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Structure of orthoreovirus RNA chaperone σNS, a component of viral replication factories.

ABSTRACT: The mammalian orthoreovirus (reovirus) σNS protein is required for formation of replication compartments that support viral genome replication and capsid assembly. Despite its functional importance, a mechanistic understanding of σNS is lacking. We conducted structural and biochemical analyses of a σNS mutant that forms dimers instead of the higher-order oligomers formed by wildtype (WT) σNS. The crystal structure shows that dimers interact with each other using N-terminal arms to form a helical assembly resembling WT σNS filaments in complex with RNA observed using cryo-EM. The interior of the helical assembly is of appropriate diameter to bind RNA. The helical assembly is disrupted by bile acids, which bind to the same site as the N-terminal arm. This finding suggests that the N-terminal arm functions in conferring context-dependent oligomeric states of σNS, which is supported by the structure of σNS lacking an N-terminal arm. We further observed that σNS has RNA chaperone activity likely essential for presenting mRNA to the viral polymerase for genome replication. This activity is reduced by bile acids and abolished by N-terminal arm deletion, suggesting that the activity requires formation of σNS oligomers. Our studies provide structural and mechanistic insights into the function of σNS in reovirus replication.

SUBMITTER: Zhao B

PROVIDER: S-EPMC10950856 | biostudies-literature | 2024 Mar

REPOSITORIES: biostudies-literature

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Structure of orthoreovirus RNA chaperone σNS, a component of viral replication factories.

Zhao Boyang B Hu Liya L Kaundal Soni S Neetu Neetu N Lee Christopher H CH Somoulay Xayathed X Sankaran Banumathi B Taylor Gwen M GM Dermody Terence S TS Venkataram Prasad B V BV

Nature communications 20240319 1

The mammalian orthoreovirus (reovirus) σNS protein is required for formation of replication compartments that support viral genome replication and capsid assembly. Despite its functional importance, a mechanistic understanding of σNS is lacking. We conducted structural and biochemical analyses of a σNS mutant that forms dimers instead of the higher-order oligomers formed by wildtype (WT) σNS. The crystal structure shows that dimers interact with each other using N-terminal arms to form a helical ...[more]

PMID: 38503747

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Project description:Within infected host cells, mammalian orthoreovirus (MRV) forms viral factories (VFs), which are sites of viral transcription, translation, assembly, and replication. The MRV nonstructural protein μNS comprises the structural matrix of VFs and is involved in recruiting other viral proteins to VF structures. Previous attempts have been made to visualize VF dynamics in live cells, but due to current limitations in recovery of replicating reoviruses carrying large fluorescent protein tags, researchers have been unable to directly assess VF dynamics from virus-produced μNS. We set out to develop a method to overcome this obstacle by utilizing the 6-amino-acid (CCPGCC) tetracysteine (TC) tag and FlAsH-EDT2 reagent. The TC tag was introduced into eight sites throughout μNS, and the capacity of the TC-μNS fusion proteins to form virus factory-like (VFL) structures and colocalize with virus proteins was characterized. Insertion of the TC tag interfered with recombinant virus rescue in six of the eight mutants, likely as a result of loss of VF formation or important virus protein interactions. However, two recombinant (r)TC-μNS viruses were rescued and VF formation, colocalization with associating virus proteins, and characterization of virus replication were subsequently examined. Furthermore, the rTC-μNS viruses were utilized to infect cells and examine VF dynamics using live-cell microscopy. These experiments demonstrate active VF movement with fusion events as well as transient interactions between individual VFs and demonstrate the importance of microtubule stability for VF fusion during MRV infection. This work provides important groundwork for future in-depth studies of VF dynamics and host cell interactions.IMPORTANCE MRV has historically been used as a model to study the double-stranded RNA (dsRNA) Reoviridae family, the members of which infect and cause disease in humans, animals, and plants. During infection, MRV forms VFs that play a critical role in virus infection but remain to be fully characterized. To study VFs, researchers have focused on visualizing the nonstructural protein μNS, which forms the VF matrix. This work provides the first evidence of recovery of replicating reoviruses in which VFs can be labeled in live cells via introduction of a TC tag into the μNS open reading frame. Characterization of each recombinant reovirus sheds light on μNS interactions with viral proteins. Moreover, utilizing the TC-labeling FlAsH-EDT2 biarsenical reagent to visualize VFs, evidence is provided of dynamic VF movement and interactions at least partially dependent on intact microtubules.

Project description:UnlabelledViral RNA-dependent RNA polymerases are considered to be low-fidelity enzymes, providing high mutation rates that allow for the rapid adaptation of RNA viruses to different host cell environments. Fidelity is tuned to provide the proper balance of virus replication rates, pathogenesis, and tissue tropism needed for virus growth. Using our structures of picornaviral polymerase-RNA elongation complexes, we have previously engineered more than a dozen coxsackievirus B3 polymerase mutations that significantly altered virus replication rates and in vivo fidelity and also provided a set of secondary adaptation mutations after tissue culture passage. Here we report a biochemical analysis of these mutations based on rapid stopped-flow kinetics to determine elongation rates and nucleotide discrimination factors. The data show a spatial separation of fidelity and replication rate effects within the polymerase structure. Mutations in the palm domain have the greatest effects on in vitro nucleotide discrimination, and these effects are strongly correlated with elongation rates and in vivo mutation frequencies, with faster polymerases having lower fidelity. Mutations located at the top of the finger domain, on the other hand, primarily affect elongation rates and have relatively minor effects on fidelity. Similar modulation effects are seen in poliovirus polymerase, an inherently lower-fidelity enzyme where analogous mutations increase nucleotide discrimination. These findings further our understanding of viral RNA-dependent RNA polymerase structure-function relationships and suggest that positive-strand RNA viruses retain a unique palm domain-based active-site closure mechanism to fine-tune replication fidelity.ImportancePositive-strand RNA viruses represent a major class of human and animal pathogens with significant health and economic impacts. These viruses replicate by using a virally encoded RNA-dependent RNA polymerase enzyme that has low fidelity, generating many mutations that allow the rapid adaptation of these viruses to different tissue types and host cells. In this work, we use a structure-based approach to engineer mutations in viral polymerases and study their effects on in vitro nucleotide discrimination as well as virus growth and genome replication fidelity. These results show that mutation rates can be drastically increased or decreased as a result of single mutations at several key residues in the polymerase palm domain, and this can significantly attenuate virus growth in vivo. These findings provide a pathway for developing live attenuated virus vaccines based on engineering the polymerase to reduce virus fitness.

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Structure of orthoreovirus RNA chaperone σNS, a component of viral replication factories.

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Structure of orthoreovirus RNA chaperone σNS, a component of viral replication factories.

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