Published under the PNAS license.Data deposition: The crystallography, atomic coordinates and structure factors have been deposited in the Protein Data Bank, www.wwpdb.org (PDB ID code 6JIM).
To better understand how positive-strand (+) RNA viruses assemble membrane-associated replication complexes (RCs) to synthesize, process, and transport viral RNA in virus-infected cells, we determined both the high-resolution structure of the core RNA replicase of chikungunya virus and the native RC architecture in its cellular context at subnanometer resolution, using in vitro reconstitution and in situ electron cryotomography, respectively. Within the core RNA replicase, the viral polymerase nsP4, which is in complex with nsP2 helicase-protease, sits in the central pore of the membrane-anchored nsP1 RNA-capping ring. 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.
Alphaviruses such as Ross River virus (RRV), chikungunya virus and Venezuelan equine encephalitis virus are mosquito-borne pathogens that can cause arthritis or encephalitis diseases. Nonstructural protein 4 (nsP4) of alphaviruses possesses RNA-dependent RNA polymerase (RdRp) activity essential for viral RNA replication. No 3D structure has been available for nsP4 of any alphaviruses despite its importance for understanding alphaviral RNA replication and for the design of antiviral drugs. Here, we report a crystal structure of the RdRp domain of nsP4 from RRV determined at a resolution of 2.6 Å. The structure of the alphavirus RdRp domain appears most closely related to RdRps from pestiviruses, noroviruses and picornaviruses. Using hydrogen-deuterium exchange mass spectrometry (HDX-MS) and nuclear magnetic resonance (NMR) methods, we showed that in-solution nsP4 is highly dynamic with an intrinsically disordered N-terminal domain. Both full-length nsP4 and the RdRp domain were able to catalyze in vitro RNA polymerization. Structure-guided mutagenesis using a trans-replicase system identified nsP4 regions critical for viral RNA replication.
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