scholarly journals Induction of Particle Polymorphism by Cucumber Necrosis Virus Coat Protein Mutants In Vivo

2007 ◽  
Vol 82 (3) ◽  
pp. 1547-1557 ◽  
Author(s):  
Kishore Kakani ◽  
Ron Reade ◽  
Umesh Katpally ◽  
Thomas Smith ◽  
D'Ann Rochon
Keyword(s):  
Coat Protein ◽  
Rna Binding ◽  
In Planta ◽  
Necrosis Virus ◽  
Protein Mutants ◽  
Cucumber Necrosis Virus ◽  
Virus Coat ◽  
R Domain

ABSTRACT The Cucumber necrosis virus (CNV) particle is a T=3 icosahedron consisting of 180 identical coat protein (CP) subunits. Plants infected with wild-type CNV accumulate a high number of T=3 particles, but other particle forms have not been observed. Particle polymorphism in several T=3 icosahedral viruses has been observed in vitro following the removal of an extended N-terminal region of the CP subunit. In the case of CNV, we have recently described the structure of T=1 particles that accumulate in planta during infection by a CNV mutant (R1+2) in which a large portion of the N-terminal RNA binding domain (R-domain) has been deleted. In this report we further describe properties of this mutant and other CP mutants that produce polymorphic particles. The T=1 particles produced by R1+2 mutants were found to encapsidate a 1.9-kb RNA species as well as smaller RNA species that are similar to previously described CNV defective interfering RNAs. Other R-domain mutants were found to encapsidate a range of specifically sized less-than-full-length CNV RNAs. Mutation of a conserved proline residue in the arm domain near its junction with the shell domain also influenced T=1 particle formation. The proportion of polymorphic particles increased when the mutation was incorporated into R-domain deletion mutants. Our results suggest that both the R-domain and the arm play important roles in the formation of T=3 particles. In addition, the encapsidation of specific CNV RNA species by individual mutants indicates that the R-domain plays a role in the nature of CNV RNA encapsidated in particles.

scholarly journals Identification of Specific Cucumber Necrosis Virus Coat Protein Amino Acids Affecting Fungus Transmission and Zoospore Attachment

2001 ◽  
Vol 75 (12) ◽  
pp. 5576-5583 ◽  
Author(s):  
Kishore Kakani ◽  
Jean-Yves Sgro ◽  
D'Ann Rochon
Keyword(s):  
Amino Acids ◽  
Coat Protein ◽  
Binding Assays ◽  
Protein Subunit ◽  
Necrosis Virus ◽  
Threefold Axis ◽  
Cucumber Necrosis Virus ◽  
Virus Coat ◽  
The Individual

ABSTRACT Cucumber necrosis virus (CNV) is naturally transmitted in the soil by zoospores of the fungal vector Olpidium bornovanus. Successful transmission requires that virus particles attach to the surface of zoospores prior to zoospore encystment on host roots. Mechanically passaged CNV was screened for mutants deficient in fungus transmission. We found six such mutants, exhibiting transmission efficiencies ranging from approximately 14 to 76% of that of wild-type (WT) CNV. Results of in vitro virus-zoospore binding assays show that each mutant binds to zoospores less efficiently than WT CNV (21 to 68%), suggesting that defects in transmission for these mutants are at least partially due to inefficient zoospore binding. Analysis of the structure of the CNV coat protein subunit and trimer indicates that affected amino acids in all of the mutants are located in the shell or protruding domain and that five of six of them are potentially exposed on the surface of the virus particle. In addition, several of the mutated sites, along with a previously identified site in a region of subunit-subunit interaction in the coat protein shell domain (M. A. Robbins, R. D. Reade, and D. M. Rochon, Virology 234:138–146, 1997), are located on the particle quasi-threefold axis, suggesting that this region of the capsid may be important in recognition of a putative zoospore receptor. The individual sites may directly affect attachment to a receptor or could indirectly affect attachment via changes in virion conformation.

scholarly journals Evaluation of the Roles of Specific Regions of the Cucumber Necrosis Virus Coat Protein Arm in Particle Accumulation and Fungus Transmission

2006 ◽  
Vol 80 (12) ◽  
pp. 5968-5975 ◽  
Author(s):  
Elizabeth Hui ◽  
D'Ann Rochon
Keyword(s):  
Coat Protein ◽  
Rna Binding ◽  
Virus Transmission ◽  
Wild Type ◽  
Necrosis Virus ◽  
Particle Accumulation ◽  
Olpidium Bornovanus ◽  
Particle Stability ◽  
Cucumber Necrosis Virus ◽  
Virus Coat

ABSTRACT The Cucumber necrosis virus (CNV) particle is a T=3 icosahedron composed of 180 identical coat protein (CP) subunits. Each CP subunit includes a 34-amino-acid (aa) arm which connects the RNA binding and shell domains. The arm is comprised of an 18-aa “β” region and a 16-aa “ε” region, with the former contributing to a β-annular structure involved in particle stability and the latter contributing to quasiequivalence and virion RNA binding. Previous work has shown that specific regions of the CNV capsid play important roles in transmission by zoospores of the fungal vector Olpidium bornovanus and that particle expansion is essential for this process. To assess the importance of the two arm regions in particle accumulation, stability, and virus transmission, five CP arm deletion mutants were constructed. Our findings indicate that β(−) mutants are capable of producing particles in plants; however, the arm(−) and ε(−) mutants are not. In addition, β(−) particles bind zoospores less efficiently than wild-type CNV and are not fungally transmissible. β(−) particles are also less thermally stable and disassemble under swelling conditions. Our finding that β(−) mutants can accumulate in plants suggests that other features of the virion, such as RNA/CP interactions, may also be important for particle stability.

scholarly journals Potato virus X RNA-mediated assembly of single-tailed ternary ‘coat protein–RNA–movement protein’ complexes

2006 ◽  
Vol 87 (9) ◽  
pp. 2731-2740 ◽  
Author(s):  
O. V. Karpova ◽  
O. V. Zayakina ◽  
M. V. Arkhipenko ◽  
E. V. Sheval ◽  
O. I. Kiselyova ◽  
...  
Keyword(s):  
Coat Protein ◽  
Potato Virus ◽  
Rna Binding ◽  
Movement Protein ◽  
Infected Plant ◽  
Potato Virus X ◽  
Translation System ◽  
Transport Form

Different models have been proposed for the nature of the potexvirus transport form that moves from cell to cell over the infected plant: (i) genomic RNA moves as native virions; or (ii) in vitro-assembled non-virion ribonucleoprotein (RNP) complexes consisting of viral RNA, coat protein (CP) and movement protein (MP), termed TGBp1, serve as the transport form in vivo. As the structure of these RNPs has not been elucidated, the products assembled in vitro from potato virus X (PVX) RNA, CP and TGBp1 were characterized. The complexes appeared as single-tailed particles (STPs) with a helical, head-like structure composed of CP subunits located at the 5′-proximal region of PVX RNA; the TGBp1 was bound to the terminal CP molecules of the head. Remarkably, no particular non-virion RNP complexes were observed. These data suggest that the CP–RNA interactions resulting in head formation prevailed over TGBp1–RNA binding upon STP assembly from RNA, CP and TGBp1. STPs could be assembled from the 5′ end of PVX RNA and CP in the absence of TGBp1. The translational ability of STPs was characterized in a cell-free translation system. STPs lacking TGBp1 were entirely non-translatable; however, they were rendered translatable by binding of TGBp1 to the end of the head. It is suggested that the RNA-mediated assembly of STPs proceeds via two steps. Firstly, non-translatable CP–RNA STPs are produced, due to encapsidation of the 5′-terminal region. Secondly, the TGBp1 molecules bind to the end of a polar head, resulting in conversion of the STPs into a translatable form.

scholarly journals Investigating the Biological Relevance of In Vitro-Identified Putative Packaging Signals at the 5′ Terminus of Satellite Tobacco Necrosis Virus 1 Genomic RNA

2019 ◽  
Vol 93 (9) ◽  
Author(s):  
Ioly Kotta-Loizou ◽  
Hadrien Peyret ◽  
Keith Saunders ◽  
Robert H. A. Coutts ◽  
George P. Lomonossoff
Keyword(s):  
Model System ◽  
Tobacco Necrosis Virus ◽  
Genomic Rna ◽  
In Planta ◽  
Necrosis Virus ◽  
Rna Sequences ◽  
Link Type ◽  
Packaging Signals

ABSTRACT Satellite tobacco necrosis virus 1 (STNV-1) is a model system for in vitro RNA encapsidation studies (N. Patel, E. C. Dykeman, R. H. A. Coutts, G. P. Lomonossoff, et al., Proc Natl Acad Sci U S A 112:2227–2232, 2015, https://doi.org/10.1073/pnas.1420812112; N. Patel, E. Wroblewski, G. Leonov, S. E. V. Phillips, et al., Proc Natl Acad Sci U S A 114:12255–12260, 2017, https://doi.org/10.1073/pnas.1706951114), leading to the identification of degenerate packaging signals (PSs) proposed to be involved in the recognition of its genome by the capsid protein (CP). The aim of the present work was to investigate whether these putative PSs can confer selective packaging of STNV-1 RNA in vivo and to assess the prospects of using decoy RNAs in antiviral therapy. We have developed an in planta packaging assay based on the transient expression of STNV-1 CP and have assessed the ability of the resulting virus-like particles (VLPs) to encapsidate mutant STNV-1 RNAs expected to have different encapsidation potential based on in vitro studies. The results revealed that >90% of the encapsidated RNAs are host derived, although there is some selectivity of packaging for STNV-1 RNA and certain host RNAs. Comparison of the packaging efficiencies of mutant STNV-1 RNAs showed that they are encapsidated mainly according to their abundance within the cells, rather than the presence or absence of the putative PSs previously identified from in vitro studies. In contrast, subsequent infection experiments demonstrated that host RNAs represent only <1% of virion content. Although selective encapsidation of certain host RNAs was noted, no direct correlation could be made between this preference and the presence of potential PSs in the host RNA sequences. Overall, the data illustrate that the differences in RNA packaging efficiency identified through in vitro studies are insufficient to explain the specific packaging of STNV-1 RNA. IMPORTANCE Viruses preferentially encapsidate their own genomic RNA, sometimes as a result of the presence of clearly defined packaging signals (PSs) in their genome sequence. Recently, a novel form of short degenerate PSs has been proposed (N. Patel, E. C. Dykeman, R. H. A. Coutts, G. P. Lomonossoff, et al., Proc Natl Acad Sci U S A 112:2227–2232, 2015, https://doi.org/10.1073/pnas.1420812112; N. Patel, E. Wroblewski, G. Leonov, S. E. V. Phillips, et al., Proc Natl Acad Sci U S A 114:12255–12260, 2017, https://doi.org/10.1073/pnas.1706951114) using satellite tobacco necrosis virus 1 (STNV-1) as a model system for in vitro studies. It has been suggested that competing with these putative PSs may constitute a novel therapeutic approach against pathogenic single-stranded RNA viruses. Our work demonstrates that the previously identified PSs have no discernible significance for the selective packaging of STNV-1 in vivo in the presence and absence of competition or replication: viral sequences are encapsidated mostly on the basis of their abundance within the cell, while encapsidation of host RNAs also occurs. Nevertheless, the putative PSs identified in STNV-1 RNA may still have applications in bionanotechnology, such as the in vitro selective packaging of RNA molecules.

scholarly journals Reappraisal of Spinach-based RNA Visualization in Plants

2020 ◽  
Author(s):  
Zhiming Yu ◽  
Fengling Mei ◽  
Haiting Yan ◽  
Qiyuan Chen ◽  
Mengqin Yao ◽  
...  
Keyword(s):  
Tandem Repeats ◽  
Rna Binding ◽  
Ex Vivo ◽  
Plant Cells ◽  
Quantitative Detection ◽  
Rna Aptamer ◽  
In Planta ◽  
Green Fluorescence

ABSTRACTRNAs can be imaged in living cells using molecular beacons, RNA-binding labeled proteins and RNA aptamer-based approaches. However, Spinach RNA-mimicking GFP (RMG) has not been successfully used to monitor cellular RNAs in plants. In this study, we re-evaluated Spinach-based RNA visualization in different plants via transient, transgenic, and virus-based expression strategies. We found that like bacterial, yeast and human cellular tRNAs, plant tRNAs such as tRNALys (K) can protect and/or stabilize the spinach RNA aptamer interaction with the fluorophore DFHBI enabling detectable levels of green fluorescence to be emitted. The tRNALys-spinach-tRNALys (KSK), once delivered into “chloroplast-free” onion epidermal cells can emit strong green fluorescence in the presence of DFHBI. Transgenic or virus-based expression of monomer KSK, in either stably transformed or virus-infected Nicotinana benthamiana plants, failed to show RMG fluorescence. However, incorporating tandem repeats of KSK into recombinant viral RNAs, enabled qualitative and quantitative detection, both in vitro and ex vivo (ex planta), of KSK-specific green fluorescence, though RMG was less obvious in vivo (in planta). These findings demonstrate Spinach-based RNA visualization has the potential for ex vivo and in vivo monitoring RNAs in plant cells.One sentence summarySpinach-based RMG technology was reevaluated to have potential for ex vivo and in vivo monitoring RNAs in plant cells.

scholarly journals Mutational analysis of the cucumber necrosis virus coat protein gene

Virology ◽  
1995 ◽  
Vol 206 (1) ◽  
pp. 38-48 ◽  
Author(s):  
Tim L. Sit ◽  
Julie C. Johnston ◽  
Melanie G. Ter Borg ◽  
Emile Frison ◽  
Morven A. McLean ◽  
...  
Keyword(s):  
Coat Protein ◽  
Coat Protein Gene ◽  
Protein Gene ◽  
Mutational Analysis ◽  
Necrosis Virus ◽  
Virus Coat Protein ◽  
Cucumber Necrosis Virus ◽  
Virus Coat
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