Molecular mechanisms in the genome replication of alphaviruses and flaviviruses

Alphaviruses and flaviviruses are two types of positive-sense RNA viruses.

Their numerous members are often spread by mosquitoes and ticks, and they cause serious diseases in humans such as chikungunya, dengue fever and tick-borne encephalitis.

Both alphaviruses and flaviviruses generate dedicated genome-replication organelles, so-called replication complexes (RCs), on the cytoplasmic face of membranes in infected cells.

Their RCs share the same membrane topology (a 60-80 nm diameter membrane bud facing away from the cytoplasm), but are generated by very different viral proteins that likely arrange differently inside the RCs.

Here, we will study the structures of RCs in infected cells and tissues using cryo-electron tomography, allowing direct visualisation of their macromolecular organisation in its cellular context.

We will test the hypothesis that RC activity is influenced by cell membrane stiffness, and we will study the biochemical basis of RC assembly and activity.

For a panel of monoclonal antibody fragments (scFvs and Fabs) that modulate the activity of the alphavirus capping enzyme, we will determine their mechanism of inhibition/activation as well as possible antiviral activity.

For flavivirus RC proteins, we will employ proteome-scale computational methods to investigate the viral proteins’ interactions with each other and host-cell proteins.

Taken together, this proposal will drastically increase our understanding of alphavirus and flavivirus genome replication.