Structure of RNA polymerase complex and genome within a dsRNA virus provides insights into the mechanisms of transcription and assembly

Wang, Xurong; Zhang, Fuxian; Su, Rui; Li, Xiaowu; Chen, Wenyuan; Chen, Qingxiu; Wang, Jiawei; Liu, Hongrong; Qin, Fang; Cheng, Lingpeng

doi:10.1073/pnas.1803885115

Cited by 46 publications

(73 citation statements)

References 54 publications

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“…Asymmetric reconstruction gave no evidence of a unique or symmetrical arrangement of VP1 with respect to the icosahedral symmetry, and we show below that the distribution of VP1 molecules has none of the symmetries that would be a subgroup of the icosahedral point group (e.g., the pseudo-D3 symmetric distribution found in cytoplasmic polyhedrosis virus and aquareovirus [8][9][10]). We conclude that VP1 binds stochastically at one of the five possible positions at each of the fivefold vertices.…”

Section: Cryo-em Reconstructions Of Vp1 Rdrp Within Rotavirus Particlesmentioning

confidence: 57%

“…In ARV particles, N-terminal arms of the five ARV CSP-A subunits around a vertex anchor the polymerase and NTPase, while the arms of the five CSP-B subunits extend to interact with two other CSP-B arms around icosahedral threefold axes [8,9]. For the two oppositely directed threefolds that are the D3 symmetry axes, the CSP-B arm from one CSP decamer also contacts the RdRp at one of the threefold related vertices, thus determining the relative orientations of the corresponding assembly units.…”

Section: Rdrp Distributionmentioning

confidence: 99%

“…1b) [7]. Since then, asymmetric reconstructions of cytoplasmic polyhedrosis virus (CPV) and of an aquareovirus (ARV) have yielded full, in situ structures of their RdRp enzymes and of a closely associated NTPase [8,9].…”

Section: Introductionmentioning

confidence: 99%

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In situstructure of rotavirus VP1 RNA-dependent RNA polymerase

Jenni

Salgado

Herrmann

et al. 2019

Preprint

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Rotaviruses, like other non-enveloped, double-strand RNA (dsRNA) viruses, package an RNAdependent RNA polymerase (RdRp) with each duplex of their segmented genomes. Rotavirus cell entry results in loss of an outer protein layer and delivery into the cytosol of an intact, inner capsid particle (the "double-layer particle" or DLP). The RdRp, designated VP1, is active inside the DLP; each VP1 achieves many rounds of mRNA transcription from its associated genome segment. Previous work has shown that one VP1 molecule lies close to each fivefold axis of the icosahedrally symmetric DLP, just beneath the inner surface of its protein shell, embedded in tightly packed RNA. We have determined a high-resolution structure for the rotavirus VP1 RdRp in situ, by local reconstruction of density around individual fivefold positions. We have analyzed intact virions ("triple-layer particles" or TLPs), non-transcribing DLPs and transcribing DLPs.Outer layer dissociation enables the DLP to synthesize RNA, in vitro as well as in vivo, but appears not to induce any detectable structural change in the RdRp. Addition of NTPs, Mg 2+ , and S-adenosyl methionine, which allows active transcription, results in conformational rearrangements, in both VP1 and the DLP capsid shell protein, that allow a transcript to exit the polymerase and the particle. The position of VP1 (among the five symmetrically related alternatives) at one vertex does not correlate with its position at other vertices. This stochastic distribution of site occupancies limits long-range order in the 11-segment, dsRNA genome.

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Section: Cryo-em Reconstructions Of Vp1 Rdrp Within Rotavirus Particlesmentioning

confidence: 57%

Section: Rdrp Distributionmentioning

confidence: 99%

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In situstructure of rotavirus VP1 RNA-dependent RNA polymerase

Jenni

Salgado

Herrmann

et al. 2019

Preprint

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“…Structural information is available for two types of purified orthoreovirus particles: the intact virus (we term similar particles that we see inside infected cells virionlike particles) and partially disassembled transcriptionally competent cores 10,15 . Cryo-EM has also allowed lower symmetry structures within the particle to be deconvoluted from the icosahedral structure of the protein shell, yielding insight into the spatial organisation of the polymerase and genome segments 12,16,17 . To link such atomic descriptions of stable purified particles to the sequential processes of virus assembly occurring inside infected cells, we grew MA104 cells on gold EM grids (Methods) and infected them with a mammalian orthoreovirus.…”

mentioning

confidence: 99%

Assembly intermediates of orthoreovirus captured in the cell

Sutton

Sun

et al. 2020

Preprint

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Traditionally molecular assembly pathways for viruses have been inferred from high resolution structures of stable intermediates purified in vitro, and from low resolution images of cell sections as well as from genetic approaches including conditionally lethal mutants. Here, we directly visualise a previously unsuspected 'single shelled' icosahedral intermediate for a mammalian orthoreovirus, in addition to the expected virions, in cryo-preserved infected cells by cryo-electron tomography of cellular lamellae 1,2 . Particle classification and averaging yielded structures at resolutions as high as 5.6 Å, sufficient to identify secondary structural elements and place known molecular structures, allowing us to produce an atomic model of the intermediate, comprising 120 copies of protein l1 and 120 copies of s2. This l1 shell is in a 'collapsed' form compared to the mature virions, with the molecules pushed inwards at the icosahedral 5-folds by ~100 Å. This grossly indented shell, although produced by a mammalian reovirus, is reminiscent of the first assembly intermediate of prokaryotic dsRNA viruses belonging to a different virus family 3 , adding weight to the supposition that these diverse viruses share a common ancestor, and suggesting mechanisms for the assembly of viruses of the Reoviridae. Such methodology holds enormous promise for the dissection of the replication cycle of many viruses.

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“…However, what the structure of BTV RdRp VP1 is and how it interacts with the genome and other proteins are both unknown, significantly limiting our understanding of the RNA transcription mechanism inside BTV. The structures of RdRp and associated NTPase proteins have recently been determined by cryoEM for 2 turreted reoviruses: cytoplasmic polyhedrosis virus (CPV) (22,23) and aquareovirus (ARV) (24,25). However, as a nonturreted member…”

mentioning

confidence: 99%

In situ structures of RNA-dependent RNA polymerase inside bluetongue virus before and after uncoating

Shivakoti

Ding

et al. 2019

Proc. Natl. Acad. Sci. U.S.A.

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Bluetongue virus (BTV), a major threat to livestock, is a multilayered, nonturreted member of the Reoviridae, a family of segmented dsRNA viruses characterized by endogenous RNA transcription through an RNA-dependent RNA polymerase (RdRp). To date, the structure of BTV RdRp has been unknown, limiting our mechanistic understanding of BTV transcription and hindering rational drug design effort targeting this essential enzyme. Here, we report the in situ structures of BTV RdRp VP1 in both the triple-layered virion and double-layered core, as determined by cryo-electron microscopy (cryoEM) and subparticle reconstruction. BTV RdRp has 2 unique motifs not found in other viral RdRps: a fingernail, attached to the conserved fingers subdomain, and a bundle of 3 helices: 1 from the palm subdomain and 2 from the N-terminal domain. BTV RdRp VP1 is anchored to the inner surface of the capsid shell via 5 asymmetrically arranged N termini of the inner capsid shell protein VP3A around the 5-fold axis. The structural changes of RdRp VP1 and associated capsid shell proteins between BTV virions and cores suggest that the detachment of the outer capsid proteins VP2 and VP5 during viral entry induces both global movements of the inner capsid shell and local conformational changes of the N-terminal latch helix (residues 34 to 51) of 1 inner capsid shell protein VP3A, priming RdRp VP1 within the capsid for transcription. Understanding this mechanism in BTV also provides general insights into RdRp activation and regulation during viral entry of other multilayered, nonturreted dsRNA viruses.

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Structure of RNA polymerase complex and genome within a dsRNA virus provides insights into the mechanisms of transcription and assembly

Cited by 46 publications

References 54 publications

In situstructure of rotavirus VP1 RNA-dependent RNA polymerase

In situstructure of rotavirus VP1 RNA-dependent RNA polymerase

Assembly intermediates of orthoreovirus captured in the cell

In situ structures of RNA-dependent RNA polymerase inside bluetongue virus before and after uncoating

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