title: Versatile Trans-Replication Systems for Chikungunya Virus Allow Functional Analysis and Tagging of Every Replicase Protein.
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.
authors.affiliation: Institute of Technology, University of Tartu, Tartu, 50411, Estonia.
authors.forename: Age
authors.initials: A
authors.lastname: Utt
authors.affiliation: Department of Food and Environmental Sciences, University of Helsinki, Helsinki, Finland.
authors.forename: Tania
authors.initials: T
authors.lastname: Quirin
authors.affiliation: Institute of Technology, University of Tartu, Tartu, 50411, Estonia.
authors.forename: Sirle
authors.initials: S
authors.lastname: Saul
authors.affiliation: Department of Food and Environmental Sciences, University of Helsinki, Helsinki, Finland.
authors.forename: Kirsi
authors.initials: K
authors.lastname: Hellström
authors.affiliation: Department of Food and Environmental Sciences, University of Helsinki, Helsinki, Finland.
authors.forename: Tero
authors.initials: T
authors.lastname: Ahola
authors.affiliation: Institute of Technology, University of Tartu, Tartu, 50411, Estonia.
authors.forename: Andres
authors.initials: A
authors.lastname: Merits
mesh_terms: D000818:Animals; D002460:Cell Line; D002646:Chikungunya virus / Q000502:physiology*; D006224:Cricetinae; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism*; D012319:RNA Polymerase II / Q000235:genetics / Q000378:metabolism*; D012367:RNA, Viral / Q000096:biosynthesis* / Q000235:genetics; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics / Q000378:metabolism*; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication
chemical_list: D012367:RNA, Viral; D014764:Viral Proteins; D012319:RNA Polymerase II; C062210:bacteriophage T7 RNA polymerase; D012324:RNA-Dependent RNA Polymerase; D012321:DNA-Directed RNA Polymerases