Amino acids 78 and 79 of Mammalian Orthoreovirus protein µNS are necessary for stress granule localization, core protein λ2 interaction, and de novo virus replication

Carroll, Kate; Hastings, Craig; Miller, Carl F.

doi:10.1016/j.virol.2013.10.009

Cited by 26 publications

(36 citation statements)

References 63 publications

(84 reference statements)

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“…Orthoreoviruses generate viral factories in the cytoplasm of infected cells [21,27,50,51,52], and PRV forms cytoplasmic globular viral factories resembling the structures produced by MRV T3D [16,19,31]. Viral factories are structures where virus replication and assembly occur, and thus where the viral proteins co-localize.…”

Section: Discussionmentioning

confidence: 99%

Viral Protein Kinetics of Piscine Orthoreovirus Infection in Atlantic Salmon Blood Cells

Haatveit

Wessel

Markussen

et al. 2017

Viruses

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Piscine orthoreovirus (PRV) is ubiquitous in farmed Atlantic salmon (Salmo salar) and the cause of heart and skeletal muscle inflammation. Erythrocytes are important target cells for PRV. We have investigated the kinetics of PRV infection in salmon blood cells. The findings indicate that PRV causes an acute infection of blood cells lasting 1–2 weeks, before it subsides into persistence. A high production of viral proteins occurred initially in the acute phase which significantly correlated with antiviral gene transcription. Globular viral factories organized by the non-structural protein µNS were also observed initially, but were not evident at later stages. Interactions between µNS and the PRV structural proteins λ1, µ1, σ1 and σ3 were demonstrated. Different size variants of µNS and the outer capsid protein µ1 appeared at specific time points during infection. Maximal viral protein load was observed five weeks post cohabitant challenge and was undetectable from seven weeks post challenge. In contrast, viral RNA at a high level could be detected throughout the eight-week trial. A proteolytic cleavage fragment of the µ1 protein was the only viral protein detectable after seven weeks post challenge, indicating that this µ1 fragment may be involved in the mechanisms of persistent infection.

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Section: Discussionmentioning

confidence: 99%

Viral Protein Kinetics of Piscine Orthoreovirus Infection in Atlantic Salmon Blood Cells

Haatveit

Wessel

Markussen

et al. 2017

Viruses

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“…The non-structural protein μNS strongly interacts with SG via amino acids 78 and 79, but does not induce or disrupt SG when expressed alone. However mutant μNS (78)(79) no longer binds core protein (λ2) and is defective in supporting virus replication (19), so the link between μNS localization to SG and virus replication remain unclear. However, PKR and the other individual eIF2α kinases are not solely required for SG induction by MRV, indicating that SG formation requires multiple eIF2α kinase signaling or another mechanism(100).…”

Section: Influenza a Virus (Iav) Blocks Sg Formation Throughout Infecmentioning

confidence: 99%

Cytoplasmic RNA Granules and Viral Infection

Tsai

Lloyd

2014

Annu. Rev. Virol.

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RNA granules are dynamic cellular structures essential for proper gene expression and homeostasis. The two principle types of cytoplasmic RNA granules are stress granules (SGs), which contain stalled translation initiation complexes, and processing bodies (P-bodies, PBs), which concentrate factors involved in mRNA degradation. RNA granules are associated with gene silencing of transcripts, thus, viruses repress RNA granule functions to favor replication. This review discusses the breadth of viral interactions with cytoplasmic RNA granules, focusing on mechanisms that modulate the functions of RNA granules and that typically promote viral replication. Currently mechanisms for virus manipulation of RNA granules can be loosely grouped into three non-exclusive categories; i) cleavage of key RNA granule factors, ii) regulation of PKR activation and iii) co-opting RNA granule factors for new roles in viral replication. Viral repression of RNA granules supports productive infection by inhibiting their gene silencing functions and counteracting their role in linking stress sensing with innate immune activation.

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“…The virus was propagated and purified as previously described (33). Primary antibodies used were as follows: monoclonal mouse anti-FLAG (␣-FLAG) antibody (F1804; Sigma-Aldrich), monoclonal mouse ␣-HA antibody (26183; ThermoFisher), polyclonal mouse ␣-NS, polyclonal rabbit ␣-NS, ␣-2, and T1L ␣-virion antibodies (9,(33)(34)(35), monoclonal mouse ␣-NS (3E10) and ␣-2 (7F4) antibodies deposited in the Developmental Studies Hybridoma Bank (DSHB) by T. S. Dermody (36,37), and rabbit ␣-MRV core (38). Secondary antibodies were Alexa 594-and 488-conjugated donkey ␣-mouse or ␣-rabbit IgG antibodies (Invitrogen Life Technologies).…”

Section: Methodsmentioning

confidence: 99%

“…CV-1 cells were infected with T1L, rTC-NS(C-term-T1L)/P5 or rTC-NS(#7-T1L)/P5 in phosphate-buffered saline (PBS) (137 mM NaCl, 3 mM KCl, 8 mM Na 2 HPO 4 , 1.5 mM KH 2 PO 4 , pH 7.4) with 2 mM MgCl 2 at an MOI of 1 for 1 h with shaking and then replenished with medium and incubated at 37°C overnight. For reverse genetics, 5.0 ϫ 10 5 BHK-T7 cells were plated on a 6-well plate (9.5 cm 2 ; Corning Inc.) and transfected as previously described (33). The cells were incubated for 6 days, at which point cells and media were subjected to three freeze/thaw cycles, and standard L929 cell plaque assays were performed (28).…”

Section: Methodsmentioning

confidence: 99%

Characterization of a Replicating Mammalian Orthoreovirus with Tetracysteine-Tagged μNS for Live-Cell Visualization of Viral Factories

Bussiere

Choudhury

Bellaire

et al. 2017

J Virol

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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 NS 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 NS. 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 NS, and the capacity of the TC-NS 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-NS viruses were rescued and VF formation, colocalization with associating virus proteins, and characterization of virus replication were subsequently examined. Furthermore, the rTC-NS 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 doublestranded 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 NS, 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 NS open reading frame. Characterization of each recombinant reovirus sheds light on NS 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.KEYWORDS Mammalian orthoreovirus, virus factories, tetracysteine tag, live cell imaging, nonstructural NS protein M ammalian orthoreovirus (MRV) is a segmented, double-stranded RNA (dsRNA) virus that has been utilized as a tool to study the life cycle of the virus family Reoviridae. The virus is also of particular interest because it is classified as an oncolytic

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Amino acids 78 and 79 of Mammalian Orthoreovirus protein µNS are necessary for stress granule localization, core protein λ2 interaction, and de novo virus replication

Cited by 26 publications

References 63 publications

Viral Protein Kinetics of Piscine Orthoreovirus Infection in Atlantic Salmon Blood Cells

Viral Protein Kinetics of Piscine Orthoreovirus Infection in Atlantic Salmon Blood Cells

Cytoplasmic RNA Granules and Viral Infection

Characterization of a Replicating Mammalian Orthoreovirus with Tetracysteine-Tagged μNS for Live-Cell Visualization of Viral Factories

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