hellstrom, Kirsi; Ahola, Tero; Merits, Andres
Contributor: University of Helsinki, Department of Food and Nutrition
University of Helsinki, Department of Food and Nutrition
University of Helsinki, Department of Food and Nutrition
Date: 2016-03-10
Language: eng
Number of pages: 27
Belongs to series: PLoS One
ISSN: 1932-6203
URI: http://hdl.handle.net/10138/161549
Abstract: Chikungunya virus (CHIKV; genus Alphavirus, family Togaviridae) has recently caused several major outbreaks affecting millions of people. There are no licensed vaccines or antivirals, and the knowledge of the molecular biology of CHIKV, crucial for development of efficient antiviral strategies, remains fragmentary. CHIKV has a 12 kb positive-strand RNA genome, which is translated to yield a nonstructural (ns) or replicase polyprotein. CHIKV structural proteins are expressed from a subgenomic RNA synthesized in infected cells. Here we have developed CHIKV trans-replication systems, where replicase expression and RNA replication are uncoupled. Bacteriophage T7 RNA polymerase or cellular RNA polymerase II were used for production of mRNAs for CHIKV ns polyprotein and template RNAs, which are recognized by CHIKV replicase and encode for reporter proteins. CHIKV replicase efficiently amplified such RNA templates and synthesized large amounts of subgenomic RNA in several cell lines. This system was used to create tagged versions of ns proteins including nsP1 fused with enhanced green fluorescent protein and nsP4 with an immunological tag. Analysis of these constructs and a matching set of replicon vectors revealed that the replicases containing tagged ns proteins were functional and maintained their subcellular localizations. When cells were co-transfected with constructs expressing template RNA and wild type or tagged versions of CHIKV replicases, formation of characteristic replicase complexes (spherules) was observed. Analysis of mutations associated with noncytotoxic phenotype in CHIKV replicons showed that a low level of RNA replication is not a pre-requisite for reduced cytotoxicity. The CHIKV trans-replicase does not suffer from genetic instability and represents an efficient, sensitive and reliable tool for studies of different aspects of CHIKV RNA replication process.
Subject: SEMLIKI-FOREST-VIRUS
MINICIRCLE DNA VECTORS
STRAND RNA-SYNTHESIS
SINDBIS-VIRUS
MINUS-STRAND
NONSTRUCTURAL PROTEIN-2
TEMPORAL REGULATION
EXPRESSION VECTORS
PLASMA-MEMBRANE
GENE-EXPRESSION
416 Food Science
Rights:


Files in this item

Total number of downloads: Loading...

Files Size Format View
journal.pone.0151616.PDF 4.390Mb PDF View/Open

This item appears in the following Collection(s)

Show full item record