Functional and structural characterization of membranous organelles supporting Chikungunya virus replication in human cells – TOMOCHIKV

The TOMOCHIKV project is based on:
1- The development of genome-wide genetic screen, relying on insertional mutagenesis in HAP1 cells
2- Development of molecular virology, cell biology and biochemistry approaches for the study of nsP1 / membrane interactions
3- The implementation of cryo-electron microscopy for the study of infected cells

Our current achievements are:
1- The identification of cellular genes necessary for the replication of CHIKV
2- Highlighting the preferential affinity of nsP1 for cholesterol-rich membrane microdomains and its consequences on the localization of the CHIKV replication complexes
3- The implementation of electron cryo-microscopy on CHIKV-infected cells

The objectives that will be pursued for the next 18 months relate to:
1- The functional study of cellular cofactors identified by genetic screen
2- Characterization of the lipid content of cell membranes supporting the formation of CHIKV replication organelles
3- The acquisition of (cryo-) tomograms and their interpretation.

1. BAKHACHE W., NEYRET A., MCKELLAR J., CLOP C., BERNARD E., WEGER-LUCARELLI J., BRIANT L. Fatty acid synthase and stearoyl-CoA desaturase-1 are conserved druggable cofactors of Old World Alphavirus genome replication. Antiviral Research. 172:104642 (2019).
www.ncbi.nlm.nih.gov/pubmed/31678479

2. BAKHACHE W., NEYRET A., BERNARD E., MERITS A., BRIANT L. Palmitoylated cysteines in Chikungunya virus nsP1 are critical for targeting to cholesterol-rich plasma membrane microdomains with functional consequences for viral genome replication. Journal of Virology JVI.02183-19 (2020).
www.ncbi.nlm.nih.gov/pubmed/32132240

3. BAKHACHE W, COUDERC É, NEYRET A, BRIANT L. Architecture and biogenesis of positive-stranded RNA virus replication organelles. Virologie. 23(3):160-175. (2019).
www.ncbi.nlm.nih.gov/pubmed/31210133

Despite intensive research efforts on the biology of the Chikungunya virus (CHIKV) our current knowledge on the replication of this medically important pathogen with high impact on quality of life and economic growth lags behind what we know on the cellular immune responses generated in infected cells. This lack of knowledge represents a barrier to the development of anti-infectious strategies.

The overall objectives of the TOMOCHIKV program are to bring new functional and structural information relative to CHIKV life cycle focusing on membranous replication organelles that foster replication of the viral genome. These compartments derived from the host plasma membrane provide a structural framework for the replication complex, concentrate viral and host factors required for optimal replication, and shield viral components from the host immune defense. They represent de facto attractive targets for the development of molecules capable to interrupt viral replication. Mechanisms accounting for the biogenesis of these organelles are unknown. TOMOCHIKV has been conceived as a transdisciplinary program, which combines a genome wide screening, functional studies in CHIKV-infected cells and (cryo-)electron tomography and (cryo-) correlative light and electron microscopy analysis to address the following aims:

(1) the identification of host-pathogen interactions required for the biogenesis of CHIKV membranous replication compartments created upon infection and hosting viral RNA replication;

(2) the role played by CHIKV nsP1 protein in the biogenesis of these compartments

(3) the 3D organization of these organelles, of associated factors and of surrounding areas during viral replication.

To achieve this goal, this program joins skills of three partners affiliated to the CNRS and INSERM, with complementary expertise in genetic screening, in molecular virology in the field of CHIKV research and in structural biology of viral complexes.

The results of the TOMOCHIKV program are expected to improve significantly the knowledge on CHIKV biology with the attractive possibility of transposing this information to the understanding of related alphavirus. They are anticipated to have broad implications for the development of therapeutic strategies targeting common mechanisms used for replication of (+)RNA viruses pathogenic for human that share properties to assemble similar membranous compartments in host cells.