Mammalian orthoreovirus core protein μ2 reorganizes host microtubule-organizing center components

Eichwald, Catherine; Ackermann, Mathias; Fraefel, Cornel

doi:10.1016/j.virol.2020.07.008

Cited by 6 publications

(5 citation statements)

References 58 publications

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“…The core-only factories transitioned towards the nucleus when cycloheximide was employed to stop further virus amplification. Previous studies found that the µ2 protein associates with microtubules [48][49][50] and the current dogma is that these interactions mediate movement of viral factories. However past studies focused on late timepoints during infection when perinuclear factories predominant, and it would be interesting to revisit microtubule-µ2 associations with respect to whether they also support the movement of core-only factories.…”

Section: Discussionmentioning

confidence: 99%

Reovirus uses temporospatial compartmentalization to orchestrate core versus outercapsid assembly

Kniert

Santos

Eaton

et al. 2022

Preprint

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Reoviridae virus family members, such as mammalian orthoreovirus (reovirus), encounter a unique challenge during replication. To hide the dsRNA from host recognition, the genome remains encapsidated in transcriptionally active proteinaceous core capsids that transcribe and release +RNA. De novo +RNAs and core proteins must repeatedly assemble into new progeny cores in order to logarithmically amplify replication. Reoviruses also produce outercapsid (OC) proteins µ1, σ3 and σ1 that assemble onto cores to create highly stable infectious full virions. Current models of reovirus replication position amplification of transcriptionally-active cores and assembly of infectious virions in shared factories, but we hypothesized that since assembly of OC proteins would halt core amplification, OC assembly is somehow regulated. Using kinetic analysis of virus +RNA, core and OC proteins, core assembly and whole virus assembly, assembly of OC proteins was found to be temporally delayed. All viral RNAs and proteins were made simultaneously, eliminating the possibility that delayed OC RNAs or proteins account for delayed OC assembly. High resolution fluorescence and electron microscopy revealed that core amplification occurred early during infection at peripheral core-only factories, while all OC proteins associated with lipid droplets (LDs) that coalesced near the nucleus in a µ1–dependent manner. Core-only factories transitioned towards the nucleus despite cycloheximide-mediated halting of new protein expression, while new core-only factories developed in the periphery. As infection progressed, OC assembly occurred at LD-and nuclear-proximal factories. Silencing of OC µ1 expression with siRNAs led to large factories that remained further from the nucleus, implicating µ1 in the transition to perinuclear factories. Moreover, late during infection, +RNA pools largely contributed to the production of de-novo viral proteins and fully-assembled infectious viruses. Altogether the results suggest an advanced model of reovirus replication with spatiotemporal segregation of core amplification, OC complexes and fully assembled virions.

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Section: Discussionmentioning

confidence: 99%

Reovirus uses temporospatial compartmentalization to orchestrate core versus outercapsid assembly

Kniert

Santos

Eaton

et al. 2022

Preprint

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“…The fluorescence signal values in the VLSs were determined similarly, as described previously by Eichwald et al ( 95 ). The intensity profile of a linear region of interest was obtained using the ImageJ plot profile tool.…”

Section: Methodsmentioning

confidence: 99%

The recruitment of TRiC chaperonin in rotavirus viroplasms correlates with virus replication

Vetter,

Papa,

Tobler

et al. 2024

mBio

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Rotavirus (RV) replication takes place in the viroplasms, cytosolic inclusions that allow the synthesis of virus genome segments and their encapsidation in the core shell, followed by the addition of the second layer of the virion. The viroplasms are composed of several viral proteins, including NSP5, which serves as the main building block. Microtubules, lipid droplets, and miRNA-7 are among the host components recruited in viroplasms. We investigated the interaction between RV proteins and host components of the viroplasms by performing a pull-down assay of lysates from RV-infected cells expressing NSP5-BiolD2. Subsequent tandem mass spectrometry identified all eight subunits of the tailless complex polypeptide I ring complex (TRiC), a cellular chaperonin responsible for folding at least 10% of the cytosolic proteins. Our confirmed findings reveal that TRiC is brought into viroplasms and wraps around newly formed double-layered particles. Chemical inhibition of TRiC and silencing of its subunits drastically reduced virus progeny production. Through direct RNA sequencing, we show that TRiC is critical for RV replication by controlling dsRNA genome segment synthesis, particularly negative-sense single-stranded RNA. Importantly, cryo-electron microscopy analysis shows that TRiC inhibition results in defective virus particles lacking genome segments and polymerase complex (VP1/VP3). Moreover, TRiC associates with VP2 and NSP5 but not with VP1. Also, VP2 is shown to be essential for recruiting TRiC in viroplasms and preserving their globular morphology. This study highlights the essential role of TRiC in viroplasm formation and in facilitating virion assembly during the RV life cycle. IMPORTANCE The replication of rotavirus takes place in cytosolic inclusions termed viroplasms. In these inclusions, the distinct 11 double-stranded RNA genome segments are co-packaged to complete a genome in newly generated virus particles. In this study, we show for the first time that the tailless complex polypeptide I ring complex (TRiC), a cellular chaperonin responsible for the folding of at least 10% of the cytosolic proteins, is a component of viroplasms and is required for the synthesis of the viral negative-sense single-stranded RNA. Specifically, TRiC associates with NSP5 and VP2, the cofactor involved in RNA replication. Our study adds a new component to the current model of rotavirus replication, where TRiC is recruited to viroplasms to assist replication.

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“…The core-only factories transitioned towards the nucleus when cycloheximide was employed to stop further virus amplification. Previous studies found that the μ2 protein associates with microtubules [50][51][52] and the current dogma is that these interactions mediate movement of viral factories. However past studies focused on late timepoints during infection when perinuclear factories predominant, and it would be interesting to revisit microtubule-μ2 associations with respect to whether they also support the movement of core-only factories.…”

Section: Plos Pathogensmentioning

confidence: 99%

Reovirus uses temporospatial compartmentalization to orchestrate core versus outercapsid assembly

et al. 2022

View full text Add to dashboard Cite

Reoviridae virus family members, such as mammalian orthoreovirus (reovirus), encounter a unique challenge during replication. To hide the dsRNA from host recognition, the genome remains encapsidated in transcriptionally active proteinaceous core capsids that transcribe and release +RNA. De novo +RNAs and core proteins must repeatedly assemble into new progeny cores in order to logarithmically amplify replication. Reoviruses also produce outercapsid (OC) proteins μ1, σ3 and σ1 that assemble onto cores to create highly stable infectious full virions. Current models of reovirus replication position amplification of transcriptionally-active cores and assembly of infectious virions in shared factories, but we hypothesized that since assembly of OC proteins would halt core amplification, OC assembly is somehow regulated. Using kinetic analysis of virus +RNA, core and OC proteins, core assembly and whole virus assembly, assembly of OC proteins was found to be temporally delayed. All viral RNAs and proteins were made simultaneously, eliminating the possibility that delayed OC RNAs or proteins account for delayed OC assembly. High resolution fluorescence and electron microscopy revealed that core amplification occurred early during infection at peripheral core-only factories, while all OC proteins associated with lipid droplets (LDs) that coalesced near the nucleus in a μ1–dependent manner. Core-only factories transitioned towards the nucleus despite cycloheximide-mediated halting of new protein expression, while new core-only factories developed in the periphery. As infection progressed, OC assembly occurred at LD-and nuclear-proximal factories. Silencing of OC μ1 expression with siRNAs led to large factories that remained further from the nucleus, implicating μ1 in the transition to perinuclear factories. Moreover, late during infection, +RNA pools largely contributed to the production of de-novo viral proteins and fully-assembled infectious viruses. Altogether the results suggest an advanced model of reovirus replication with spatiotemporal segregation of core amplification, OC complexes and fully assembled virions.

show abstract

Mammalian orthoreovirus core protein μ2 reorganizes host microtubule-organizing center components

Cited by 6 publications

References 58 publications

Reovirus uses temporospatial compartmentalization to orchestrate core versus outercapsid assembly

Reovirus uses temporospatial compartmentalization to orchestrate core versus outercapsid assembly

The recruitment of TRiC chaperonin in rotavirus viroplasms correlates with virus replication

Reovirus uses temporospatial compartmentalization to orchestrate core versus outercapsid assembly

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