Regulation of rotavirus polymerase activity by inner capsid proteins

Gridley, C.L.; Patton, John T.

doi:10.1016/j.coviro.2014.08.008

Cited by 14 publications

(11 citation statements)

References 34 publications

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“…On the other hand, for some dsRNA viruses that conduct semi-conservative transcription, in which the nascent strand is part of the dsRNA genome (e.g., bacteriophage φ6 21 and picobirnavirus 22 ), RdRp is completely functional for replication in vitro. However, exactly how CSP regulates 23 RdRp’s activities in rotaviruses remains unknown. Also, unlike other RdRps that conduct semi-conservative transcription, reovirus’s RdRp can conduct both replication and transcription and switch between the two states directly after polymerization.…”

Section: Introductionmentioning

confidence: 99%

In situ structures of rotavirus polymerase in action and mechanism of mRNA transcription and release

et al. 2019

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Transcribing and replicating a double-stranded genome require protein modules to unwind, transcribe/replicate nucleic acid substrates, and release products. Here we present in situ cryo-electron microscopy structures of rotavirus dsRNA-dependent RNA polymerase (RdRp) in two states pertaining to transcription. In addition to the previously discovered universal “hand-shaped” polymerase core domain shared by DNA polymerases and telomerases, our results show the function of N- and C-terminal domains of RdRp: the former opens the genome duplex to isolate the template strand; the latter splits the emerging template-transcript hybrid, guides genome reannealing to form a transcription bubble, and opens a capsid shell protein (CSP) to release the transcript. These two “helicase” domains also extensively interact with CSP, which has a switchable N-terminal helix that, like cellular transcriptional factors, either inhibits or promotes RdRp activity. The in situ structures of RdRp, CSP, and RNA in action inform mechanisms of not only transcription, but also replication.

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Section: Introductionmentioning

confidence: 99%

In situ structures of rotavirus polymerase in action and mechanism of mRNA transcription and release

et al. 2019

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“…In addition to enclosing the viral genome and anchoring TECs, the capsid shell protein (CSP) also regulates polymerase activity in dsRNA viruses 22-25 . In particular, the CSP N-terminal fragment plays roles in genome replication, mRNA transcription and capping 23,26,27 .…”

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confidence: 99%

In situ structures of the segmented genome and RNA polymerase complex inside a dsRNA virus

Zhang

Ding

et al. 2015

Nature

111

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Summary Viruses in the Reoviridae, like the triple-shelled human rotavirus and the single-shelled insect cytoplasmic polyhedrosis virus (CPV), all package a genome of segmented dsRNAs inside the viral capsid and carry out endogenous mRNA synthesis through a transcriptional enzyme complex (TEC). By direct electron-counting cryoEM and asymmetric reconstruction, we have determined the organization of the dsRNA genome inside quiescent CPV (q-CPV) and the in situ atomic structures of TEC within CPV in both quiescent and transcribing (t-CPV) states. We show that the total 10 segmented dsRNAs in CPV are organized with 10 TECs in a specific, non-symmetric manner, with each dsRNA segment attached directly to a TEC. TEC consists of two extensively-interacting subunits: an RNA-dependent RNA polymerase (RdRP) and an NTPase VP4. We find that the bracelet domain of RdRP undergoes significant conformational change when converted from q-CPV to t-CPV, leading to formation of the RNA template entry channel and access to the polymerase active site. An N-terminal helix from each of two subunits of the capsid shell protein (CSP) interacts with VP4 and RdRP. These findings establish the link between sensing of environmental cues by the external proteins and activation of endogenous RNA transcription by the TEC inside the virus.

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“…This is achieved through a variety of mechanisms. For example, viruses with dsRNA genomes assemble particles around positive-sense RNA [(ϩ) RNA] and generate dsRNA only within enclosed capsids (1)(2)(3). In contrast, (ϩ) RNA viruses replicate their genomes in association with highly modified host membranes (reviewed in references 4 and 5), generating convoluted single-and doublemembrane vesicles (e.g., coronaviruses and hepaciviruses) or relatively simple 50-to-70-nm-diameter membrane invaginations called spherules (first discovered in alphaviruses [6,7]).…”

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confidence: 99%

Role of Mitochondrial Membrane Spherules in Flock House Virus Replication

Short

Speir

Gopal

et al. 2016

J Virol

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Viruses that generate double-stranded RNA (dsRNA) during replication must overcome host defense systems designed to detect this infection intermediate. All positive-sense RNA viruses studied to date modify host membranes to help facilitate the sequestration of dsRNA from host defenses and concentrate replication factors to enhance RNA production. Flock House virus (FHV) is an attractive model for the study of these processes since it is well characterized and infects Drosophila cells, which are known to have a highly effective RNA silencing system. During infection, FHV modifies the outer membrane of host mitochondria to form numerous membrane invaginations, called spherules, that are ϳ50 nm in diameter and known to be the site of viral RNA replication. While previous studies have outlined basic structural features of these invaginations, very little is known about the mechanism underlying their formation. Here we describe the optimization of an experimental system for the analysis of FHV host membrane modifications using crude mitochondrial preparations from infected Drosophila cells. These preparations can be programmed to synthesize both single-and double-stranded FHV RNA. The system was used to demonstrate that dsRNA is protected from nuclease digestion by virus-induced membrane invaginations and that spherules play an important role in stimulating RNA replication. Finally, we show that spherules generated during FHV infection appear to be dynamic as evidenced by their ability to form or disperse based on the presence or absence of RNA synthesis. IMPORTANCEIt is well established that positive-sense RNA viruses induce significant membrane rearrangements in infected cells. However, the molecular mechanisms underlying these rearrangements, particularly membrane invagination and spherule formation, remain essentially unknown. How the formation of spherules enhances viral RNA synthesis is also not understood, although it is assumed to be partly a result of evading host defense pathways. To help interrogate some of these issues, we optimized a cell-free replication system consisting of mitochondria isolated from Flock House virus-infected Drosophila cells for use in biochemical and structural studies. Our data suggest that spherules generated during Flock House virus replication are dynamic, protect double-stranded RNA, and enhance RNA replication in general. Cryo-electron microscopy suggests that the samples are amenable to detailed structural analyses of spherules engaged in RNA synthesis. This system thus provides a foundation for understanding the molecular mechanisms underlying spherule formation, maintenance, and function during positive-sense viral RNA replication.

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Regulation of rotavirus polymerase activity by inner capsid proteins

Cited by 14 publications

References 34 publications

In situ structures of rotavirus polymerase in action and mechanism of mRNA transcription and release

In situ structures of rotavirus polymerase in action and mechanism of mRNA transcription and release

In situ structures of the segmented genome and RNA polymerase complex inside a dsRNA virus

Role of Mitochondrial Membrane Spherules in Flock House Virus Replication

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