rus
Issue #5/2024
A.I.Akhmetova, N.A.Nikitin, M.V.Arkhipenko, O.V.Karpova, I.V.Yaminsky
VISUALIZATION OF TOBACCO MOSAIC VIRUS BY ATOMIC FORCE AND ELECTRON MICROSCOPY
VISUALIZATION OF TOBACCO MOSAIC VIRUS BY ATOMIC FORCE AND ELECTRON MICROSCOPY
DOI: https://doi.org/10.22184/1993-8578.2024.17.5.302.310
For nanoparticle synthesis, viruses have many advantages over other types of biomolecules, as they occur in a wide range of shapes and sizes and have diverse chemical functionalities. It is important that plant viruses are harmless to humans, and therefore are widely used in biotechnology. Tobacco mosaic virus is emerging as an interesting target for use as a nanotemplate and delivery vehicle due to its high aspect ratio, narrow size distribution, diverse biochemical functionalities on the surface, and compatibility through chemical conjugation. In addition, it is quite easy to synthesize tobacco mosaic virus, and its properties can be manipulated through genetic modification or heat treatment.
For nanoparticle synthesis, viruses have many advantages over other types of biomolecules, as they occur in a wide range of shapes and sizes and have diverse chemical functionalities. It is important that plant viruses are harmless to humans, and therefore are widely used in biotechnology. Tobacco mosaic virus is emerging as an interesting target for use as a nanotemplate and delivery vehicle due to its high aspect ratio, narrow size distribution, diverse biochemical functionalities on the surface, and compatibility through chemical conjugation. In addition, it is quite easy to synthesize tobacco mosaic virus, and its properties can be manipulated through genetic modification or heat treatment.
Теги: bionanoscopy physics of living systems scanning probe microscopy tobacco mosaic virus virion бионаноскопия вирион вирус табачной мозаики сканирующая зондовая микроскопия физика живых систем
INTRODUCTION
Plant viruses have different shapes: cowpea chlorotic mottle virus, cowpea mosaic virus and bark mosaic virus form icosahedral structures ranging in size from 18 to 30 nm, while tobacco mosaic virus (TMV) and barley streak mosaic virus have stick-like shapes up to 300 nm [1]. This diversity of shapes makes plant viruses an interesting target as a platform for development of nanomaterials that can be used in biomedicine as a delivery vehicle, in electronics and energy to create catalysts, sensors, battery anodes, and semiconductor digital memory devices.
Viral particles consist of self-assembling capsid proteins and nucleic acids that encode viral proteins. Capsid proteins have diverse biochemical functionalities due to amino acid residues that can interact with metals in solution [2, 3]. These residues can be conjugated with other compounds to synthesise various nanomaterials with new functional properties.
ТМV was the first virus visualised in an electron microscope. Microphotographs of ТМV published by Gustav A. Kausche, Edgar Pfankuch, and Helmut Ruska in 1939 showed individual rod-shaped particles measuring 330 × 15 nanometres [4]. While Francis Crick was completing his doctoral thesis on X-ray diffraction methods for proteins [5], James Watson was trying to determine the ТМV structure [6]. D.Watson describes in a very fascinating way how they unravelled the DNA structure. He wanted to do DNA research, and the study of ТМV was the perfect cover, as the key component of the virus is nucleic acid. The fact that the virus contained RNA and not DNA was even better, since by unravelling the RNA, one could get a clue to the DNA structure. Watson did not fully understand crystallographic theory by his own admission, but guessed that some of the spots in the X-ray diffraction of the ТМV that puzzled John Desmond Bernal and Isidor Funkyuchen could be explained by the fact that the ТМV had a helical configuration.
After obtaining his own X-ray images of ТМV paracrystals, Watson confirmed his conjecture. Based on his new data, he argued that the ТМV is a helix repeating every three turns with a period of 68 Å. He also suggested that the viral RNA is located in the centre of this helix, similar to its location in spherical viruses: turnip yellow mosaic virus and bacteriophage T2 [7]. His paper on ТМV was submitted to the journal Biochimica et Biophysical Acta a week before his famous note with Crick on the double-stranded structure of DNA appeared in Nature [8]. Thus, Watson’s familiarity with the theory of helical diffraction was very helpful in understanding the full significance of Rosalind Franklin’s "Photograph 51".
Currently, tobacco mosaic virus is one of the most well-studied model objects. Atomic force microscopy can be used to obtain new structural data on particles morphology under normal conditions and under changes in ambient temperature [9]. The shape and size of the particles depend on the methods of isolation, purification, sample storage conditions, procedures for depositing the sample on the substrate and substrate nature. It was previously shown that a substrate can affect the virus by causing partial destruction of the protein shell [10].
MATERIALS AND METHODS
Tobacco mosaic virus (TMV), strain U1 from the Department of Virology collection, Moscow State University, was accumulated in tobacco plants (Nicotiana tabacum L.) of the Samsun variety. N. tabacum plants were grown to the stage of 5–6 large leaves formation in a greenhouse with additional lighting (high-pressure sodium lamps) at 22–25 °C. Plants were infected by mechanical inoculation. A suspension of previously isolated and purified virus at a concentration of 50 µg/ml was used as infectious material. In 1–2 weeks after infection, symptoms of systemic plant damage develop: mosaic symptoms in the form of alternating light-green and dark-green areas, often accompanied by the appearance of abnormalities in the form of localised swellings, clearly observed when compared with a healthy control plant. Leaves of infected plants were collected three weeks after infection, packed and frozen (–18 °C). TMV was isolated and purified by differential centrifugation as described previously [11].
Viral precipitate was dissolved in 0.01 M Tris-HCl1 pH 7.8. The TMV solution was clarified by low-speed centrifugation at 10000g for 15 minutes. A 0.05 ml sample was taken to determine TMV concentration and preparation purity.
Quality of the isolated preparation of TMV (presence of impurities, morphology and particle size) was controlled by spectrophotometry, electrophoretic analysis and transmission electron microscopy (TEM). For TEM, the preparation of TMV was sorbed on copper grids for electron microscopy coated with formvar, negatively contrasted with 1% phosphotungstic acid solution and analysed using a Leo 912 electron microscope (Zeiss).
3D morphology of viral particles was studied using FemtoScan SPM in air on graphite and mica substrates in resonance mode, NSG10 cantilever was used, image processing was performed in FemtoScan Online software [12].
RESULTS
Fig.1 shows the TEM image of the virus. The characteristic dimensions of the tobacco mosaic virus are 300 nm in length and 18 nm in height. Both single particles and particles lined up end-to-end with each other, thus forming long string of particles up to 1 µm, are present in the image. Individual particles are rod-shaped.
Figure 2 shows a sample of virus obtained in the resonance mode of AFM. A few microliters of the sample was applied to the surface of freshly pierced mica and dried.
Both 300 nm particles and other lengths, including very small particles of less than 100 nm, are present in the frame. Long particles are also present in the sample when viruses are lined up side by side. The variation in particle size can be explained by separation and purification methods, which results in virus fragments appearance and mica surface segments. By means of AFM it is also possible to determine the character of particle distribution on the surface – viruses do not gather in one place, but tend to spread on the surface. The underestimated height value in AFM is a consequence of interaction with the substrate.
Figure 3 shows a sample of tobacco mosaic virus, where almost all particles tend to line up end-to-end, giving the impression of branching particles. According to the histogram, the characteristic height is 14 nm. The image indicates degradation of the virus protein envelope, as a result, a variation in the cross section of the VTM particles is observed. Due to the loss of mechanical rigidity, the particles loose the rectilinear shape characteristic of intact particles. Partial destruction of the particles is also indicated by the observed underestimated height size of 14.5 nm.
With AFM, detailed characterisation of all objects in the frame can be obtained. FemtoScan Online software can automatically select objects and calculate geometric characteristics of each structure, as well as compare the particle size distribution of two samples:
P – perimeter;
S – square;
V – voilume;
RMS – root mean square value of the object height (roughness);
form factor 1 is the ratio of the radius of a circle of equivalent area to the radius of a circle of equivalent perimeter. For a circular object, this Form Factor 2 is equal to one. The more rugged the perimeter of the object, the closer its value is to zero;
form Factor 2 is the ratio of the doubled length of the object to its perimeter. For a thin thread this ratio is equal to one, for a circle it is equal to zero,
H – maximum object height;
<H> – average height of the object.
The above set of 8 parameters is sufficient to reliably assess the differences in the prepared samples.
From the data in the table, it can be seen that the second sample has much higher values of perimeter, area and volume of particles, while the height of objects and RMS roughness are almost the same.
It is interesting to evaluate the form factor – the particles in the second sample are more prone to form elongated filamentous structures in contrast to the first, which may be a consequence of sample application and drying on the substrate. It is also likely that during storage and preparation of the sample for AFM examination, the particles may break and are less likely to line up one after another on the substrate.
Table 1 shows averaged particle parameters for two cases of sample preparation (sample 1 and 2). The particles of sample 2 are more inclined to an ordered face to face arrangement. This is indicated by the increased values of the parameters P, S and V for the second sample. The proportional increase in the perimeter and volume of the particles by a factor of 2 of the second sample indicates a characteristic organisation one behind the other.
The virus sample in Fig.4 was heated to 90 °C at a concentration of 1 mg/ml for 1 min. There is a significant difference in the shape of particles from the previous two samples: all particles seem to try to merge together not only end-to-end but also side by side, almost no single particles are found. According to the data of geometrical sizes of objects in sample № 3 we can see underestimation of the total height of objects, increase in the volume of objects, while the values of form factors do not differ from sample No.2.
CONCLUSIONS
Studies of TMV have significantly contributed to the basic understanding of viruses, promoted intensive development of molecular genetics and understanding of the nature of infectious diseases. TMV served as a model for studying such human pathogens as influenza and poliomyelitis [13, 14].
Within the framework of this work, data on samples of TMV were obtained, the particles were visualised using AFM and TEM. The geometrical characteristics of particles were obtained from AFM data, and different adsorption patterns of particles on mica substrate were shown depending on the sample, which may be a consequence of sample preparation, application and storage conditions of the TMV sample. A change in particle shape and disposition on mica due to heating up to 90 °C was shown. Using the example of tobacco mosaic virus, the possibilities of using the atomic force microscopy method to determine the morphology and nature of adsorption of viral particles on the substrate by AFM are shown.
ACKNOWLEDGEMENTS
This work was supported within the framework of Interdisciplinary Scientific and Educational Schools of Moscow University, Project No. 23-Sh04-04.
PEER REVIEW INFO
Editorial board thanks the anonymous reviewer(s) for their contribution to the peer review of this work. It is also grateful for their consent to publish papers on the journal’s website and SEL eLibrary eLIBRARY.RU.
Declaration of Competing Interest. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Plant viruses have different shapes: cowpea chlorotic mottle virus, cowpea mosaic virus and bark mosaic virus form icosahedral structures ranging in size from 18 to 30 nm, while tobacco mosaic virus (TMV) and barley streak mosaic virus have stick-like shapes up to 300 nm [1]. This diversity of shapes makes plant viruses an interesting target as a platform for development of nanomaterials that can be used in biomedicine as a delivery vehicle, in electronics and energy to create catalysts, sensors, battery anodes, and semiconductor digital memory devices.
Viral particles consist of self-assembling capsid proteins and nucleic acids that encode viral proteins. Capsid proteins have diverse biochemical functionalities due to amino acid residues that can interact with metals in solution [2, 3]. These residues can be conjugated with other compounds to synthesise various nanomaterials with new functional properties.
ТМV was the first virus visualised in an electron microscope. Microphotographs of ТМV published by Gustav A. Kausche, Edgar Pfankuch, and Helmut Ruska in 1939 showed individual rod-shaped particles measuring 330 × 15 nanometres [4]. While Francis Crick was completing his doctoral thesis on X-ray diffraction methods for proteins [5], James Watson was trying to determine the ТМV structure [6]. D.Watson describes in a very fascinating way how they unravelled the DNA structure. He wanted to do DNA research, and the study of ТМV was the perfect cover, as the key component of the virus is nucleic acid. The fact that the virus contained RNA and not DNA was even better, since by unravelling the RNA, one could get a clue to the DNA structure. Watson did not fully understand crystallographic theory by his own admission, but guessed that some of the spots in the X-ray diffraction of the ТМV that puzzled John Desmond Bernal and Isidor Funkyuchen could be explained by the fact that the ТМV had a helical configuration.
After obtaining his own X-ray images of ТМV paracrystals, Watson confirmed his conjecture. Based on his new data, he argued that the ТМV is a helix repeating every three turns with a period of 68 Å. He also suggested that the viral RNA is located in the centre of this helix, similar to its location in spherical viruses: turnip yellow mosaic virus and bacteriophage T2 [7]. His paper on ТМV was submitted to the journal Biochimica et Biophysical Acta a week before his famous note with Crick on the double-stranded structure of DNA appeared in Nature [8]. Thus, Watson’s familiarity with the theory of helical diffraction was very helpful in understanding the full significance of Rosalind Franklin’s "Photograph 51".
Currently, tobacco mosaic virus is one of the most well-studied model objects. Atomic force microscopy can be used to obtain new structural data on particles morphology under normal conditions and under changes in ambient temperature [9]. The shape and size of the particles depend on the methods of isolation, purification, sample storage conditions, procedures for depositing the sample on the substrate and substrate nature. It was previously shown that a substrate can affect the virus by causing partial destruction of the protein shell [10].
MATERIALS AND METHODS
Tobacco mosaic virus (TMV), strain U1 from the Department of Virology collection, Moscow State University, was accumulated in tobacco plants (Nicotiana tabacum L.) of the Samsun variety. N. tabacum plants were grown to the stage of 5–6 large leaves formation in a greenhouse with additional lighting (high-pressure sodium lamps) at 22–25 °C. Plants were infected by mechanical inoculation. A suspension of previously isolated and purified virus at a concentration of 50 µg/ml was used as infectious material. In 1–2 weeks after infection, symptoms of systemic plant damage develop: mosaic symptoms in the form of alternating light-green and dark-green areas, often accompanied by the appearance of abnormalities in the form of localised swellings, clearly observed when compared with a healthy control plant. Leaves of infected plants were collected three weeks after infection, packed and frozen (–18 °C). TMV was isolated and purified by differential centrifugation as described previously [11].
Viral precipitate was dissolved in 0.01 M Tris-HCl1 pH 7.8. The TMV solution was clarified by low-speed centrifugation at 10000g for 15 minutes. A 0.05 ml sample was taken to determine TMV concentration and preparation purity.
Quality of the isolated preparation of TMV (presence of impurities, morphology and particle size) was controlled by spectrophotometry, electrophoretic analysis and transmission electron microscopy (TEM). For TEM, the preparation of TMV was sorbed on copper grids for electron microscopy coated with formvar, negatively contrasted with 1% phosphotungstic acid solution and analysed using a Leo 912 electron microscope (Zeiss).
3D morphology of viral particles was studied using FemtoScan SPM in air on graphite and mica substrates in resonance mode, NSG10 cantilever was used, image processing was performed in FemtoScan Online software [12].
RESULTS
Fig.1 shows the TEM image of the virus. The characteristic dimensions of the tobacco mosaic virus are 300 nm in length and 18 nm in height. Both single particles and particles lined up end-to-end with each other, thus forming long string of particles up to 1 µm, are present in the image. Individual particles are rod-shaped.
Figure 2 shows a sample of virus obtained in the resonance mode of AFM. A few microliters of the sample was applied to the surface of freshly pierced mica and dried.
Both 300 nm particles and other lengths, including very small particles of less than 100 nm, are present in the frame. Long particles are also present in the sample when viruses are lined up side by side. The variation in particle size can be explained by separation and purification methods, which results in virus fragments appearance and mica surface segments. By means of AFM it is also possible to determine the character of particle distribution on the surface – viruses do not gather in one place, but tend to spread on the surface. The underestimated height value in AFM is a consequence of interaction with the substrate.
Figure 3 shows a sample of tobacco mosaic virus, where almost all particles tend to line up end-to-end, giving the impression of branching particles. According to the histogram, the characteristic height is 14 nm. The image indicates degradation of the virus protein envelope, as a result, a variation in the cross section of the VTM particles is observed. Due to the loss of mechanical rigidity, the particles loose the rectilinear shape characteristic of intact particles. Partial destruction of the particles is also indicated by the observed underestimated height size of 14.5 nm.
With AFM, detailed characterisation of all objects in the frame can be obtained. FemtoScan Online software can automatically select objects and calculate geometric characteristics of each structure, as well as compare the particle size distribution of two samples:
P – perimeter;
S – square;
V – voilume;
RMS – root mean square value of the object height (roughness);
form factor 1 is the ratio of the radius of a circle of equivalent area to the radius of a circle of equivalent perimeter. For a circular object, this Form Factor 2 is equal to one. The more rugged the perimeter of the object, the closer its value is to zero;
form Factor 2 is the ratio of the doubled length of the object to its perimeter. For a thin thread this ratio is equal to one, for a circle it is equal to zero,
H – maximum object height;
<H> – average height of the object.
The above set of 8 parameters is sufficient to reliably assess the differences in the prepared samples.
From the data in the table, it can be seen that the second sample has much higher values of perimeter, area and volume of particles, while the height of objects and RMS roughness are almost the same.
It is interesting to evaluate the form factor – the particles in the second sample are more prone to form elongated filamentous structures in contrast to the first, which may be a consequence of sample application and drying on the substrate. It is also likely that during storage and preparation of the sample for AFM examination, the particles may break and are less likely to line up one after another on the substrate.
Table 1 shows averaged particle parameters for two cases of sample preparation (sample 1 and 2). The particles of sample 2 are more inclined to an ordered face to face arrangement. This is indicated by the increased values of the parameters P, S and V for the second sample. The proportional increase in the perimeter and volume of the particles by a factor of 2 of the second sample indicates a characteristic organisation one behind the other.
The virus sample in Fig.4 was heated to 90 °C at a concentration of 1 mg/ml for 1 min. There is a significant difference in the shape of particles from the previous two samples: all particles seem to try to merge together not only end-to-end but also side by side, almost no single particles are found. According to the data of geometrical sizes of objects in sample № 3 we can see underestimation of the total height of objects, increase in the volume of objects, while the values of form factors do not differ from sample No.2.
CONCLUSIONS
Studies of TMV have significantly contributed to the basic understanding of viruses, promoted intensive development of molecular genetics and understanding of the nature of infectious diseases. TMV served as a model for studying such human pathogens as influenza and poliomyelitis [13, 14].
Within the framework of this work, data on samples of TMV were obtained, the particles were visualised using AFM and TEM. The geometrical characteristics of particles were obtained from AFM data, and different adsorption patterns of particles on mica substrate were shown depending on the sample, which may be a consequence of sample preparation, application and storage conditions of the TMV sample. A change in particle shape and disposition on mica due to heating up to 90 °C was shown. Using the example of tobacco mosaic virus, the possibilities of using the atomic force microscopy method to determine the morphology and nature of adsorption of viral particles on the substrate by AFM are shown.
ACKNOWLEDGEMENTS
This work was supported within the framework of Interdisciplinary Scientific and Educational Schools of Moscow University, Project No. 23-Sh04-04.
PEER REVIEW INFO
Editorial board thanks the anonymous reviewer(s) for their contribution to the peer review of this work. It is also grateful for their consent to publish papers on the journal’s website and SEL eLibrary eLIBRARY.RU.
Declaration of Competing Interest. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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