Combining Transient Expression and Cryo-EM to Obtain High-Resolution Structures of Luteovirid Particles

Byrne, Matthew J.; Steele, John F. C.; Hesketh, Emma L.; Walden, Miriam; Thompson, Rebecca; Lomonossoff, George P.; Ranson, Neil A.

doi:10.1016/j.str.2019.09.010

Cited by 26 publications

(21 citation statements)

References 58 publications

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“…Here, however, we observed that VLPs that expressed in plants have a heterogeneous structure and aggregation-prone particles ( Figure S4). A similar phenomenon was observer when PEMV1 CP was expressed in plants [30].…”

Section: Discussionsupporting

confidence: 71%

Rapid High-Yield Transient Expression of Swine Hepatitis E ORF2 Capsid Proteins in Nicotiana benthamiana Plants and Production of Chimeric Hepatitis E Virus-Like Particles Bearing the M2e Influenza Epitope

Zahmanova

Mazalovska

Takova

et al. 2019

Plants

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The Hepatitis E virus (HEV) is a causative agent of acute hepatitis, mainly transmitted by the fecal-oral route or zoonotic. Open reading frame (ORF) 2 encodes the viral capsid protein, which is essential for virion assembly, host interaction, and inducing neutralizing antibodies. In this study, we investigated whether full-length and N- and C-terminally modified versions of the capsid protein transiently expressed in N. benthamiana plants could assemble into highly-immunogenic, virus-like particles (VLPs). We also assessed whether such VLPs can act as a carrier of foreign immunogenic epitopes, such as the highly-conserved M2e peptide from the Influenza virus. Plant codon-optimized HEV ORF2 capsid genes were constructed in which the nucleotides coding the N-terminal, the C-terminal, or both parts of the protein were deleted. The M2e peptide was inserted into the P2 loop after the residue Gly556 of HEV ORF2 protein by gene fusion, and three different chimeric constructs were designed. Plants expressed all versions of the HEV capsid protein up to 10% of total soluble protein (TSP), including the chimeras, but only the capsid protein consisting of aa residues 110 to 610 (HEV 110–610) and chimeric M2 HEV 110–610 spontaneously assembled in higher order structures. The chimeric VLPs assembled into particles with 22–36 nm in diameter and specifically reacted with the anti-M2e antibody.

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Section: Discussionsupporting

confidence: 71%

Rapid High-Yield Transient Expression of Swine Hepatitis E ORF2 Capsid Proteins in Nicotiana benthamiana Plants and Production of Chimeric Hepatitis E Virus-Like Particles Bearing the M2e Influenza Epitope

Zahmanova

Mazalovska

Takova

et al. 2019

Plants

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“…However, in vitro assembly is only feasible on a small scale with a virus, such as TMV, that has been extensively studied. While expression of the coat protein (CP) and self-assembly into VLPs has been very effective with isometric plant viruses [ 5 , 9 , 10 , 11 , 12 ], expression of the coat protein alone in the case of helical viruses generally results in either low yields, very heterogeneous material, or both. For example, the non-replicating pEAQ- HT system [ 13 ] has been used for the production of turnip mosaic virus VLPs by expression of coat protein alone [ 14 ], and tobacco mosaic virus VLPs by co-expression of coat protein with RNA bearing a TMV origin of assembly (OAS; [ 15 ]); however only relatively low yields were reported.…”

Section: Introductionmentioning

confidence: 99%

A Replicating Viral Vector Greatly Enhances Accumulation of Helical Virus-Like Particles in Plants

Thuenemann

Byrne

Peyret

et al. 2021

Viruses

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The production of plant helical virus-like particles (VLPs) via plant-based expression has been problematic with previous studies suggesting that an RNA scaffold may be necessary for their efficient production. To examine this, we compared the accumulation of VLPs from two potexviruses, papaya mosaic virus and alternanthera mosaic virus (AltMV), when the coat proteins were expressed from a replicating potato virus X- based vector (pEff) and a non-replicating vector (pEAQ-HT). Significantly greater quantities of VLPs could be purified when pEff was used. The pEff system was also very efficient at producing VLPs of helical viruses from different virus families. Examination of the RNA content of AltMV and tobacco mosaic virus VLPs produced from pEff revealed the presence of vector-derived RNA sequences, suggesting that the replicating RNA acts as a scaffold for VLP assembly. Cryo-EM analysis of the AltMV VLPs showed they had a structure very similar to that of authentic potexvirus particles. Thus, we conclude that vectors generating replicating forms of RNA, such as pEff, are very efficient for producing helical VLPs.

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“…The particles of the obtained chimeric virus may be a fascinating object of study under cryo-electron microscopy [49] taking into consideration that the structure of the ryegrass mottle virus (RMV) capsid was resolved at 2.9 angstroms [50] and that most non-enveloped icosahedral viruses have the same type of icosahedral capsids.…”

Section: Discussionmentioning

confidence: 99%

Chimeric Virus Made from crTMV RNA and the Coat Protein of Potato Leafroll Virus is Targeted to the Nucleolus and Can Infect Nicotiana benthamiana Mechanically

et al. 2020

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A genetically engineered chimeric virus crTMV-CP-PLRV composed of the crucifer-infecting tobacco mosaic virus (crTMV) RNA and the potato leafroll virus (PLRV) coat protein (CP) was obtained by agroinfiltration of Nicotiana benthamiana with the binary vector pCambia-crTMV-CPPLRV. The significant levels of the chimeric virus enabled direct visualization of crTMV-CP-PLRV in the cell and to investigate the mechanism of the pathogenesis. Localization of the crTMV-CP-PLRV in plant cells was examined by immunoblot techniques, as well as light, and transmission electron microscopy. The chimera can transfer between vascular and nonvascular tissues. The chimeric virus inoculum is capable to infect N. benthamiana mechanically. The distinguishing feature of the chimeric virus, the RNA virus with the positive genome, was found to localize in the nucleolus. We also investigated the role of the N-terminal sequence of the PLRV P3 coat protein in the cellular localization of the virus. We believe that the gene of the PLRV CP can be substituted with genes from other challenging-to-study plant pathogens to produce other useful recombinant viruses.

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Combining Transient Expression and Cryo-EM to Obtain High-Resolution Structures of Luteovirid Particles

Cited by 26 publications

References 58 publications

Rapid High-Yield Transient Expression of Swine Hepatitis E ORF2 Capsid Proteins in Nicotiana benthamiana Plants and Production of Chimeric Hepatitis E Virus-Like Particles Bearing the M2e Influenza Epitope

Rapid High-Yield Transient Expression of Swine Hepatitis E ORF2 Capsid Proteins in Nicotiana benthamiana Plants and Production of Chimeric Hepatitis E Virus-Like Particles Bearing the M2e Influenza Epitope

A Replicating Viral Vector Greatly Enhances Accumulation of Helical Virus-Like Particles in Plants

Chimeric Virus Made from crTMV RNA and the Coat Protein of Potato Leafroll Virus is Targeted to the Nucleolus and Can Infect Nicotiana benthamiana Mechanically

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