Structural and phenotypic analysis of Chikungunya virus RNA replication elements

Catherine Kendall,Marietta Müller,Henna Khalid,Nicola J Stonehouse,Andrew Tuplin,Dominic H Banda,Alain Kohl,Andres Merits

doi:10.1093/nar/gkz640

Abstract

Chikungunya virus (CHIKV) is a re-emerging, pathogenic Alphavirus transmitted to humans by Aedes spp. mosquitoes. We have mapped the RNA structure of the 5′ region of the CHIKV genome using selective 2′-hydroxyl acylation analysed by primer extension (SHAPE) to investigate intramolecular base-pairing at single-nucleotide resolution. Taking a structure-led reverse genetic approach, in both infectious virus and sub-genomic replicon systems, we identified six RNA replication elements essential to efficient CHIKV genome replication - including novel elements, either not previously analysed in other alphaviruses or specific to CHIKV. Importantly, through a reverse genetic approach we demonstrate that the replication elements function within the positive-strand genomic copy of the virus genome, in predominantly structure-dependent mechanisms during efficient replication of the CHIKV genome. Comparative analysis in human and mosquito-derived cell lines reveal that a novel element within the 5′UTR is essential for efficient replication in both host systems, while those in the adjacent nsP1 encoding region are specific to either vertebrate or invertebrate host cells. In addition to furthering our knowledge of fundamental aspects of the molecular virology of this important human pathogen, we foresee that results from this study will be important for rational design of a genetically stable attenuated vaccine.

Highlights

Chikungunya virus (CHIKV) is a member of the Alphavirus genus within the Togaviridae family and has become an increasingly important arbovirus in tropical and sub-tropical regions, responsible for a range of febrile and both acute and chronic arthralgic symptoms in humans
These results were in concordance with SHAPEconstrained in silico thermodynamic folding predictions, which again predicted that RNA structure within this region of the virus genome was independent of differences in permissive temperature between vertebrate and invertebrate hosts (Figure 1B and Supplementary Figure S1)
We describe novel RNA structural elements, within the positive-strand genomic copy of the CHIKV genome, that function in host independent and dependent mechanisms during efficient replication of the virus genome

Summary

Introduction

Chikungunya virus (CHIKV) is a member of the Alphavirus genus within the Togaviridae family and has become an increasingly important arbovirus in tropical and sub-tropical regions, responsible for a range of febrile and both acute and chronic arthralgic symptoms in humans. CHIKV is transmitted by Aedes spp. mosquitos - predominantly Aedes aegypti in tropical/sub-tropical regions but increasingly via Aedes albopictus, which has a wider geographical distribution, including across more temperate regions. CHIKV is a small, enveloped, virus with a positive-sense RNA genome ∼11.8 kb in length. ORF-1 encodes the non-structural proteins nsP1– 4, which form distinct modules of the viral replicase complex that is responsible for CHIKV RNA synthesis. Proteolytic processing of the non-structural protein precursors in the replicase complex, favours its association with the negative-strand and subsequent replication of positive-sense full-length genomic transcripts. Subgenomic (26S) ORF-2 transcripts, encoding the structural proteins (capsid, envelope glycoproteins and 6K viroporin channel), are synthesized from a sub-genomic promoter in the negative strand [8]

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Results

Conclusion