Phase separation of both a plant virus movement protein and cellular factors support virus-host interactions

Mapping Intimacies ◽

10.1101/2021.05.11.443547 ◽

2021 ◽

Author(s):

Shelby L Brown ◽

Jared P. May

Keyword(s):

Phase Separation ◽

Mosaic Virus ◽

Self Assembly ◽

Electrostatic Interactions ◽

Movement Protein ◽

Virus Movement ◽

In Planta ◽

Ribonucleoprotein Complexes

Phase separation concentrates biomolecules, which should benefit RNA viruses that must sequester viral and host factors during an infection. Here, the p26 movement protein from Pea enation mosaic virus 2 (PEMV2) was found to phase separate and partition in nucleoli and G3BP stress granules (SGs) in vivo . Electrostatic interactions drive p26 phase separation as mutation of basic (R/K-G) or acidic (D/E-G) residues either blocked or reduced phase separation, respectively. During infection, p26 must partition inside the nucleolus and interact with fibrillarin (Fib2) as a pre-requisite for systemic trafficking of viral RNAs. Partitioning of p26 in pre-formed Fib2 droplets was dependent on p26 phase separation suggesting that phase separation of viral movement proteins supports nucleolar partitioning and virus movement. Furthermore, viral ribonucleoprotein complexes containing p26, Fib2, and PEMV2 RNA were formed via phase separation in vitro and could provide the basis for self-assembly in planta . Interestingly, both R/K-G and D/E-G p26 mutants failed to support systemic trafficking of a Tobacco mosaic virus (TMV) vector in Nicotiana benthamiana suggesting that p26 phase separation, proper nucleolar partitioning, and systemic movement are intertwined. p26 also partitioned in SGs and G3BP over-expression restricted PEMV2 accumulation >20-fold. Expression of phase separation-deficient G3BP only restricted PEMV2 5-fold, demonstrating that G3BP phase separation is critical for maximum antiviral activity.

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Phase separation of a plant virus movement protein and cellular factors support virus-host interactions

PLoS Pathogens ◽

10.1371/journal.ppat.1009622 ◽

2021 ◽

Vol 17 (9) ◽

pp. e1009622

Author(s):

Shelby L. Brown ◽

Dana J. Garrison ◽

Jared P. May

Keyword(s):

Phase Separation ◽

Mosaic Virus ◽

Movement Protein ◽

Droplet Formation ◽

Virus Movement ◽

Genomic Rnas ◽

Systemic Movement ◽

Ribonucleoprotein Complexes ◽

Rna Accumulation

Both cellular and viral proteins can undergo phase separation and form membraneless compartments that concentrate biomolecules. The p26 movement protein from single-stranded, positive-sense Pea enation mosaic virus 2 (PEMV2) separates into a dense phase in nucleoli where p26 and related orthologues must interact with fibrillarin (Fib2) as a pre-requisite for systemic virus movement. Using in vitro assays, viral ribonucleoprotein complexes containing p26, Fib2, and PEMV2 genomic RNAs formed droplets that may provide the basis for self-assembly in planta. Mutating basic p26 residues (R/K-G) blocked droplet formation and partitioning into Fib2 droplets or the nucleolus and prevented systemic movement of a Tobacco mosaic virus (TMV) vector in Nicotiana benthamiana. Mutating acidic residues (D/E-G) reduced droplet formation in vitro, increased nucleolar retention 6.5-fold, and prevented systemic movement of TMV, thus demonstrating that p26 requires electrostatic interactions for droplet formation and charged residues are critical for nucleolar trafficking and virus movement. p26 readily partitioned into stress granules (SGs), which are membraneless compartments that assemble by clustering of the RNA binding protein G3BP following stress. G3BP is upregulated during PEMV2 infection and over-expression of G3BP restricted PEMV2 RNA accumulation >20-fold. Deletion of the NTF2 domain that is required for G3BP condensation restored PEMV2 RNA accumulation >4-fold, demonstrating that phase separation enhances G3BP antiviral activity. These results indicate that p26 partitions into membraneless compartments with either proviral (Fib2) or antiviral (G3BP) factors.

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Tobacco Mosaic Virus Movement Protein Functions as a Structural Microtubule-Associated Protein

Journal of Virology ◽

10.1128/jvi.00540-06 ◽

2006 ◽

Vol 80 (17) ◽

pp. 8329-8344 ◽

Cited By ~ 67

Author(s):

Jamie Ashby ◽

Emmanuel Boutant ◽

Mark Seemanpillai ◽

Adrian Sambade ◽

Christophe Ritzenthaler ◽

...

Keyword(s):

Tobacco Mosaic Virus ◽

Mosaic Virus ◽

Movement Protein ◽

Cell Extract ◽

Transport Processes ◽

In Vitro Assays ◽

Protein Functions ◽

Intercellular Spread

ABSTRACT The cell-to-cell spread of Tobacco mosaic virus infection depends on virus-encoded movement protein (MP), which is believed to form a ribonucleoprotein complex with viral RNA (vRNA) and to participate in the intercellular spread of infectious particles through plasmodesmata. Previous studies in our laboratory have provided evidence that the vRNA movement process is correlated with the ability of the MP to interact with microtubules, although the exact role of this interaction during infection is not known. Here, we have used a variety of in vivo and in vitro assays to determine that the MP functions as a genuine microtubule-associated protein that binds microtubules directly and modulates microtubule stability. We demonstrate that, unlike MP in whole-cell extract, microtubule-associated MP is not ubiquitinated, which strongly argues against the hypothesis that microtubules target the MP for degradation. In addition, we found that MP interferes with kinesin motor activity in vitro, suggesting that microtubule-associated MP may interfere with kinesin-driven transport processes during infection.

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Gene-Activated Matrix with Self-Assembly Anionic Nano-Device Containing Plasmid DNAs for Rat Cranial Bone Augmentation

Materials ◽

10.3390/ma14227097 ◽

2021 ◽

Vol 14 (22) ◽

pp. 7097

Author(s):

Masahito Hara ◽

Yoshinori Sumita ◽

Yukinobu Kodama ◽

Mayumi Iwatake ◽

Hideyuki Yamamoto ◽

...

Keyword(s):

Self Assembly ◽

Electrostatic Interactions ◽

Cranial Bone ◽

Bone Augmentation ◽

Mesenchymal Progenitor Cells ◽

Anionic Polymer ◽

Morphogenetic Protein ◽

Low Cytotoxicity

We have developed nanoballs, a biocompatible self-assembly nano-vector based on electrostatic interactions that arrange anionic macromolecules to polymeric nanomaterials to create nucleic acid carriers. Nanoballs exhibit low cytotoxicity and high transfection efficiently in vivo. This study investigated whether a gene-activated matrix (GAM) composed of nanoballs containing plasmid (p) DNAs encoding bone morphogenetic protein 4 (pBMP4) could promote bone augmentation with a small amount of DNA compared to that composed of naked pDNAs. We prepared nanoballs (BMP4-nanoballs) constructed with pBMP4 and dendrigraft poly-L-lysine (DGL, a cationic polymer) coated by γ-polyglutamic acid (γ-PGA; an anionic polymer), and determined their biological functions in vitro and in vivo. Next, GAMs were manufactured by mixing nanoballs with 2% atelocollagen and β-tricalcium phosphate (β-TCP) granules and lyophilizing them for bone augmentation. The GAMs were then transplanted to rat cranial bone surfaces under the periosteum. From the initial stage, infiltrated macrophages and mesenchymal progenitor cells took up the nanoballs, and their anti-inflammatory and osteoblastic differentiations were promoted over time. Subsequently, bone augmentation was clearly recognized for up to 8 weeks in transplanted GAMs containing BMP4-nanoballs. Notably, only 1 μg of BMP4-nanoballs induced a sufficient volume of new bone, while 1000 μg of naked pDNAs were required to induce the same level of bone augmentation. These data suggest that applying this anionic vector to the appropriate matrices can facilitate GAM-based bone engineering.

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Mimovirus: a Novel Form of Vaccine That Induces Hepatitis B Virus-Specific Cytotoxic T-Lymphocyte Responses In Vivo

Journal of Virology ◽

10.1128/jvi.76.20.10264-10269.2002 ◽

2002 ◽

Vol 76 (20) ◽

pp. 10264-10269 ◽

Cited By ~ 12

Author(s):

Yu-Zhang Wu ◽

Jian-Ping Zhao ◽

Ying Wan ◽

Zheng-Cai Jia ◽

Wei Zhou ◽

...

Keyword(s):

Self Assembly ◽

Mammalian Cells ◽

Electrostatic Interactions ◽

Cytotoxic T Lymphocyte ◽

Interleukin 12 ◽

Antigen Delivery ◽

Intracellular Pathogens ◽

Virus Size

ABSTRACT CD8+ cytotoxic T lymphocytes (CTLs) are now recognized as important mediators of immunity against intracellular pathogens, including human immunodeficiency virus and tumors. How to efficiently evoke antigen-specific CTL responses in vivo has become a crucial problem in the development of modern vaccines. Here, we developed a completely novel CTL vaccine—mimovirus, which is a kind of virus-size particulate antigen delivery system. It was formed by the self-assembly of a cationic peptide containing 18 lysines and a CTL-epitope peptide of HBsAg28-39, with a plasmid encoding mouse interleukin-12 (IL-12) through electrostatic interactions. We examined the formation of mimovirus by DNA retardation assay, DNase I protection assay, and transmission electron microscopy and demonstrated that mimovirus could efficiently transfer the plasmid encoding IL-12 into mammalian cells such as P815 cells in vitro. Furthermore, it was proved that mimovirus could induce an HBsAg28-39-specific CTL response in vivo. Considering its effectiveness, flexibility, and defined composition, mimovirus is potentially a novel system for vaccination against intracellular pathogens and tumors.

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Self-assembly of multi-component mitochondrial nucleoids via phase separation

10.1101/822858 ◽

2019 ◽

Author(s):

Marina Feric ◽

Tyler G. Demarest ◽

Jane Tian ◽

Deborah L. Croteau ◽

Vilhelm A. Bohr ◽

...

Keyword(s):

Phase Separation ◽

Mitochondrial Dysfunction ◽

Self Assembly ◽

Size Control ◽

Premature Aging ◽

List Type ◽

Mitochondrial Nucleoids ◽

Hutchinson Gilford Progeria Syndrome

SummaryMitochondria contain an autonomous and spatially segregated genome. The organizational unit of their genome is the nucleoid, which consists of mitochondrial DNA (mtDNA) and associated architectural proteins. Here, we show that phase separation is the primary physical mechanism for assembly and size-control of the mitochondrial nucleoid. The major mtDNA-binding protein TFAM spontaneously phase separates in vitro via weak, multivalent interactions into viscoelastic droplets with slow internal dynamics. In combination, TFAM and mtDNA form multiphase, gel-like structures in vitro, which recapitulate the in vivo dynamic behavior of mt-nucleoids. Enlarged, phase-separated, yet transcriptionally active, nucleoids are present in mitochondria from patients with the premature aging disorder Hutchinson-Gilford Progeria Syndrome (HGPS) and are associated with mitochondrial dysfunction. These results point to phase separation as an evolutionarily conserved mechanism of genome organization.HighlightsMitochondrial genomes are organized by phase separation.The main packaging protein TFAM and mtDNA combine to form viscoelastic, multiphase droplets in vitro.Mitochondrial nucleoids exhibit phase behavior in vivo, including dynamic rearrangements and heterogenous organization.Coalescence and enlargement of mt-nucleoids occur upon loss of mitochondrial homeostasis as well as in prematurely aged cells and are associated with mitochondrial dysfunction.

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Dimerization of Recombinant Tobacco Mosaic Virus Movement Protein

Journal of Virology ◽

10.1128/jvi.78.5.3372-3377.2004 ◽

2004 ◽

Vol 78 (7) ◽

pp. 3372-3377 ◽

Cited By ~ 23

Author(s):

Laurence M. Brill ◽

Songpon Dechongkit ◽

Byron DeLaBarre ◽

Jonathon Stroebel ◽

Roger N. Beachy ◽

...

Keyword(s):

Tobacco Mosaic Virus ◽

Mosaic Virus ◽

Electrostatic Interactions ◽

Analytical Ultracentrifugation ◽

Movement Protein ◽

Hydrophobic Interactions ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

In Planta ◽

Native Page

ABSTRACT The p30 movement protein (MP) is essential for cell-to-cell spread of tobacco mosaic virus in planta. We used anion-exchange chromatography and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to obtain highly purified 30-kDa MP, which migrated as a single band in native PAGE. Analytical ultracentrifugation suggested that the protein was monodisperse and dimeric in the nonionic detergent n-octyl-β-d-glucopyranoside. Circular dichroism (CD) spectroscopy showed that the detergent-solubilized protein contained significant α-helical secondary structure. Proteolysis of the C-tail generated a trypsin-resistant core that was a mixture of primarily monomers and some dimers. We propose that MP dimers are stabilized by electrostatic interactions in the C terminus as well as hydrophobic interactions between putative transmembrane α-helical coiled coils.

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Molecular determinants of phase separation forDrosophilaDNA replication licensing factors

10.1101/2021.04.12.439485 ◽

2021 ◽

Author(s):

Matthew W. Parker ◽

Jonchee Kao ◽

Alvin Huang ◽

James M. Berger ◽

Michael R. Botchan

Keyword(s):

Phase Separation ◽

Self Assembly ◽

A Priori ◽

Initiation Factor ◽

Sequence Composition ◽

Intrinsically Disordered ◽

Intrinsically Disordered Regions ◽

Partner Proteins

ABSTRACTLiquid-liquid phase separation (LLPS) of intrinsically disordered regions (IDRs) in proteins can drive the formation of membraneless compartments in cells. Phase-separated structures enrich for specific partner proteins and exclude others. We have shown that the IDRs of metazoan DNA replication initiators drive DNA-dependent phase separationin vitroand chromosome bindingin vivo, and that initiator condensates selectively recruit specific partner proteins. How initiator IDRs facilitate LLPS and maintain compositional specificity is unknown. UsingD. melanogaster (Dm)Cdt1 as a model initiation factor, we show that phase separation results from a synergy between electrostatic DNA-bridging interactions and hydrophobic inter-IDR contacts. Both sets of interactions depend on sequence composition (but not sequence order), are resistant to 1,6- hexanediol, and do not depend on aromaticity. These findings demonstrate that distinct sets of interactions drive self-assembly and condensate specificity across different phase-separating systems and advance efforts to predict IDR LLPS propensity and specificitya priori.

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Thermodynamic and sequential characteristics of phase separation and droplet formation for an intrinsically disordered region/protein ensemble

PLoS Computational Biology ◽

10.1371/journal.pcbi.1008672 ◽

2021 ◽

Vol 17 (3) ◽

pp. e1008672

Author(s):

Wen-Ting Chu ◽

Jin Wang

Keyword(s):

Phase Separation ◽

High Temperatures ◽

Electrostatic Interactions ◽

Sequence Length ◽

Intrinsically Disordered ◽

Dynamics Simulations ◽

Conventional Behavior ◽

Liquid Liquid Phase Separation

Liquid–liquid phase separation (LLPS) of some IDPs/IDRs can lead to the formation of the membraneless organelles in vitro and in vivo, which are essential for many biological processes in the cell. Here we select three different IDR segments of chaperon Swc5 and develop a polymeric slab model at the residue-level. By performing the molecular dynamics simulations, LLPS can be observed at low temperatures even without charge interactions and disappear at high temperatures. Both the sequence length and the charge pattern of the Swc5 segments can influence the critical temperature of LLPS. The results suggest that the effects of the electrostatic interactions on the LLPS behaviors can change significantly with the ratios and distributions of the charged residues, especially the sequence charge decoration (SCD) values. In addition, three different forms of swc conformation can be distinguished on the phase diagram, which is different from the conventional behavior of the free IDP/IDR. Both the packed form (the condensed-phase) and the dispersed form (the dilute-phase) of swc chains are found to be coexisted when LLPS occurs. They change to the fully-spread form at high temperatures. These findings will be helpful for the investigation of the IDP/IDR ensemble behaviors as well as the fundamental mechanism of the LLPS process in bio-systems.

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Disruption of Microtubule Organization and Centrosome Function by Expression of Tobacco Mosaic Virus Movement Protein

Journal of Virology ◽

10.1128/jvi.00254-06 ◽

2006 ◽

Vol 80 (12) ◽

pp. 5807-5821 ◽

Cited By ~ 41

Author(s):

Jacqueline Ferralli ◽

Jamie Ashby ◽

Monika Fasler ◽

Vitaly Boyko ◽

Manfred Heinlein

Keyword(s):

Tobacco Mosaic Virus ◽

Mosaic Virus ◽

Cell Biology ◽

Mammalian Cells ◽

Movement Protein ◽

Infected Plant ◽

In Vivo Studies ◽

Microtubule Organization

ABSTRACT The movement protein (MP) of Tobacco mosaic virus mediates the cell-to-cell transport of viral RNA through plasmodesmata, cytoplasmic cell wall channels for direct cell-to-cell communication between adjacent cells. Previous in vivo studies demonstrated that the RNA transport function of the protein correlates with its association with microtubules, although the exact role of microtubules in the movement process remains unknown. Since the binding of MP to microtubules is conserved in transfected mammalian cells, we took advantage of available mammalian cell biology reagents and tools to further address the interaction in flat-growing and transparent COS-7 cells. We demonstrate that neither actin, nor endoplasmic reticulum (ER), nor dynein motor complexes are involved in the apparent alignment of MP with microtubules. Together with results of in vitro coprecipitation experiments, these findings indicate that MP binds microtubules directly. Unlike microtubules associated with neuronal MAP2c, MP-associated microtubules are resistant to disruption by microtubule-disrupting agents or cold, suggesting that MP is a specialized microtubule binding protein that forms unusually stable complexes with microtubules. MP-associated microtubules accumulate ER membranes, which is consistent with a proposed role for MP in the recruitment of membranes in infected plant cells and may suggest that microtubules are involved in this process. The ability of MP to interfere with centrosomal γ-tubulin is independent of microtubule association with MP, does not involve the removal of other tested centrosomal markers, and correlates with inhibition of centrosomal microtubule nucleation activity. These observations suggest that the function of MP in viral movement may involve interaction with the microtubule-nucleating machinery.

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