The organization of double-stranded RNA in the chikungunya virus replication organelle.

Alphaviruses are mosquito-borne, positive-sense single-stranded RNA viruses. Amongst the alphaviruses, chikungunya virus is notable as a large source of human illness, especially in tropical and subtropical regions. When they invade a cell, alphaviruses generate dedicated organelles for viral genome replication, so-called spherules. Spherules form as outward-facing buds at the plasma membrane, and it has recently been shown that the thin membrane neck that connects this membrane bud with the cytoplasm is guarded by a two-megadalton protein complex that contains all the enzymatic functions necessary for RNA replication. The lumen of the spherules contains a single copy of the negative-strand template RNA, present in a duplex with newly synthesized positive-sense RNA. Less is known about the organization of this double-stranded RNA as compared to the protein components of the spherule. Here, we analyzed cryo-electron tomograms of chikungunya virus spherules in terms of the organization of the double-stranded RNA replication intermediate. We find that the double-stranded RNA has a shortened apparent persistence length as compared to unconstrained double-stranded RNA. Around half of the genome is present in either of five conformations identified by subtomogram classification, each representing a relatively straight segment of ~25-32 nm. Finally, the RNA occupies the spherule lumen at a homogeneous density, but has a preferred orientation to be perpendicular to a vector pointing from the membrane neck towards the spherule center. Taken together, this analysis lays another piece of the puzzle of the highly coordinated alphavirus genome replication.