Molecular Architecture of the Chikungunya Virus Replication Complex

Abstract

All positive-strand (+) RNA viruses assemble membrane-associated replication complexes (RCs) for viral RNA synthesis in virus-infected cells. However, how these multi-component RCs assemble and function in synthesizing, processing, and transporting viral RNAs to the cytosol remains poorly defined. Here, we determined both the structure of the core RNA replicase of chikungunya virus (family Togaviridae) at a near-atomic level and the native RC architecture in its cellular context at the subnanometer resolution, using in vitro reconstitution and in situ electron cryotomography, respectively. Within the core RNA replicase (nsP1+2+4), the viral RNA-dependent RNA polymerase nsP4, in complex with nsP2 helicase-protease, was found to co-fold with the membrane-anchored nsP1 RNA-capping dodecameric ring and is located asymmetrically within nsP1 central pore. This complex forms the minimal core RNA replicase, while the addition of a large cytoplasmic ring next to the C-terminus of nsP1 forms the holo-RNA-RC as observed at the neck of spherules formed in virus-infected cells. These results represent a major conceptual advance in elucidating the molecular mechanisms of RNA virus replication and the principles underlying the molecular architecture of RCs, likely to be shared with many pathogenic (+) RNA viruses. At last, our study will direct the needed development of antiviral therapies targeting RCs of pathogenic viruses.

Positive strand RNA viruses differ in the constraints they place on the folding of their negative strand

“…The simplest model explaining this observation is that the[-] RNA varies in the extent to which it is double stranded in different viral RCs and it is this that correlates with both cleavage and the suppression of replication. Certainly, recent electron cryotomography studies on CHIKV replication spherules would support the notion that this viruses [-] RNA is sequestered away in a double stranded state, consistent with the lack of cleavage we observed in our studies(Laurent et al 2022;Tan et al 2022). Interestingly, these same studies suggest that the viral helicase nsp3 is either not associated with the replicase proteins found at the neck of the spherule, or if it is then this association in on the cytoplasmic rather than the lumen face.…”

Molecular basis of specific viral RNA recognition and 5′-end capping by the Chikungunya virus nsP1

“…Given the strict sequence conservation, all alphaviral nsP1 are likely to share the same structure and function (Figure S3). As many members of alphavirus are known human pathogens causing (re)emerging epidemics across the world (Queyriaux et al, 2008;Schwartz and Albert, 2010;Strauss and Strauss, 1994), our work, together with the recently published alphavirus replication complex (Tan et al, 2022) structure, will guide the development of specific antiviral drugs targeting nsP1, the viral-RNA-capping process, and the alphavirus replication complex (Delang et al, 2016;Feibelman et al, 2018;Ferron et al, 2012).…”

“…Recently, nsP4 has been implicated in viral RNA template recognition and replication efficiency (Lello et al, 2021). The recently elucidated alphavirus replication complex shows that replication is tightly protected in the spherule, allowing for deliberate coupling of ssRNA capping by nsP1 and nsP2 before exporting it into the cytosol (Tan et al, 2022). The dynamics of alphaviral RNA replication and transcription require the interplay between viral RNA and all four essential members of the alphavirus replication enzymes (Ahola and Merits, 2016;Pietila et al, 2017;Rupp et al, 2015).…”

The Tetraspanin CD81 Is a Host Factor for Chikungunya Virus Replication

“…Our data suggest that CD81 promotes membrane curvature, thereby facilitating CHIKV replication at the plasma membrane. Illustration created with BioRender.com and modified from reference 80 and 105 .…”