Spherical Plant Viruses Research Articles

Application of Plant Viruses as a Biotemplate for Nanomaterial Fabrication.

Viruses are widely used to fabricate nanomaterials in the field of nanotechnology. Plant viruses are of great interest to the nanotechnology field because of their symmetry, polyvalency, homogeneous size distribution, and ability to self-assemble. This homogeneity can be used to obtain the high uniformity of the templated material and its related properties. In this paper, the variety of nanomaterials generated in rod-like and spherical plant viruses is highlighted for the cowpea chlorotic mottle virus (CCMV), cowpea mosaic virus (CPMV), brome mosaic virus (BMV), and tobacco mosaic virus (TMV). Their recent studies on developing nanomaterials in a wide range of applications from biomedicine and catalysts to biosensors are reviewed.

Empty Turnip yellow mosaic virus capsids as delivery vehicles to mammalian cells

Turnip yellow mosaic virus (TYMV) was able to enter animal cells when the spherical plant virus was conjugated with Tat, a cell penetrating peptide (CPP). Tat was chemically attached to the surface lysine residues of TYMV using hydrazone chemistry. Baby hamster kidney (BHK) cells were incubated with either unmodified or Tat-conjugated TYMV and examined by flow cytometry and confocal microscopic analyses. Tat conjugation was shown to be more efficient than Lipofectamine in allowing TYMV to enter the mammalian cells. Tat-assisted-transfection was also associated with less loss of cell viability than lipofection. Among the CPPs tested (Tat, R8, Pep-1 and Pen), it was observed that R8 and Pen were also effective while Pep-1 was not. We also examined if the internal space of TYMV can be used to load fluorescein dye as a model cargo. When TYMV is treated by freezing and thawing, the virus is known to convert into a structure with a 6–8 nm hole and release viral RNA. When the resultant pot-like particles were reacted with fluorescein-5-maleimide using interior sulfhydryl groups as conjugation sites, about 145 fluorescein molecules were added per particle. The fluorescein-loaded TYMV particles were conjugated with Tat and introduced into BHK cells, again with higher transfection efficiency compared to lipofection. Our studies demonstrate the potential of modified TYMV as an efficient system for therapeutic cargo delivery to mammalian cells.

In Vitro-Reassembled Plant Virus-Like Particles of Hibiscus Chlorotic Ringspot Virus (HCRSV) as Nano-Protein Cages for Drugs.

Spherical shaped plant viruses require a precise quantity, size, and shape of their coat protein subunits to assemble into virions of identical dimensions. The capsid of spherical plant virus particles typically consists of a precisely shaped protein cage, which in many cases is assembled from identical coat protein subunits. In addition to packaging the viral genome, such protein cages may have the capacity to load foreign compounds, either large molecules (e.g., polymers) or small molecules (e.g., anticancer chemotherapy drugs). Therefore, reassembled protein cages of suitable viruses can serve as carriers for cargo loading, which is what makes them an attractive platform for drug delivery. Here we describe methods to reassemble plant virus-like particles of hibiscus chlorotic ringspot virus (HCRSV) as nano-protein cages including the techniques to purify coat protein, prepare virus-like particles, and load them with foreign compounds.

Stability of Cucumber Necrosis Virus at the Quasi-6-Fold Axis Affects Zoospore Transmission.

Cucumber necrosis virus (CNV) is a member of the genus Tombusvirus and has a monopartite positive-sense RNA genome. CNV is transmitted in nature via zoospores of the fungus Olpidium bornovanus As with other members of the Tombusvirus genus, the CNV capsid swells when exposed to alkaline pH and EDTA. We previously demonstrated that a P73G mutation blocks the virus from zoospore transmission while not significantly affecting replication in plants (K. Kakani, R. Reade, and D. Rochon, J Mol Biol 338:507-517, 2004, https://doi.org/10.1016/j.jmb.2004.03.008). P73 lies immediately adjacent to a putative zinc binding site (M. Li et al., J Virol 87:12166-12175, 2013, https://doi.org/10.1128/JVI.01965-13) that is formed by three icosahedrally related His residues in the N termini of the C subunit at the quasi-6-fold axes. To better understand how this buried residue might affect vector transmission, we determined the cryo-electron microscopy structure of wild-type CNV in the native and swollen state and of the transmission-defective mutant, P73G, under native conditions. With the wild-type CNV, the swollen structure demonstrated the expected expansion of the capsid. However, the zinc binding region at the quasi-6-fold at the β-annulus axes remained intact. By comparison, the zinc binding region of the P73G mutant, even under native conditions, was markedly disordered, suggesting that the β-annulus had been disrupted and that this could destabilize the capsid. This was confirmed with pH and urea denaturation experiments in conjunction with electron microscopy analysis. We suggest that the P73G mutation affects the zinc binding and/or the β-annulus, making it more fragile under neutral/basic pH conditions. This, in turn, may affect zoospore transmission.IMPORTANCECucumber necrosis virus (CNV), a member of the genus Tombusvirus, is transmitted in nature via zoospores of the fungus Olpidium bornovanus While a number of plant viruses are transmitted via insect vectors, little is known at the molecular level as to how the viruses are recognized and transmitted. As with many spherical plant viruses, the CNV capsid swells when exposed to alkaline pH and EDTA. We previously demonstrated that a P73G mutation that lies inside the capsid immediately adjacent to a putative zinc binding site (Li et al., J Virol 87:12166-12175, 2013, https://doi.org/10.1128/JVI.01965-13) blocks the virus from zoospore transmission while not significantly affecting replication in plants (K. Kakani, R. Reade, and D. Rochon, J Mol Biol 338:507-517, 2004, https://doi.org/10.1016/j.jmb.2004.03.008). Here, we show that the P73G mutant is less stable than the wild type, and this appears to be correlated with destabilization of the β-annulus at the icosahedral 3-fold axes. Therefore, the β-annulus appears not to be essential for particle assembly but is necessary for interactions with the transmission vector.

Replication and packaging of Turnip yellow mosaic virus RNA containing Flock house virus RNA1 sequence

Turnip yellow mosaic virus (TYMV) is a spherical plant virus that has a single 6.3 kb positive strand RNA as a genome. In this study, RNA1 sequence of Flock house virus (FHV) was inserted into the TYMV genome to test whether TYMV can accommodate and express another viral entity. In the resulting construct, designated TY-FHV, the FHV RNA1 sequence was expressed as a TYMV subgenomic RNA. Northern analysis of the Nicotiana benthamiana leaves agroinfiltrated with the TY-FHV showed that both genomic and subgenomic FHV RNAs were abundantly produced. This indicates that the FHV RNA1 sequence was correctly expressed and translated to produce a functional FHV replicase. Although these FHV RNAs were not encapsidated, the FHV RNA having a TYMV CP sequence at the 3’-end was efficiently encapsidated. When an eGFP gene was inserted into the B2 ORF of the FHV sequence, a fusion protein of B2-eGFP was produced as expected. [BMB Reports 2014; 47(6): 330-335]

Genome Size Constraint in Replication and Packaging of Turnip Yellow Mosaic Virus

Turnip yellow mosaic virus (TYMV) is a spherical plant virus that has a single 6.3 kb positive strand RNA as a genome. Previously, we have made the recombinant TYMV construct containing a 0.7 kb eGFP gene or a 1.8 kb GUS gene. The genomic RNAs from these constructs were efficiently encapsidated. To examine in more detail whether size constraint exists for replication and packaging of TYMV, we have inserted into the TY-GUS an extra sequence derived from either eGFP or GUS. We also made a recombinant containing RNA1 sequence of Flock house virus. These TYMV recombinants were introduced into Nicotiana benthamiana leaves by agroinfiltration. Northern blot analysis of the viral RNAs in the agroinfiltrated leaves showed that the genomic RNA band from the recombinant TYMV became weaker as longer sequence was inserted. The result also showed that the efficiency of genomic RNA encapsidation decreased sharply when an extra sequence of 2.2 kb or more was inserted. In contrast, the recombinant subgenomic RNA containing an extra sequence of up to 3.2 kb was efficiently encapsidated. Overall, these results show that size constraint exists for replication and encapsidation of TYMV RNA.

Torradoviruses are transmitted in a semi-persistent and stylet-borne manner by three whitefly vectors

Members of the genus Torradovirus (family Secoviridae, type species Tomato torrado virus, ToTV) are spherical plant viruses transmitted by the whitefly species Trialeurodes vaporariorum and Bemisia tabaci. Knowledge on the mode of vector transmission is lacking for torradoviruses. Here, the mode of transmission was determined for Tomato marchitez virus (ToMarV). A minimal acquisition access period (AAP) and inoculation access period (IAP) of approximately 2h each was required for its transmission by T. vaporariorum, while optimal transmission required an AAP and IAP of at least 16h and 8h, respectively. Whiteflies could retain the virus under non-feeding conditions for at least 8h without loss of transmission efficiency, but upon feeding on a non-host plant in between the AAP and IAP they retained the virus for no more than 8h. Similar conditions supported transmission of isolates of ToTV and Tomato chocolàte virus (ToChV) by T. vaporariorum and B. tabaci. Additionally, similar experiments revealed the banded-winged whitefly (Trialeurodes abutilonea) as a vector for all three virus species. The results are congruent with acquisition and retention periods for semi-persistent virus transmission. RT-PCR detection analysis of ToTV and ToMarV in the vector's body revealed their presence in the stylet, but not in the head where the pharynx of the foregut is located. The results altogether indicate a semi-persistent stylet-borne mode of vector transmission for torradoviruses. Additionally, this is the first group of spherical viruses transmitted by at least three different species of whiteflies.

Encapsidation of DNA, a protein and a fluorophore into virus-like particles by the capsid protein of cucumber mosaic virus

An important property of some spherical plant viruses is their ability to reassemble in vitro from native capsid protein (CP) and RNA into infectious virus-like particles (VLPs). Virions of cucumber mosaic virus (CMV) are stabilized by protein-RNA interactions and the nucleic acid is essential for assembly. This study demonstrated that VLPs will form in the presence of both ssDNA and dsDNA oligonucleotides, and with a lower size limit of 20 nt. Based on urea disruption assays, assembled VLPs from CMV CP and RNA (termed ReCMV) exhibited a level of stability similar to that of virions purified from plants, whilst VLPs from CMV CP and a 20mer exhibited comparable or greater stability. Fluorescent labelling of VLPs was achieved by the encapsidation of an Alexa Fluor 488-labelled 45mer oligonucleotide (ReCMV-Alexa488-45) and confirmed by transmission electron and confocal microscopy. Using ssDNA as a nucleating factor, encapsidation of fluorescently labelled streptavidin (53 kDa) conjugated to a biotinylated oligonucleotide was observed. The biological activity and stability of ReCMV and ReCMV-Alexa488-45 was confirmed in infectivity assays and insect vector feeding assays. This work demonstrates the utility of CMV CP as a protein cage for use in the growing repertoire of nanotechnological applications.

Tomato bushy stunt viruses (TBSV) in nanotechnology investigated by scanning force and scanning electron microscopy

Spherical plant viruses like the tomato bushy stunt virus (TBSV) allow for multiple applications in nanotechnology due to their shape. In this article, different types of the virus were created by extending coat protein (CP) at carboxylic termini with 2 different charged amino acids by point mutation. The obtained CPs carried 6 aspartic acid (negative charge) and 4 histamine (positive charge) residues. The ability of TBSV to form self assembled monolayers with large ordered areas on native and chemically modified mica will be presented. The structural differences between layers formed by the wild type and by the genetically modified types will be discussed in detail.

A Sequence in Coat Protein Open Reading Frame Is Required for Turnip Yellow Mosaic Virus Replication

Turnip yellow mosaic virus (TYMV) is a spherical plant virus that has a single 6.3 kb positive strand RNA genome. Information for TYMV replication is limited, except that the 3’-terminal sequence and 5’-untranslated region are required for genome replication. When a foreign sequence was inserted at the position upstream of the coat protein (CP) open reading frame (ORF), replication of the recombinant TYMV was comparable to wild type, as long as an RNAi suppressor was provided. In contrast, when the foreign sequence was inserted between the CP ORF and the 3’-terminal tRNA-like structure, replication of the recombinant virus was not detected. This result suggests that the CP ORF contains an essential replication element which should be appropriately spaced with respect to the 3’-end. Analysis of TYMV constructs containing a part or a full additional CP ORF indicates that the 3’ quarter of the CP ORF is required for TYMV replication.

The Pro/Hel region is indispensable for packaging non-replicating turnip yellow mosaic virus RNA, but not replicating viral RNA.

Turnip yellow mosaic virus (TYMV) is a spherical plant virus that has a single 6.3 kb positive strand RNA. The genomic RNA has a tRNA-like structure (TLS) at the 3'-end. The 3'-TLS and hairpins in the 5'-untranslated region supposedly serve as packaging signals; however, recent studies have shown that they do not play a role in TYMV RNA packaging. In this study, we focused on packaging signals by examining a series of deletion mutants of TYMV. Analysis of encapsidated viral RNA after agroinfiltration of the deletion constructs into Nicotiana benthamiana showed that the mutant RNA lacking the protease (Pro)/helicase (Hel) region was not encapsidated by the coat proteins provided in trans, implicating that a packaging signal lies in the Pro/Hel region. Examination of two Pro(-)Hel(-) mutants showed that protein activity from the Pro/Hel domains was dispensable for the packaging of the non-replicating TYMV RNA. In contrast, the mutant TYMV RNA lacking the Pro/Hel region was efficiently encapsidated when the mutant TYMV was co-introduced with a wild-type TYMV, suggesting that packaging mechanisms might differ depending on whether the virus is replicating or not.

Elastic Properties of Viruses

Viruses are compact biological nanoparticles whose elastic and dynamical properties are hardly known. Inelastic (Brillouin) light scattering was used to characterize these properties, from microcrystals of the Satellite Tobacco Mosaic Virus, a nearly spherical plant virus of 17-nm diameter. Longitudinal sound velocities in wet and dry Satellite Tobacco Mosaic Virus crystals were determined and compared to that of the well-known protein crystal, lysozyme. Localized vibrational modes of the viral particles (i.e., particle modes) were sought in the relevant frequency ranges, as derived assuming the viruses as full free nanospheres. Despite very favorable conditions, regarding virus concentration and expected low damping in dry microcrystals, no firm evidence of virus particle modes could be detected.

The role of inter-subunit ionic interactions in the assembly of Physalis mottle tymovirus

Physalis mottle tymovirus (PhMV) is a small spherical plant virus with its RNA genome encapsidated in a protein shell made of 180 identical coat protein (CP) subunits. The amino acid residues involved in two interfacial salt bridges, Asp-83/Arg-159 and Arg-68/Asp-150 and Lys-153, were targeted for mutagenesis with a view to delineate the role of interfacial ionic interactions in the subunit folding and assembly of the virus. R159A and D83A-R159A recombinant CP (rCP) mutants formed stable T = 3 capsids, indicating that the D83-R159 interfacial salt bridge is dispensable for the folding and assembly of PhMV. However, D150A and R68Q-D150A mutant rCPs were present in the insoluble fraction, suggesting that the R68-D150 interfacial salt bridge is crucial for subunit folding and assembly. Similarly, K153Q, D83A-K153Q, and H69A-K153Q mutant rCPs were present in the insoluble fraction. Interestingly, the R68Q-D150A, D83A-K153Q, and H69A-K153Q double mutant rCPs could be refolded into partially folded soluble heterogeneous aggregates of 14-16 S. The results further confirm our earlier observation that subunit folding and assembly are concerted events in PhMV.

Spherical plant viruses: interactions in solution, phase diagrams and crystallization of brome mosaic virus.

Brome mosaic virus (BMV) is a small icosahedral plant virus of mean diameter 268 A. Interactions between BMV particles in solution were studied by means of small-angle X-ray scattering in order to find crystallization conditions. The interactions between biomacromolecules as large as these viruses have not yet been systematically studied by this method. As it is known that usually proteins crystallize in, or close to, attractive regimes, the interactions between BMV particles in solution were studied as a function of pH, type of salt and size and concentration of polyethylene glycol. An unexpected result of these studies is that the precipitates obtained upon addition of PEG alone or PEG combined with salt were in fact made of microcrystals, which were all characterized by the same series of diffraction peaks, with positions close to those of a centered cubic space group. A phase diagram of the virus as a function of PEG concentration was established by means of microbatch experiments. From the precipitation zones, conditions for crystallization were tested from 5 to 40 mg ml(-1) virus with 3-10%(w/v) PEG 8000 or PEG 20,000. Small crystals were obtained in several conditions after a few days and continued growing for several weeks.

Genome structure of cucumber leaf spot virus: sequence analysis suggests it belongs to a distinct species within the Tombusviridae

The complete nucleotide sequence of cucumber leaf spot virus (CLSV) has been determined and the sizes and locations of predicted viral proteins deduced. The genome consists of 4432 nucleotides and contains five long ORFs. The 5′ proximal ORF encodes a 25 kDa product that terminates in an amber codon which may be readthrough to produce an 84 kDa protein (ORF 2). ORF 3 codes for the 41 kDa coat protein (CP). ORFs 4 and 5 are completely overlapping at the 3′ terminus and code for 27 and 17 kDa products, respectively. The CLSV genome structure is similar to that of tombusviruses and nearly identical to pothos latent virus (PoLV), a newly proposed, atypical, member of the Tombusviridae. It is proposed that CLSV and PoLV be considered strains of a new tombusvirus species. Amino acid sequence comparisons of the CLSV CP and the CPs of several small spherical plant viruses suggest that CLSV is most closely related to melon necrotic spot carmovirus (MNSV), red clover necrotic mosaic dianthovirus (RCNMV) and cucumber necrosis tombusvirus (CNV). These viruses, like CLSV, are transmitted by the soil inhabiting fungus, Olpidium bornovanus.

Containerless protein crystallization in floating drops: application to crystal growth monitoring under reduced nucleation conditions

A micromethod was developed for the batch crystallization of proteins under conditions were the solution has no contact with the container walls. Drops of crystallization solutions (5 to 100 μl) are placed at the interface between two layers of inert and non-miscible silicone fluids contained in square glass or plastic cuvettes. The densities of the fluids are either lower or higher than those of the major precipitating agents of macromolecules, including aqueous solutions containing salts, polyethylene glycols or alcohols. Several proteins and a spherical plant virus were crystallized in the temperature range 4°C–20°C using this set-up. A thermostated device was built for the dynamic control of the temperature of crystallization drops and the monitoring of crystal growth by video-microscopy. In all cases, the habit of the crystals grown in floating drops are identical to those of controls grown in sealed glass tubes without silicone fluid. The comparison of the number of crystals in drops kept under one layer of fluid and in floating drops of the same volume indicates that heterogeneous nucleation is minimized when protein crystallization is performed in floating drops. The advantages and limitations of this novel containerless crystallization method are discussed.

In Vitro Assembly of Cowpea Chlorotic Mottle Virus from Coat Protein Expressed in Escherichia coli and in Vitro-Transcribed Viral cDNA

The small spherical plant virus, cowpea chlorotic mottle virus (CCMV), provides an ideal system to examine spherical virus assembly. We have modified the CCMV in vitro assembly system to produce virions from coat protein expressed in Escherichia coli and viral RNA transcribed in vitro from full-length cDNAs. Examination of the in vitro assembled particles with cryoelectron microscopy and image reconstruction techniques demonstrates that the particles are indistinguishable from plant purified particles at 2.5 nm resolution. Mutational analysis of the coat protein N- and C-terminal extensions demonstrate their respective roles in virus assembly. The N-terminus is required for assembly of RNA containing particles but not for the assembly of empty virions. The C-terminus is essential for coat protein dimer formation and particle assembly.

Degree of linear polarization method for determination of intrinsic optical anisotropy of southern bean mosaic virus

AbstractSouthern bean mosaic virus (SBMV) is a spherical plant virus. It possesses an intrinsic (structural) anisotropy due to the orientation of valence bonds of the amino acid and/or nucleic acid residues. According to the light scattering theory of Rayleigh and Gans for optically anisotropic spheres, the degree of linear polarization of the scattered light depends on the intrinsic anisotropy, the relative refractive index of the sphere to the solvent, the scattering angle, and the inclination angle of linearly polarized incident light to the scattering plane. By using the theoretical expression of the degree of linear polarization, the intrinsic anisotropy parameter of southern bean mosaic virus in the compact spherical state was calculated with the appropriate experimental values. This novel method should be useful in elucidating the internal structure of spherical viruses and other spherical complexes of macromolecules. © 1993 John Wiley & Sons, Inc.

Viruslike particles associated with ringfleck mosaic of mountain ash and a mosaic disease of raspberry in the Bavarian Forest

Two types of spherical viruslike particles measuring ca. 25–30 nm and 80 nm in diameter, respec-tively, were found both in ringfleck mosaic affected mountain ash and adjacent raspberry bushes in the Bavarian Forest. The smaller particles were also found in symptomless mountain ash in the same forest. The sap from infected mountain ash reacted with an antiserum to tomato spotted wilt virus in agar-gel-diffusion test. No reactions were obtained with raspberry or mountain ash sap when tested against antisera to a number of small spherical plant viruses.

Use of Agarose Gel Electrophoresis to Monitor Conformational Changes of Some Small, Spherical Plant Viruses

Several small, spherical plant viruses undergo conformational changes (swell) under conditions that deprotonate carboxyl groups and/or remove divalent cations. In this study, swollen, ribonuclease A susceptible turnip crinkle carmovirus (TCV) and tomato bushy stunt tombusvirus (TBSV) particles migrated more slowly than contracted virions during agarose gel electrophoresis, and swollen TCV virions stained more intensely with ethidium bromide than did contracted virions (...)