scholarly journals The movement protein encoded by gene 3 of rice transitory yellowing virus is associated with virus particles

2012 ◽  
Vol 93 (10) ◽  
pp. 2290-2298 ◽  
Author(s):  
Akihiro Hiraguri ◽  
Hiroyuki Hibino ◽  
Takaharu Hayashi ◽  
Osamu Netsu ◽  
Takumi Shimizu ◽  
...  
Keyword(s):  
Movement Protein ◽  
Cell Movement ◽  
Immunogold Labelling ◽  
Tomato Mosaic Virus ◽  
Structural Protein ◽  
Western Blots ◽  
Thin Sections ◽  
Virus Particles ◽  
E Coli ◽  
Yellowing Virus

Gene 3 in the genomes of several plant-infecting rhabdoviruses, including rice transitory yellowing virus (RTYV), has been postulated to encode a cell-to-cell movement protein (MP). Trans-complementation experiments using a movement-defective tomato mosaic virus and the P3 protein of RTYV, encoded by gene 3, facilitated intercellular transport of the mutant virus. In transient-expression experiments with the GFP-fused P3 protein in epidermal leaf cells of Nicotiana benthamiana, the P3 protein was associated with the nucleus and plasmodesmata. Immunogold-labelling studies of thin sections of RTYV-infected rice plants using an antiserum against Escherichia coli-expressed His6-tagged P3 protein indicated that the P3 protein was located in cell walls and on virus particles. In Western blots using antisera against E. coli-expressed P3 protein and purified RTYV, the P3 protein was detected in purified RTYV, whilst antiserum against purified RTYV reacted with the E. coli-expressed P3 protein. After immunogold labelling of crude sap from RTYV-infected rice leaves, the P3 protein, as well as the N protein, was detected on the ribonucleocapsid core that emerged from partially disrupted virus particles. These results provide evidence that the P3 protein of RTYV, which functions as a viral MP, is a viral structural protein and seems to be associated with the ribonucleocapsid core of virus particles.

scholarly journals Actin Cytoskeleton Is Involved in Targeting of a Viral Hsp70 Homolog to the Cell Periphery

2005 ◽  
Vol 79 (22) ◽  
pp. 14421-14428 ◽  
Author(s):  
Alexey I. Prokhnevsky ◽  
Valera V. Peremyslov ◽  
Valerian V. Dolja
Keyword(s):  
Actin Cytoskeleton ◽  
Tobacco Mosaic Virus ◽  
Mosaic Virus ◽  
Movement Protein ◽  
Fluorescent Proteins ◽  
Cell Movement ◽  
Plant Viruses ◽  
Cell Periphery ◽  
Virus Particles ◽  
Shock Proteins

ABSTRACT The cell-to-cell movement of plant viruses involves translocation of virus particles or nucleoproteins to and through the plasmodesmata (PDs). As we have shown previously, the movement of the Beet yellows virus requires the concerted action of five viral proteins including a homolog of cellular ∼70-kDa heat shock proteins (Hsp70h). Hsp70h is an integral component of the virus particles and is also found in PDs of the infected cells. Here we investigate subcellular distribution of Hsp70h using transient expression of Hsp70h fused to three spectrally distinct fluorescent proteins. We found that fluorophore-tagged Hsp70h forms motile granules that are associated with actin microfilaments, but not with microtubules. In addition, immobile granules were observed at the cell periphery. A pairwise appearance of these granules at the opposite sides of cell walls and their colocalization with the movement protein of Tobacco mosaic virus indicated an association of Hsp70h with PDs. Treatment with various cytoskeleton-specific drugs revealed that the intact actomyosin motility system is required for trafficking of Hsp70h in cytosol and its targeting to PDs. In contrast, none of the drugs interfered with the PD localization of Tobacco mosaic virus movement protein. Collectively, these findings suggest that Hsp70h is translocated and anchored to PDs in association with the actin cytoskeleton.

scholarly journals The Tomato brown rugose fruit virus movement protein overcomes Tm-22 resistance in tomato while attenuating viral transport

2021 ◽  
Author(s):  
Hagit Hak ◽  
Ziv Spiegelman
Keyword(s):  
Mosaic Virus ◽  
Movement Protein ◽  
Cell Movement ◽  
Systemic Infection ◽  
Movement Analysis ◽  
Tomato Mosaic Virus ◽  
Resistance Response ◽  
Global Epidemic ◽  
High Durability ◽  
C Terminus

Tomato brown rugose fruit virus (ToBRFV) is a new virus of the Tobamovirus genus, causing substantial damage to tomato crops. Reports of recent ToBRFV outbreaks from around the world indicate an emerging global epidemic. ToBRFV overcomes all tobamovirus resistances in tomato, including the durable Tm-22 resistance gene, which had been effective against multiple tobamoviruses. Here, we show that the ToBRFV movement protein (MPToBRFV) enables the virus to evade Tm-22 resistance. Transient expression of MPToBRFV failed to activate the Tm-22 resistance response. Replacement of the original MP sequence of Tomato mosaic virus (ToMV) with MPToBRFV enabled this recombinant virus to infect Tm-22 resistant plants. Using hybrid protein analysis, we show that the elements required to evade Tm-22 are located between MPToBRFV amino acids 1 and 216, and not the C terminus as previously assumed. Analysis of ToBRFV systemic infection in tomato revealed that ToBRFV spreads slower compared to ToMV. Interestingly, replacement of Tobacco mosaic virus (TMV) and ToMV MPs with MPToBRFV caused an attenuation of systemic infection of both viruses. Cell-to-cell movement analysis showed that MPToBRFV moves less effectively compared to the TMV MP (MPTMV). These findings suggest that overcoming Tm-22 is associated with attenuated MP function. This may explain the high durability of Tm-22 resistance, which had remained unbroken for over 60 years.

scholarly journals Identification of distinct steps during tubule formation by the movement protein of Cowpea mosaic virus

2003 ◽  
Vol 84 (12) ◽  
pp. 3485-3494 ◽  
Author(s):  
Jeroen Pouwels ◽  
Noortje Kornet ◽  
Nikkie van Bers ◽  
Teun Guighelaar ◽  
Jan van Lent ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Mosaic Virus ◽  
Movement Protein ◽  
Cell Movement ◽  
Cowpea Mosaic Virus ◽  
Tubule Formation ◽  
Virus Particles ◽  
Cell Localization ◽  
Support Cell ◽  
Infected Cells

The movement protein (MP) of Cowpea mosaic virus (CPMV) forms tubules through plasmodesmata in infected plants thus enabling virus particles to move from cell to cell. Localization studies of mutant MPs fused to GFP in protoplasts and plants identified several functional domains within the MP that are involved in distinct steps during tubule formation. Coinoculation experiments and the observation that one of the C-terminal deletion mutants accumulated uniformly in the plasma membrane suggest that dimeric or multimeric MP is first targeted to the plasma membrane. At the plasma membrane the MP quickly accumulates in peripheral punctuate spots, from which tubule formation is initiated. One of the mutant MPs formed tubules containing virus particles on protoplasts, but could not support cell-to-cell movement in plants. The observations that this mutant MP accumulated to a higher level in the cell than wt MP and did not accumulate in the cell wall opposite infected cells suggest that breakdown or disassembly of tubules in neighbouring, uninfected cells is required for cell-to-cell movement.

scholarly journals Tm-22 Resistance in Tomato Requires Recognition of the Carboxy Terminus of the Movement Protein of Tomato Mosaic Virus

1998 ◽  
Vol 11 (6) ◽  
pp. 498-503 ◽  
Author(s):  
Hans Weber ◽  
Artur J. P. Pfitzner
Keyword(s):  
Mosaic Virus ◽  
Resistance Gene ◽  
Movement Protein ◽  
Cell Movement ◽  
Tomato Mosaic Virus ◽  
Wild Type ◽  
Virus Transport ◽  
Tomato Plants ◽  
Movement Proteins ◽  
Carboxy Terminal

The Tm-22 resistance gene is used in most commercial tomato cultivars for protection against infection with tomato mosaic virus (ToMV). It has been suggested that Tm-22 resistance interferes with viral cell-to-cell movement in plants; ToMV strain ToMV-22 requires two amino acid (aa) exchanges in the carboxy-terminal region of the viral 30-kDa movement protein (at positions 238 and 244) to overcome Tm-22 resistance. For further analysis of this region of the 30-kDa protein, two stop codons were introduced into ToMV movement proteins at aa positions 235 and 237, leading to deletion of the terminal 30 aa. The mutant virus strains were able to infect wild-type tomato plants systemically, suggesting the carboxy-terminal portion of the ToMV 30-kDa protein is dispensable for virus transport in tomato. Even more important, the deletion mutants overcame the Tm-22 resistance gene. These data indicate the carboxy-terminal domain of the ToMV movement protein serves as a recognition target in the context of the Tm-22 resistance gene. Furthermore, expression of the 30-kDa movement protein from wild-type ToMV, but not from ToMV-22, in transgenic tomato plants with the Tm-22 resistance gene led to elicitation of a necrotic reaction in tomato seedlings, showing that the 30-kDa protein on its own is able to induce the plant's defense reaction.

scholarly journals The Tomato brown rugose fruit virus movement protein overcomes Tm-22 resistance while attenuating viral transport

2020 ◽  
Author(s):  
Hagit Hak ◽  
Ziv Spiegelman
Keyword(s):  
Mosaic Virus ◽  
Movement Protein ◽  
Cell Movement ◽  
Systemic Infection ◽  
Movement Analysis ◽  
Viral Fitness ◽  
Tomato Mosaic Virus ◽  
Resistance Response ◽  
Global Epidemic ◽  
C Terminus

Tomato brown rugose fruit virus (ToBRFV) is a new virus of the Tobamovirus genus, causing substantial damage to tomato crops in the Middle East. Reports of recent ToBRFV outbreaks from around the world indicate an emerging global epidemic. ToBRFV overcomes all tobamovirus resistances in tomato, including the durable Tm-22 resistance gene. Here, we show that the ToBRFV movement protein (MPToBRFV) is the cause for overcoming Tm-22 resistance. Transient expression of MPToBRFV failed to activate the Tm-22 resistance response. Replacement of the original MP sequences of Tomato mosaic virus (ToMV) with MPToBRFV enabled this recombinant virus to overcome Tm-22 resistance. Hybrid protein analysis revealed that the resistance-breaking elements are located between MPToBRFV amino acids 1 and 216, and not the C terminus as previously assumed. Interestingly, replacement of Tobacco mosaic virus (TMV) and ToMV MPs with MPToBRFV caused an attenuation of systemic infection of both viruses. Cell-to-cell movement analysis revealed that MPToBRFV moves less effectively compared to the TMV MP (MPTMV). These findings suggest that overcoming Tm-22 is associated with attenuated MP function. This viral fitness cost may explain the high durability of Tm-22 resistance, which had remained unbroken for over 60 years.

scholarly journals Tobamoviral Movement Protein Transiently Expressed in a Single Epidermal Cell Functions Beyond Multiple Plasmodesmata and Spreads Multicellularly in an Infection-Coupled Manner

2001 ◽  
Vol 14 (2) ◽  
pp. 126-134 ◽  
Author(s):  
Atsushi Tamai ◽  
Tetsuo Meshi
Keyword(s):  
Mosaic Virus ◽  
Fluorescent Protein ◽  
Movement Protein ◽  
Microprojectile Bombardment ◽  
Cell Movement ◽  
Tomato Mosaic Virus ◽  
Protein Variant ◽  
Minute Amount ◽  
Cell Functions ◽  
Infected Cells

Cell-to-cell movement of a plant virus requires expression of the movement protein (MP). It has not been fully elucidated, however, how the MP functions in primary infected cells. With the use of a microprojectile bombardment-mediated DNA infection system for Tomato mosaic virus (ToMV), we found that the cotransfected ToMV MP gene exerts its effects in the initially infected cells and in their surrounding cells to achieve multicellular spread of movement-defective ToMV. Five other tobamoviral MPs examined also transcomplemented the movement-defective phenotype of ToMV, but the Cucumber mosaic virus 3a MP did not. Together with the cell-to-cell movement of the mutant virus, a fusion between the MP and an enhanced green fluorescent protein variant (EGFP) expressed in trans was distributed multicellularly and localized primarily in plasmodesmata between infected cells. In contrast, in noninfected sites the MP-EGFP fusion accumulated predominantly inside the bombarded cells as irregularly shaped aggregates, and only a minute amount of the fusion was found in plasmodesmata. Thus, the behavior of ToMV MP is greatly modulated in the presence of a replicating virus and it is highly likely that the MP spreads in the infection sites, coordinating with the cell-to-cell movement of the viral genome.

Studies of a Defective Measles Virus

1968 ◽  
Vol 26 ◽  
pp. 208-209
Author(s):  
R. M. McCombs ◽  
M. Benyesh-Melnick ◽  
J. P. Brunschwig
Keyword(s):  
Inclusion Bodies ◽  
Measles Virus ◽  
Giant Cells ◽  
Helical Structures ◽  
Thin Sections ◽  
Virus Particles ◽  
Extracellular Virus ◽  
Culture Cells ◽  
Infected Cells ◽  
Eosinophilic Inclusions

Measles virus is an agent that is capable of replicating in a number of different culture cells and generally causes the formation of multinucleated giant cells. As a result of infection, virus is released from the cells into the culture fluids and reinfection can be initiated by this cell-free virus. The extracellular virus has been examined by negative staining with phosphotungstic acid and has been shown to be a rather pleomorphic particle with a diameter of about 140 mμ. However, no such virus particles have been detected in thin sections of the infected cells. Rather, the only virus-induced structures present in the giant cells are eosinophilic inclusions (intracytoplasmic or intranuclear). These inclusion bodies have been shown to contain helical structures, resembling the nucleocapsid observed in negatively stained preparations.

Rapid, Refined, and Robust Method for Expression, Purification, and Characterization of Recombinant Human Amyloid-beta M1-42

2019 ◽  
Author(s):  
Priya Prakash ◽  
Travis Lantz ◽  
Krupal P. Jethava ◽  
Gaurav Chopra
Keyword(s):  
Amyloid Beta ◽  
Western Blots ◽  
Force Microscopy ◽  
E Coli ◽  
Atomic Force ◽  
Set Up ◽  
Short Time

Amyloid plaques found in the brains of Alzheimer’s disease (AD) patients primarily consists of amyloid beta 1-42 (Ab42). Commercially, Ab42 is synthetized using peptide synthesizers. We describe a robust methodology for expression of recombinant human Ab(M1-42) in Rosetta(DE3)pLysS and BL21(DE3)pLysS competent E. coli with refined and rapid analytical purification techniques. The peptide is isolated and purified from the transformed cells using an optimized set-up for reverse-phase HPLC protocol, using commonly available C18 columns, yielding high amounts of peptide (~15-20 mg per 1 L culture) in a short time. The recombinant Ab(M1-42) forms characteristic aggregates similar to synthetic Ab42 aggregates as verified by western blots and atomic force microscopy to warrant future biological use. Our rapid, refined, and robust technique to purify human Ab(M1-42) can be used to synthesize chemical probes for several downstream in vitro and in vivo assays to facilitate AD research.

scholarly journals Ultrafiltration Process in Disinfection and Advanced Treatment of Tertiary Treated Wastewater

Membranes ◽  
2021 ◽  
Vol 11 (3) ◽  
pp. 221
Author(s):  
Rafał Tytus Bray ◽  
Katarzyna Jankowska ◽  
Eliza Kulbat ◽  
Aneta Łuczkiewicz ◽  
Aleksandra Sokołowska
Keyword(s):  
Wastewater Treatment ◽  
Wastewater Treatment Plants ◽  
Microscopic Analysis ◽  
Fecal Coliforms ◽  
Treated Wastewater ◽  
Chemical Parameters ◽  
Virus Particles ◽  
E Coli ◽  
The One ◽  
Physical And Chemical

The paper presents the results of research on the use of ultrafiltration, using membranes of 200 and 400 kDa separation, for disinfection of municipal treated wastewater. The research was conducted on a fractional technical scale using real municipal treated wastewater from two large wastewater treatment plants treating most of the wastewater over the one-million polycentric Gdańsk agglomeration (1.2 million inhabitants). UF 200 kDa and UF 400 kDa processes enabled further improvement of the physical and chemical parameters of treated wastewater. Total phosphorus (to below 0.2 mg/L–UF 200 kDa, 0.13 mg/L–UF 400 kDa) and turbid substances (to below 0.2 mg/L, both membranes) were removed in the highest degree. COD was reduced efficiently (to below 25.6 mgO2/L–UF 200 kDa, 26.8 mgO2/L–UF 400 kDa), while total nitrogen was removed to a small extent (to 7.12 mg/L–UF 200 kDa and 5.7 mg/L–UF 400 kDa. Based on the reduction of indicator bacteria; fecal coliforms including E. coli (FC) and fecal enterococci (FE) it was found that the ultrafiltration is an effective method of disinfection. Not much indicator bacterial were observed in the permeate after processes (UF 200 kDa; FC—5 CFU/L; FE—1 CFU/L and UF 400 kDa; FC—70 CFU/L; FE—10 CFU/L. However, microscopic analysis of prokaryotic cells and virus particles showed their presence after the application of both membrane types; TCN 3.0 × 102 cells/mL–UF 200 kDa, 5.0 × 103 cells/mL–UF 400 kDa, VP 1.0 × 105/mL. The presence of potentially pathogenic, highly infectious virus particles means that ultrafiltration cannot be considered a sufficient disinfection method for treated wastewater diverted for reuse or discharged from high load wastewater treatment plants to recreational areas. For full microbiological safety it would be advisable to apply an additional disinfection method (e.g., ozonation).

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