The Use of a Replicating Virus Vector For in Planta Generation of Tobacco Mosaic Virus Nanorods Suitable For Metallization

Saunders, Keith; Thuenemann, Eva C.; Shah, Sachin N.; Peyret, Hadrien; Kristianingsih, Ruth; Lopez, Sergio G.; Richardson, Jeremy; Lomonossoff, George P.

doi:10.3389/fbioe.2022.877361

Cited by 8 publications

(11 citation statements)

References 38 publications

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“…Previous work showed that pEff-generated potexvirus and virgavirus VLPs contain RNAs derived from the replication of the vector, reinforcing the idea that an RNA scaffold was important for helical particle formation 37 , 41 . To assess whether this also applies for pEff-produced potyvirid VLPs, we analysed both types of purified SPFMV and SPMMV VLPs.…”

Section: Resultsmentioning

confidence: 73%

“…Observations on the distribution of VLP lengths do not fit exactly with the expected sizes of the most abundant RNAs derived from the replicative pEff-vector constructs. In other cases the predicted equivalences were more evident, as found with the tobamovirus TMV 41 and with the potexvirus AltMV 37 , suggesting that for the sweet potato potyvirids the encapsidation of RNAs into VLPs was less strictly regulated. Alternatively, we cannot exclude the possibility that the conditions required for purification might result in partial breakdown of VLPs due to shearing forces.…”

Section: Discussionmentioning

confidence: 96%

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CryoEM and stability analysis of virus-like particles of potyvirus and ipomovirus infecting a common host

et al. 2023

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Sweet potato feathery mottle virus (SPFMV) and Sweet potato mild mottle virus (SPMMV) are members of the genera Potyvirus and Ipomovirus, family Potyviridae, sharing Ipomoea batatas as common host, but transmitted, respectively, by aphids and whiteflies. Virions of family members consist of flexuous rods with multiple copies of a single coat protein (CP) surrounding the RNA genome. Here we report the generation of virus-like particles (VLPs) by transient expression of the CPs of SPFMV and SPMMV in the presence of a replicating RNA in Nicotiana benthamiana. Analysis of the purified VLPs by cryo-electron microscopy, gave structures with resolutions of 2.6 and 3.0 Å, respectively, showing a similar left-handed helical arrangement of 8.8 CP subunits per turn with the C-terminus at the inner surface and a binding pocket for the encapsidated ssRNA. Despite their similar architecture, thermal stability studies reveal that SPMMV VLPs are more stable than those of SPFMV.

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Section: Resultsmentioning

confidence: 73%

Section: Discussionmentioning

confidence: 96%

CryoEM and stability analysis of virus-like particles of potyvirus and ipomovirus infecting a common host

et al. 2023

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“…For the expression of the SAP-M2e recombinant protein in N. bethamiana plants, we used the self-replicating viral vector pEff genome [42] that was used for the production of different recombinant proteins [40,[54][55][56]. The 19S_SAP_Sp_4M2eh protein was successfully expressed at the level of about 100 µg/g of fresh leaf biomass, and the purification yield was about 60 µg per gram.…”

Section: Discussionmentioning

confidence: 99%

“…All these genetic elements are located in the T-DNA region of a binary vector that could be maintained in both E. coli and A. tumefaciens. This pEff vector was previously used for the fast high-level expression of recombinant proteins in plant cells [40,55,56,59].…”

Section: Expression Vectormentioning

confidence: 99%

Plant-Produced Nanoparticles Based on Artificial Self-Assembling Peptide Bearing the Influenza M2e Epitope

Blokhina

Mardanova

Зыкова

et al. 2023

Plants

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Despite advances in vaccine development, influenza remains a persistent global health threat and the search for a broad-spectrum recombinant vaccine against influenza continues. The extracellular domain of the transmembrane protein M2 (M2e) of the influenza A virus is highly conserved and can be used to develop a universal vaccine. M2e is a poor immunogen by itself, but it becomes highly immunogenic when linked to an appropriate carrier. Here, we report the transient expression of a recombinant protein comprising four tandem copies of M2e fused to an artificial self-assembling peptide (SAP) in plants. The hybrid protein was efficiently expressed in Nicotiana benthamiana using the self-replicating potato virus X-based vector pEff. The protein was purified using metal affinity chromatography under denaturing conditions. The hybrid protein was capable of self-assembly in vitro into spherical particles 15–30 nm in size. The subcutaneous immunization of mice with M2e-carrying nanoparticles induced high levels of M2e-specific IgG antibodies in serum and mucosal secretions. Immunization provided mice with protection against a lethal influenza A virus challenge. SAP-based nanoparticles displaying M2e peptides can be further used to develop a recombinant “universal” vaccine against influenza A produced in plants.

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“…All these genetic elements are located in the T-DNA region of a binary vector that could be maintained in both Escherichia coli and Agrobacterium tumefaciens . This pEff vector was previously used for the fast high-level expression of recombinant proteins in plant cells [ 5 , 24 , 45 , 56 , 77 ].…”

Section: Methodsmentioning

confidence: 99%

High-Yield Production of Chimeric Hepatitis E Virus-Like Particles Bearing the M2e Influenza Epitope and Receptor Binding Domain of SARS-CoV-2 in Plants Using Viral Vectors

Mardanova

Kotlyarov

Stuchinskaya

et al. 2022

IJMS

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Capsid protein of Hepatitis E virus (HEV) is capable of self-assembly into virus-like particles (VLPs) when expressed in Nicotiana benthamiana plants. Such VLPs could be used as carriers of antigens for vaccine development. In this study, we obtained VLPs based on truncated coat protein of HEV bearing the M2e peptide of Influenza A virus or receptor-binding domain of SARS-CoV-2 spike glycoprotein (RBD). We optimized the immunogenic epitopes’ presentation by inserting them into the protruding domain of HEV ORF2 at position Tyr485. The fusion proteins were expressed in Nicotiana benthamiana plants using self-replicating potato virus X (PVX)-based vector. The fusion protein HEV/M2, targeted to the cytosol, was expressed at the level of about 300–400 μg per gram of fresh leaf tissue and appeared to be soluble. The fusion protein was purified using metal affinity chromatography under native conditions with the final yield about 200 μg per gram of fresh leaf tissue. The fusion protein HEV/RBD, targeted to the endoplasmic reticulum, was expressed at about 80–100 μg per gram of fresh leaf tissue; the yield after purification was up to 20 μg per gram of fresh leaf tissue. The recombinant proteins HEV/M2 and HEV/RBD formed nanosized virus-like particles that could be recognized by antibodies against inserted epitopes. The ELISA assay showed that antibodies of COVID-19 patients can bind plant-produced HEV/RBD virus-like particles. This study shows that HEV capsid protein is a promising carrier for presentation of foreign antigen.

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The Use of a Replicating Virus Vector For in Planta Generation of Tobacco Mosaic Virus Nanorods Suitable For Metallization

Cited by 8 publications

References 38 publications

CryoEM and stability analysis of virus-like particles of potyvirus and ipomovirus infecting a common host

CryoEM and stability analysis of virus-like particles of potyvirus and ipomovirus infecting a common host

Plant-Produced Nanoparticles Based on Artificial Self-Assembling Peptide Bearing the Influenza M2e Epitope

High-Yield Production of Chimeric Hepatitis E Virus-Like Particles Bearing the M2e Influenza Epitope and Receptor Binding Domain of SARS-CoV-2 in Plants Using Viral Vectors

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