How does Chikungunya virus regulate the switch between genome translation and replication?
Lead Research Organisation:
University of Leeds
Department Name: Sch of Molecular & Cellular Biology
Abstract
Chikungunya virus (CHIKV) is an arbovirus, transmitted by Aedes aegypti and Aedes albopictus mosquitos. Since its re-emergence in 2004, it has caused numerous epidemics across expanding geographical ranges within sub-/tropical areas of Africa, Asia and the Americas and increasingly across temperate regions of Europe, Asian and North America. The main risk factor for spread of CHIKV is change in global mosquito vector distribution - as a consequence of climate change associated seasonal variations and increasing mean temperatures, globalisation and changes in land use. Invasive populations of Ae. albopictus have been identified in the UK and temperature rises are expected to increase their months of activity and range.
Chikungunya disease is associated with severe debilitating joint pain for months or years and exacerbation of co-morbidities. High mortality rates are observed among elderly patients with pre-existing conditions, such as hypertension or diabetes. Despite increasing numbers of epidemics and their expanding geographical spread, there remains no specific antiviral therapy or licenced vaccine.
The mechanism and interactions by which CHIKV controls translation and replication of its genome are unclear. We have shown that CHIKV requires host protein MSI2 to replicate its genome in human cells. We and others have also shown that host protein DHX9 is required for efficient CHIKV translation, while simultaneously inhibiting replication of the virus genome. We demonstrated that MSI2 and DHX9 interact specifically with the 5' region of the CHIKV genome and identified an MSI2 RNA binding site within this region of the virus genome. We previously published a study demonstrating that the 5' region of the virus genome forms complex RNA structures, essential for CHIKV replication. We have also produced preliminary data consistent these RNA structures regulating the MSI2 binding to the CHIKV genome. Therefore, the scientific objective of the project is to generate a detailed functional and structural model of how these different factors interact, to regulate CHIKV translation and genome replication.
We will address the following objectives: (i) We will identify MSI2 and DHX9 RNA binding sites within the CHIKV genome and the role of RNA structure in MSI2 and DHX9 binding. (ii) Confocal imaging will be used to determine changes in localisation, co-localisation, expression and degradation of MSI2, DHX9 and nsPs during CHIKV translation and genome replication. Using the same approach, we will determine the influence of individual RNA structures on these changes and interactions. (iii) Our previously published studies demonstrate that RNA structures in the 5' region of the CHIKV genome are essential for virus genome replication. Therefore, we will use in vitro and in cell approaches to determine the RNA structure of the CHIKV genome following interaction with MSI2 and DHX9. We will also determine the RNA structure specifically associated with CHIKV genome replication and different stages of virus translation. (iv) To complete our understanding of the interactions between MSI2, DHX9 and the 5' region of CHIKV genome we use Cryo Electron Microscopy to generate high-resolution 3D models of unbound and protein-bound RNA tertiary structures. With results from the other aims, this will allow us to produce a detailed model of the regulation of CHIKV translation and genome replication.
As well as furthering our understanding of how CHIKV controls translation and replication of its genome, results will provide a model for related human pathogenic viruses. An increased understanding of this fundamental mechanism will provide insight towards novel therapeutic targets and attenuated vaccine design.
Chikungunya disease is associated with severe debilitating joint pain for months or years and exacerbation of co-morbidities. High mortality rates are observed among elderly patients with pre-existing conditions, such as hypertension or diabetes. Despite increasing numbers of epidemics and their expanding geographical spread, there remains no specific antiviral therapy or licenced vaccine.
The mechanism and interactions by which CHIKV controls translation and replication of its genome are unclear. We have shown that CHIKV requires host protein MSI2 to replicate its genome in human cells. We and others have also shown that host protein DHX9 is required for efficient CHIKV translation, while simultaneously inhibiting replication of the virus genome. We demonstrated that MSI2 and DHX9 interact specifically with the 5' region of the CHIKV genome and identified an MSI2 RNA binding site within this region of the virus genome. We previously published a study demonstrating that the 5' region of the virus genome forms complex RNA structures, essential for CHIKV replication. We have also produced preliminary data consistent these RNA structures regulating the MSI2 binding to the CHIKV genome. Therefore, the scientific objective of the project is to generate a detailed functional and structural model of how these different factors interact, to regulate CHIKV translation and genome replication.
We will address the following objectives: (i) We will identify MSI2 and DHX9 RNA binding sites within the CHIKV genome and the role of RNA structure in MSI2 and DHX9 binding. (ii) Confocal imaging will be used to determine changes in localisation, co-localisation, expression and degradation of MSI2, DHX9 and nsPs during CHIKV translation and genome replication. Using the same approach, we will determine the influence of individual RNA structures on these changes and interactions. (iii) Our previously published studies demonstrate that RNA structures in the 5' region of the CHIKV genome are essential for virus genome replication. Therefore, we will use in vitro and in cell approaches to determine the RNA structure of the CHIKV genome following interaction with MSI2 and DHX9. We will also determine the RNA structure specifically associated with CHIKV genome replication and different stages of virus translation. (iv) To complete our understanding of the interactions between MSI2, DHX9 and the 5' region of CHIKV genome we use Cryo Electron Microscopy to generate high-resolution 3D models of unbound and protein-bound RNA tertiary structures. With results from the other aims, this will allow us to produce a detailed model of the regulation of CHIKV translation and genome replication.
As well as furthering our understanding of how CHIKV controls translation and replication of its genome, results will provide a model for related human pathogenic viruses. An increased understanding of this fundamental mechanism will provide insight towards novel therapeutic targets and attenuated vaccine design.
Technical Summary
We have identified RNA stem-loops and a pseudoknot within the 5' region of the Chikungunya virus (CHIKV) genome and show that they are required for replicating the virus genome. Furthermore, we have identified two host proteins, Musashi RNA binding protein-2 (MSI2) and DExH-Box helicase-9 (DHX9), that interact specifically with the same 5' region of the CHIKV genome. We demonstrated that MSI2 binding is required for CHIKV genome replication and that binding efficiency is dependent on RNA the stem-loop structures. We and others have also shown that DHX9 has the opposite effect, upregulating CHIKV translation and down-regulating genome replication.
Our results are consistent with a hypothesis in which the switch between CHIKV translation and genome replication is controlled by dynamic interactions between the 5' RNA stem-loops, host proteins MSI2 and DHX9 and CHIKV non-structural proteins (nsPs). To determine how these RNA/protein interactions regulate the switch between CHIKV translation and genome replication, we will use reverse genetic, biochemical, cell biology, in-cell SHAPE and cryoEM approaches to investigate our proposed model through the following objectives:
- The influence of RNA structure on MSI2 and DHX9 RNA binding and identification of binding motifs.
- MSI2, DHX9 and nsP localisation, expression and degradation and the influence of RNA structure, during CHIKV translation and genome replication.
- RNA secondary structure associated with MSI2 and DHX9 binding, translation and genome replication.
- High resolution tertiary RNA structure bound and unbound to MSI2 and DHX
Results from this study will determine how these RNA/protein interactions regulate the switch between CHIKV translation and genome replication and provide a model for other pathogenic alphaviruses and related +veRNA viruses. An increased understanding of this fundamental mechanism will provide insight towards novel therapeutic targets and attenuated vaccine design.
Our results are consistent with a hypothesis in which the switch between CHIKV translation and genome replication is controlled by dynamic interactions between the 5' RNA stem-loops, host proteins MSI2 and DHX9 and CHIKV non-structural proteins (nsPs). To determine how these RNA/protein interactions regulate the switch between CHIKV translation and genome replication, we will use reverse genetic, biochemical, cell biology, in-cell SHAPE and cryoEM approaches to investigate our proposed model through the following objectives:
- The influence of RNA structure on MSI2 and DHX9 RNA binding and identification of binding motifs.
- MSI2, DHX9 and nsP localisation, expression and degradation and the influence of RNA structure, during CHIKV translation and genome replication.
- RNA secondary structure associated with MSI2 and DHX9 binding, translation and genome replication.
- High resolution tertiary RNA structure bound and unbound to MSI2 and DHX
Results from this study will determine how these RNA/protein interactions regulate the switch between CHIKV translation and genome replication and provide a model for other pathogenic alphaviruses and related +veRNA viruses. An increased understanding of this fundamental mechanism will provide insight towards novel therapeutic targets and attenuated vaccine design.
