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

Zhang, Kuo; Law, Michelle Cheok Yien; Nguyen, Tam D.; Tan, Yen Nee; Wirawan, Melissa; Law, Yee-Song; Jeong, Lak Shin; Luo, Dahai

doi:10.1016/j.celrep.2022.111133

Cited by 14 publications

(25 citation statements)

References 47 publications

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“…S5, A to C). The PM-associated crown ring closely matches the reported cryo-EM structures of the recombinant nsP1 dodecamer (10,11,20). While the crown ring contains a plug-like density in the 12-fold symmetric average, the cytoplasmic ring features a cylinder-like density with a notably wide and mostly empty cavity (15.6-nm diameter and ~10-nm height) (Fig.…”

Section: Molecular Organization Of Chikv Rc In Situsupporting

confidence: 82%

“…The ultrastructure of the distantly related Flock House nodavirus (FHV) RC provided the first structural insight into RC assembly in the form of a dodecameric crown-shaped scaffold anchored at the neck of spherule packed with viral RNA condensate (9,18). Similarly, multimeric ring-shaped ultrastructures were recently reported in both coronavirus RC (19) and alphavirus nsP1 (10,11,20). However, these ultrastructures have yet to explain the organization of multicomponent RCs and the molecular mechanism by which RCs ultimately achieve viral RNA synthesis and transport to the cytosol.…”

Section: Introductionmentioning

confidence: 98%

“…The RC likely creates a favorable compartment for viral RNA synthesis that minimizes the host immune response to dsRNA intermediates and possesses multifunctional enzymes capable of orchestrating rapid and efficient synthesis of genomic and subgenomic viral RNAs (4)(5)(6)(7)(8)(9). nsP1 to nsP4 are structurally known to localize to the RCs and are essential to viral RNA replication through their own distinct enzymatic and nonenzymatic functions: nsP1 [Protein Data Bank (PDB) 7DOP] displays methyl-and guanylyltransferase activities required for viral RNA 5′ cap synthesis and plasma membrane (PM) anchoring ability (10,11); nsP2 consists of an N-terminal superfamily 1 RNA helicase (PDB 6JIM) and a cysteine protease at the C-terminal region (PDB 3TRK) responsible for polyprotein autoprocessing (12,13); nsP3 (PDB 3GPG) contains an adenosine diphosphateribosyl binding and hydrolase domain and a disordered region as the host factor interaction hub (14,15); last, nsP4 (PDB 7VB4/7F0S) is the RNA-dependent RNA polymerase (RdRp) for viral RNA synthesis (16,17). Despite having all the high-resolution individual structures of the nsP1 to nsP4, the overall architecture and assembly mechanisms of the alphavirus RC remains unresolved.…”

Section: Introductionmentioning

confidence: 99%

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Molecular architecture of the Chikungunya virus replication complex

et al. 2022

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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.

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Section: Molecular Organization Of Chikv Rc In Situsupporting

confidence: 82%

Section: Introductionmentioning

confidence: 98%

Section: Introductionmentioning

confidence: 99%

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Molecular architecture of the Chikungunya virus replication complex

et al. 2022

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“…Thus, the capping reaction is specific for Alphavirus RNAs short enough to prevent the formation of secondary structures. This results are consistent with the recent identification of the U as the nucleotide recognized by the nsP1 through specific interactions with residues at the Nterminal extension of the capping domain (30).…”

Section: The Nsp1 Rna Capping Reaction Is Structure and Sequence Spec...supporting

confidence: 93%

“…While this manuscript was in preparation Zhang et al (30) have reported an independent study presenting structures of some of states of the capping reaction provided here with nsP1 capping pores produced in mammalian cells. The expression and purification of capping pores in mammalian cells delivers complexes with an expanded pore conformation.…”

Section: Discussionmentioning

confidence: 99%

Structural basis and dynamics of Chikungunya alphavirus RNA capping by the nsP1 capping pores

Jones

Hons

Rabah

et al. 2022

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Alphaviruses are emerging positive stranded RNA virus which replicate and transcribe their genomes in membranous organelles formed in the cell cytoplasm. The non-structural protein 1 (nsP1) is responsible for RNA capping and the gating of replication organelles by assembling into monotopic membrane-associated dodecameric pores (Jones R. et al. Nature 2021). The capping path is unique for Alphavirus; beginning with the N7 methylation of a GTP molecule, followed by the covalent linkage of a m7GMP group to a conserved histidine in nsP1 and the transfer of this cap structure to a diphosphate RNA (Ahola T. et al. PNAS 1995). Here we provide structural snapshots of different stages of the reaction pathway showing how nsP1 pores recognize the substrates of the methyl-transfer reaction, GTP and SAM, how it reaches a metastable post-methylation state with SAH and m7GTP in the active site, the subsequent covalent transfer of m7GMP to nsP1 and post-reaction conformational changes triggering the opening of the pore. In addition, we biochemically characterize the capping reaction, demonstrating specificity for the RNA substrate and the reversibility of the cap transfer resulting in decapping activity and the release of intermediates of the reaction. Our data identify the molecular determinants allowing each pathway transition, provide explanation for the need for the SAM methyl donor all along the pathway and new clues about the conformational rearrangements associated to the enzymatic activity of nsP1. Together our results set new ground for the structural and functional understanding of alphavirus RNA-capping and the design of antivirals.

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