Director’s guest: Justine Girard, CBS Montpellier – “In situ characterization of the CHIKV replication organelles by cryo-electron tomography”

Abstract

Chikungunya virus (CHIKV) is a human pathogen transmitted by mosquito bites from the Aedes family, which can lead to severe disease. In the last ten years, a dramatic increase in the number of cases was observed all over the world, driven by global warming, increasing the presence of its mosquito vector in temperate regions. In the absence of effective treatments, CHIKV is becoming an important public health issue. Alphaviruses such as CHIKV are known to be able to remodel cellular membranes to favor viral replication, and filopodia-like structures are commonly observed upon infection. Importantly, similarly to other (+) ss RNA viruses, CHIKV replication takes place in viral-induced compartments named replications organelles (ROs). It is assumed that these ROs provide a favorable environment for viral genome replication but also help to avoid the cellular RNA degradation machinery and innate immune detection. In CHIKV-infected cells, ROs are readily detected at the plasma membrane (PM), where CHIKV nsPs and its viral genome will form a replication complex (RC). Based on results obtained for similar viruses and recent publications using purified proteins, it was proposed that nsP1 is driving the formation of RC. However, there is to date, no published validation of the different RC model formations obtained from infected cells. In this regard, we therefore aimed to validate the different models by studying the tridimensional architecture of ROs directly from HEK293T cells infected by CHIKV using cryo-electron tomography (cryo-ET) approaches. The identification of the different viral and cellular partners involved in CHIKV replication could lead to the development of new therapeutic targets. In this work, we were able to determine the appropriate infection conditions allowing us to observe and confirm the presence of RO, first by confocal and electron microscopy (EM). Our work, like others, was also able to demonstrate the presence of filopodia-like structures upon infection, where, for the first time, it was possible to show that ROs and viral particles coexist. Cryo-ET analyses further validate our previous observations and help us shed light on an unexpected diversity of ROs suggesting that their formation and interaction with the near environment is a highly dynamic process. Importantly, ROs observed in our tomograms all showed the presence of a ‘foot’ at their neck. Sub-tomogram averaging methods demonstrated that this platform is formed of nsP1 proteins that assemble into a dodecameric ring to form a pore that could control the entry and exit of cellular and/or viral factors, thus validating previous models that suggest that nsP1 oligomerizes into a ring, located at the neck of the ROs. These results lead us to propose a model linking viral replication and viral-induced cellular deformations.