Macromolecular organization of the chikungunya virus replication organelle

Abstract: The chikungunya virus is a positive-sense RNA virus responsible for the crippling chikungunya fever. It is transmitted through the bites of two species of mosquitoes: Aedes aegypti and Aedes albopictus. A key feature of this virus is that it is able to remodel the plasma membrane to form replication organelles called “spherules” in which the viral genomic RNA is replicated. There are four non-structural proteins in charge of the replication of the genome: nsP1, the capping enzyme, nsP2 the helicase, NTPase and protease, nsP3, a protein modulating the host-cell response to the infection and the RNA-dependent RNA polymerase nsP4. When I started my PhD, spherules had only been imaged using resin-embedding electron microscopy, which does not preserve macromolecular structure. It was unknown how the different non-structural proteins interacted with each other. The process leading to formation and maintenance of spherules at the plasma membrane was also not known. Using cryo-electron tomography, we could image spherules and unveil their macromolecular organization. We could identify a previously unreported two megadalton protein complex sitting at the neck of spherules, serving as an interface between the lumen of spherules and the cytoplasm. We found that nsP1 binds to negatively charged lipids at the plasma membrane. We also report that the host factor CD81, known to bind cholesterol at the plasma membrane, is a key element for the virus replication.We could establish a mathematical model highlighting the way those spherules form and are maintained at the plasma membrane.  We quantified the amount of genomic RNA present in each spherule and found that a single copy was present as a double-stranded replication intermediate. We further studied the spatial organization of the viral genome in spherules and found that it occupies homogenously the lumen of these replication organelles and has a moderate preferential folding inside spherules.We aimed to characterize further the ATPase and helicase activities of nsP2 and nsP2 associated to nsP1 or nsP3 as polyproteins. These polyproteins are present in the early stages of the viral RNA replication. We estimated the kinetic parameters of the ATPase function of these proteins and showed that nsp2 had a helicase activity however; the helicase functions of P12 and P23 were severely reduced. We could show that P12 and P23 exhibited instead an ATP-independent chaperoning activity, able to partially unwind double-stranded RNA.

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