Versatile trans-replication systems for Chikungunya virus allow functional analysis and tagging of every replicase protein
Autor(es): Utt Age, Quirin Tania, Saul Sirle, Hellström Kirsi, Ahola Tero, Merits Andres
Resumo: Chikungunya virus (CHIKV; genus Alphavirus, family Togaviridae) has recently caused several major outbreaks affecting millions of people. There are no licensed vaccines or antivirals, - the knowledge of the molecular biology of CHIKV, crucial for development of efficient antiviral strategies, remains fragmentary. CHIKV has a 12 kb positive-str- 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 - RNA replication are uncoupled. Bacteriophage T7 RNA polymerase or cellular RNA polymerase II were used for production of mRNAs for CHIKV ns polyprotein - template RNAs, which are recognized by CHIKV replicase - encode for reporter proteins. CHIKV replicase efficiently amplified such RNA templates - 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 - nsP4 with an immunological tag. Analysis of these constructs - a matching set of replicon vectors revealed that the replicases containing tagged ns proteins were functional - maintained their subcellular localizations. When cells were co-transfected with constructs expressing template RNA - 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 - represents an efficient, sensitive - reliable tool for studies of different aspects of CHIKV RNA replication process.
Imprenta: PloS One, v. 11, n. 3, p. e0151616, 2016
Identificador do objeto digital: 10.1371/journal.pone.0151616
Descritores: Chikungunya virus - Cell ; Chikungunya virus - Genome ; Chikungunya virus - Proteins ; Chikungunya virus - RNA ; Chikungunya Virus - Virus ; Chikungunya virus - Vaccine
Data de publicação: 2016
