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

PLoS One. 2016 Mar 10;11(3):e0151616. doi: 10.1371/journal.pone.0151616. eCollection 2016.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cell Line
  • Chikungunya virus / physiology*
  • Cricetinae
  • DNA-Directed RNA Polymerases / genetics
  • DNA-Directed RNA Polymerases / metabolism*
  • RNA Polymerase II / genetics
  • RNA Polymerase II / metabolism*
  • RNA, Viral / biosynthesis*
  • RNA, Viral / genetics
  • RNA-Dependent RNA Polymerase / genetics
  • RNA-Dependent RNA Polymerase / metabolism*
  • Viral Proteins / genetics
  • Viral Proteins / metabolism*
  • Virus Replication*

Substances

  • RNA, Viral
  • Viral Proteins
  • RNA Polymerase II
  • bacteriophage T7 RNA polymerase
  • RNA-Dependent RNA Polymerase
  • DNA-Directed RNA Polymerases

Grants and funding

This work was supported by institutional research funding (IUT20-27) of the Estonian Ministry of Education and Research, the Estonian Science Foundation (grant 9400), the European Union 7th Framework Integrated Chikungunya Research project (grant agreement number 261202), the European Union through the European Regional Development Fund (grant agreement 10.1-9/1008) and via the Center of Excellence in Chemical Biology (to AM). This work was also supported by Academy of Finland grant number 1265997 (to TA). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.