Another long-term project in the lab has come to completion – this time with a publication in eLife. Our PhD student Tim Laurent was incredibly brave to start an empty-canvas project in 2018, studying the structure of the alphavirus replication organelles (a.k.a. “spherules”). Arguably the worst alphavirus in terms of global disease burden is chikungunya virus (CHIKV), and Tim chose this virus in spite of its BLS3 rating. In the first of many collaborative aspect to the project, Tim’s work on CHIKV in the lower BSL2 setting was enabled since the group of Andreas Merits in Tartu were so expedient in sharing a viral replicon particle system (essentially a “single cycle” CHIKV that cannot spread). Tim used cryo-electron tomography to study the genome-replicating organelles of CHIKV at the plasma membrane of infected cells. He uncovered a protein complex sitting at the membrane neck and used subtomogram averaging to obtain a first structure of it.
Another important part of this paper came from our previous postdoc Pravin Kumar. Pravin used biochemical reconstitution methods to show how the “neck complex” assembles on membranes: nsP1 anchors to the membranes dependent on monovalent anionic lipids, and can recruit the helicase-protease nsP2 which on its own has no membrane affinity.
In a further collaborative aspect, we were blessed to work together with two mathematicians with a keen interest in membrane remodelling: Andreas Carlson and Susanne Liese at Oslo University. They heard about our system during an interdisciplinary workshop organised by Andreas at Geilo, Norway. We told them about our suspicion that membrane remodelling might be driven by the polymerisation of viral RNA, which would be a novel mechanism of membrane budding. Tim and I (Lars) returned to Umeå from the workshop in early March 2020, so other things (i.e. other viruses) were on our minds. Our jaws dropped a few months later when Susanne and Andreas contacted us and basically said “we’ve solved it”. They had created a mathematical framework that modelled the membrane as a Helfrich-type elastic surface, and the polymerising RNA as a confined worm-like chain. Calibrating the model against experimental data, it first recapitulated early stage intermediates that we didn’t have in the original data set, and then delivered a clear answer to the question we had posed: indeed, RNA polymerisation can provide sufficient energy to remodel the replication organelle’s membrane into its characteristic balloon shape. In quantitative terms, at the peak “resistance” of the inflating membrane it requires about ten percent of the energy released by RNA polymerisation.
Congratulations to all co-authors which also includes two former Master’s thesis students: Mattias Jonasson and Farnaz Zare.
And since the article was published in eLife it is free for everyone to read here: