Morfologia das partículas do vírus do anel do pimentão

Kitajima, E. W.; Oliveira, A. R.; Costa, A. S.

doi:10.1590/S0006-87051969000100001

Resumos

Partículas tubulares, ca. 25 m¼. em diâmetro externo e com um canal axial de 4 m¼ de largura, foram observadas em preparações rápidas ou purificadas e no interior de tecidos de plantas infetadas pelo vírus do anel do pimentão (VAP). Dois tipos de partículas, quanto ao comprimento normal, respectivamente 55 e 200 m¼, foram notadas em preparações in vitro, e com infetividade associada somente às partículas longas. Partículas, de preparações purificadas, quando incubadas com ribo-nuclease ou tripsina, não se alteravam, embora em algumas das experiências a tripsinização houvesse induzido uma agregação terminal das partículas. Quando aquecidas a temperaturas superiores a 65-70°C/10' há, paralelamente à redução da infetividade, uma decomposição dos virions, que se transformam em material pulverulento. Em secções ultra-finas de tecidos infetados foram encontrados agregados ordenados de partículas similares àquelas encontradas in vitro, no citoplasma. Essas inclusões constituiam-se somente de partículas longas (ca. 200 m¼), e a ausência de grupos de partículas curtas características sugere que estas representem fragmentos aproximadamente iguais a 1/4 das longas.

Tubular particles, about 25 m¼ wide, with an axial channel 4 m¼ in diameter, were found in leaf dip or purified preparations and also within tissues, from plants infected with pepper ringspot virus (PRSV), but not in uninoculated control plants. The particle length determinations showed two prevalent types of particles in vitro, respectively with ca. 55 m¼ and 200 m¼ in normal length. These values were independent of the virus isolate, host plant or preparative procedures for electron microscopy employed. In thin section profiles, a dense rim was observed bordering the axial channel, in cross section of particles stained with uranyl acetate, which might represent the location of the viral nucleic acid in the virion. The long, 200 m¼, particles can be separated from the short, 55 m¼ particles through sucrose gradient density ultracentrifugation, when it can be demonstrated that only the longer particles are infective. Incubation of purified preparations with ribonuclease and/or trypsin, did not affect infectivity or the structure of the virions. However, in some experiments, the trypsin treatment induced an end-to-end aggregation of the particles. Purified preparations lose infectivity after being heated for 10 minutes at 65-70°C. Simultaneously a generalized degradation of virions is noticeable. The end product of this degradation is a powdery material, composed of granules 30-40 Aº in diameter. In ultrathin sections of PRSV-infected tissues, ordered aggregates of particles, similar to those found in vitro,were observed in the cytoplasm. Such inclusions consisted of long (200 m¼) particles only. In no instance, groups of typical 55 m¼, short particles were found, and this suggests that they are fragments of the longer, 200 m¼. particles, rather than products of self multiplication.

Morfologia das partículas do vírus do anel do pimentão^* * Parte da tese para doutoramento apresentada pelo autor principal. Parcialmente subvencionada pela Fundação de Amparo à Pesquisa do Estado de São Paulo (C. Agron. 66/107) e pelo Conselho Nacional de Pesquisas (TC 4827).

Morphology of pepper ringspot virus (Brazilian tobacco rattle virus) particles

E. W. Kitajima; A. R. Oliveira; A. S. Costa

Engenheiros-agrônomos, Seção de Virologia, Instituto Agronômico

SINOPSE

Partículas tubulares, ca. 25 mμ. em diâmetro externo e com um canal axial de 4 mμ de largura, foram observadas em preparações rápidas ou purificadas e no interior de tecidos de plantas infetadas pelo vírus do anel do pimentão (VAP). Dois tipos de partículas, quanto ao comprimento normal, respectivamente 55 e 200 mμ, foram notadas em preparações in vitro, e com infetividade associada somente às partículas longas.

Partículas, de preparações purificadas, quando incubadas com ribo-nuclease ou tripsina, não se alteravam, embora em algumas das experiências a tripsinização houvesse induzido uma agregação terminal das partículas. Quando aquecidas a temperaturas superiores a 65-70°C/10' há, paralelamente à redução da infetividade, uma decomposição dos virions, que se transformam em material pulverulento.

Em secções ultra-finas de tecidos infetados foram encontrados agregados ordenados de partículas similares àquelas encontradas in vitro, no citoplasma. Essas inclusões constituiam-se somente de partículas longas (ca. 200 mμ), e a ausência de grupos de partículas curtas características sugere que estas representem fragmentos aproximadamente iguais a 1/4 das longas.

SUMMARY

Tubular particles, about 25 mμ wide, with an axial channel 4 mμ in diameter, were found in leaf dip or purified preparations and also within tissues, from plants infected with pepper ringspot virus (PRSV), but not in uninoculated control plants. The particle length determinations showed two prevalent types of particles in vitro, respectively with ca. 55 mμ and 200 mμ in normal length. These values were independent of the virus isolate, host plant or preparative procedures for electron microscopy employed. In thin section profiles, a dense rim was observed bordering the axial channel, in cross section of particles stained with uranyl acetate, which might represent the location of the viral nucleic acid in the virion. The long, 200 mμ, particles can be separated from the short, 55 mμ particles through sucrose gradient density ultracentrifugation, when it can be demonstrated that only the longer particles are infective.

Incubation of purified preparations with ribonuclease and/or trypsin, did not affect infectivity or the structure of the virions. However, in some experiments, the trypsin treatment induced an end-to-end aggregation of the particles.

Purified preparations lose infectivity after being heated for 10 minutes at 65-70°C. Simultaneously a generalized degradation of virions is noticeable. The end product of this degradation is a powdery material, composed of granules 30-40 Aº in diameter.

In ultrathin sections of PRSV-infected tissues, ordered aggregates of particles, similar to those found in vitro,were observed in the cytoplasm. Such inclusions consisted of long (200 mμ) particles only. In no instance, groups of typical 55 mμ, short particles were found, and this suggests that they are fragments of the longer, 200 mμ. particles, rather than products of self multiplication.

Texto completo disponível apenas em PDF.

Full text available only in PDF format.

LITERATURA CITADA

Recebida para publicação em 7 de outubro de 1967.

1. BRANDES, J. Eine elektronenmikroskopische Schnellmethode zum Nachweis fäden- und stäbchenförmiger Viren, insbesonders in Kartoffeldunkelkeimen. NachrBl. dtsch. PflSchDienst (Braunschweig) 9:151-152, 1957.
2. ________& WETTER, C. Classification of elongated plant viruses on the basis of particle morphology. Virology 8:99-115, 1959.
3. ________; PHILLIPE, M. R. & THORNBERRY, H. H. Electron microscopy of particles associated with soil-borne wheat mosaic. Phytopath. Z. 50:181-190, 1964.
4. BRENNER, S. & HORNE, R. W. A negative staining method for high resolution electron microscopy of viruses. Biochim. Biophys. Acta 34:103-110, 1959.
5. CADMAN, C. H. Evidence for association of tobacco rattle virus nucleic acid with a cell component. Nature 193:49-52, 1962.
6. ________& HARRISON, B. D. Studies on the properties of soil-borne viruses of the tobacco rattle type occurring in Scotland. Ann. Appl. Biol. 47:542-556, 1959.
7. COMMONER, B. Linear biosynthesis of tobacco mosaic virus: development and test of a model. Proc. Natl. Acad. Sci., U.S. 48:2076-2085, 1962.
8. COSTA, A. S.; KITAJIMA, E. W. & OLIVEIRA, A. R. Virus do anel do pimentăo: um integrante do grupo do virus do "rattle" do fumo. (Dados năo publicados)
9. DE ZOETEN, G. A. California tobacco rattle virus, its intracellular appearance and the cytology of the infected cells. Phytopathology 56:744-754, 1966.
10. FINCH, T. Resolution of the substructure of tobacco mosaic virus in the electron microscope. J. Mol. Biol. 8:872-874, 1964.
11. ________. Preliminary X-ray diffraction studies of tobacco rattle and barley stripe mosaic viruses. J. Mol. Biol. 12:612-619, 1965.
12. FROST, R. R.; HARRISON, B. D. & WOODS, R. D. Apparent symbiotic interaction between particles of tobacco rattle virus. J. Gen. Virol. 1:57-70, 1967.
13. HARRISON, B. D. & NIXON, H. L. Separation and properties of particles of tobacco rattle virus with different length. J. Gen. Microbiol. 21:569-581, 1959.
14. HARRISON, B. D. & WOODS, R. D. Serotypes and particle dimensions of tobacco rattle virus from Europe to America. Virology 28:610-620, 1966.
15. HART, R. G. Morphological changes accompanying thermal denaturation of tobacco mosaic virus. Biochim. Biophys. Acta. 30:388-389, 1956.
16. HOLMES, K. C. & FRANKLIN, R. The radial distribution in some strains of tobacco mosaic virus. Virology 6:328-336, 1958.
17. HORNE, R. W. & WILDY, P. Virus structure revealed by negative staining. Adv. Virus Res. 10:101-170, 1963.
18. ________; RUSSEL, G. E. & TRIM, A. R. High resolution electron microscopy of beet yellows virus filaments. J. Mol. Biol. 1:234-236, 1959.
19. HUXLEY, H. E. & ZUBAY, G. Preferential staining of nucleic acid containing structures for electron microscopy. J. Biophys. Biochem. Cytol. 11:273-296, 1961.
20. KARNOVSKI, M. J. Simple methods for staining with lead at high pH in electron microscopy. J. Biophys. Biochem. Cytol. 11:729-732, 1961.
21. KITAJIMA, E. W. A rapid method to detect particles of some spherical plant viruses in fresh preparations. J. Electron-microscopy (Tokyo) 14:119-121, 1965.
22. ________; SILVA, D. M.; OLIVEIRA, A. R.; MÜLLER, G. W. & COSTA, A. S. Electron microscope investigations on tristeza. In: Proc. 3rd. Conf. Int. Org. Citrus Virologists, 1965. p. 1-9.
23. KLIMENTKO, S. M.; UVAROV, V. N. & GUAJDAMOVICH, S. J. Spatial arrangement of ribonucleoprotein strand of vesicular stomatitis virus. In: Proc. 6th Int. Conf. Electron Microscopy (Kyoto), 1966. v. 2. p. 183-184.
24. KÖHLER, E. Versuch einer Deutung der Partikellángen pflanzlicher Virusarten. Naturwissenschaften 43:230-231, 1956.
25. LISTER, R. M. Possible relationship of virus-specific products of tobacco rattle virus infection. Virology 28:350-353, 1966.
26. LUFT, J. H. Improvements in epoxy resin embedding methods. J. Biophys. Biochem. Cytol. 9:409-414, 1961.
27. MATTERN, C. F. T. Electron microscopic observations of tobacco mosaic virus structure. Virology 17:76-83, 1962.
28. MILLONIG, G. Studio sui fattori che determinano la preservazione della ultrastruttura. In: From molecule to cell-Symposium on electron microscopy. Buff a, P., ed. Roma, C.N.R., 1964. p. 347-362.
29. NIXON, H. L. & HARRISON, B. D. Electron microscopic evidence on the structure of the particles of tobacco rattle virus. J. Gen. Microbiol. 21:582-590, 1959.
30. OFFORD, R. E. Electron microscopic investigations on the structure of the particles of tobacco rattle virus. J. Mol. Biol. 17:370-375, 1966.
31. OLIVEIRA, A. R. Serologia aplicada ao estudo do virus do anel do pimentăo. (Tese para doutoramento, E.S.A. "Luiz de Queiroz", Piracicaba, 1967) 40fls.
32. REYNOLDS, E. S. The use of lead citrate at high pH as an electron opaque stain in electron microscopy. J. Cell Biol. 17:208-211, 1963.
33. SÄNGER, H. L. & BRANDENBURG, E. über die Gewinnung von infektiősen Pressaft aus "Wintertype" Pflanzen des Tabak-Rattle-Virus durch Phenol Extraktionen. Naturwissenschften 48:391, 1961.
34. SILVA, D. M. Estudos sôbre purificaçăo, composiçăo e algumas propriedades do vírus do anel do pimentăo. (Tese para livre docęncia, E.S.A. "Luiz de Queiroz", Piracicaba, 1965) 106fls.
35. SIMPSON, R. W. & HAUSER, R. W. Structural components of vesicular stomatitis virus. Virology 29:654-667, 1966.
36. TAKAHASHI, W.N. The effect of the high pH on the linear aggregation of the tobacco mosaic virus. Am. J. Bot. 36:642-645, 1949.
37. WATSON, M. L. Straining of tissue sections for electron microscopy with heavy metals. J. Biophys. Biochem. Cytol. 4:475-478, 1958.

*

Parte da tese para doutoramento apresentada pelo autor principal. Parcialmente subvencionada pela Fundação de Amparo à Pesquisa do Estado de São Paulo (C. Agron. 66/107) e pelo Conselho Nacional de Pesquisas (TC 4827).

Datas de Publicação

Publicação nesta coleção
24 Mar 2009
Data do Fascículo
Jan 1969

Histórico

Recebido
07 Out 1967

This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.

[1] 1. BRANDES, J. Eine elektronenmikroskopische Schnellmethode zum Nachweis fäden- und stäbchenförmiger Viren, insbesonders in Kartoffeldunkelkeimen. NachrBl. dtsch. PflSchDienst (Braunschweig) 9:151-152, 1957.

[2] 2. ________& WETTER, C. Classification of elongated plant viruses on the basis of particle morphology. Virology 8:99-115, 1959.

[3] 3. ________; PHILLIPE, M. R. & THORNBERRY, H. H. Electron microscopy of particles associated with soil-borne wheat mosaic. Phytopath. Z. 50:181-190, 1964.

[4] 4. BRENNER, S. & HORNE, R. W. A negative staining method for high resolution electron microscopy of viruses. Biochim. Biophys. Acta 34:103-110, 1959.

[5] 5. CADMAN, C. H. Evidence for association of tobacco rattle virus nucleic acid with a cell component. Nature 193:49-52, 1962.

[6] 6. ________& HARRISON, B. D. Studies on the properties of soil-borne viruses of the tobacco rattle type occurring in Scotland. Ann. Appl. Biol. 47:542-556, 1959.

[7] 7. COMMONER, B. Linear biosynthesis of tobacco mosaic virus: development and test of a model. Proc. Natl. Acad. Sci., U.S. 48:2076-2085, 1962.

[8] 8. COSTA, A. S.; KITAJIMA, E. W. & OLIVEIRA, A. R. Virus do anel do pimentăo: um integrante do grupo do virus do "rattle" do fumo. (Dados năo publicados)

[9] 9. DE ZOETEN, G. A. California tobacco rattle virus, its intracellular appearance and the cytology of the infected cells. Phytopathology 56:744-754, 1966.

[10] 10. FINCH, T. Resolution of the substructure of tobacco mosaic virus in the electron microscope. J. Mol. Biol. 8:872-874, 1964.

[11] 11. ________. Preliminary X-ray diffraction studies of tobacco rattle and barley stripe mosaic viruses. J. Mol. Biol. 12:612-619, 1965.

[12] 12. FROST, R. R.; HARRISON, B. D. & WOODS, R. D. Apparent symbiotic interaction between particles of tobacco rattle virus. J. Gen. Virol. 1:57-70, 1967.

[13] 13. HARRISON, B. D. & NIXON, H. L. Separation and properties of particles of tobacco rattle virus with different length. J. Gen. Microbiol. 21:569-581, 1959.

[14] 14. HARRISON, B. D. & WOODS, R. D. Serotypes and particle dimensions of tobacco rattle virus from Europe to America. Virology 28:610-620, 1966.

[15] 15. HART, R. G. Morphological changes accompanying thermal denaturation of tobacco mosaic virus. Biochim. Biophys. Acta. 30:388-389, 1956.

[16] 16. HOLMES, K. C. & FRANKLIN, R. The radial distribution in some strains of tobacco mosaic virus. Virology 6:328-336, 1958.

[17] 17. HORNE, R. W. & WILDY, P. Virus structure revealed by negative staining. Adv. Virus Res. 10:101-170, 1963.

[18] 18. ________; RUSSEL, G. E. & TRIM, A. R. High resolution electron microscopy of beet yellows virus filaments. J. Mol. Biol. 1:234-236, 1959.

[19] 19. HUXLEY, H. E. & ZUBAY, G. Preferential staining of nucleic acid containing structures for electron microscopy. J. Biophys. Biochem. Cytol. 11:273-296, 1961.

[20] 20. KARNOVSKI, M. J. Simple methods for staining with lead at high pH in electron microscopy. J. Biophys. Biochem. Cytol. 11:729-732, 1961.

[21] 21. KITAJIMA, E. W. A rapid method to detect particles of some spherical plant viruses in fresh preparations. J. Electron-microscopy (Tokyo) 14:119-121, 1965.

[22] 22. ________; SILVA, D. M.; OLIVEIRA, A. R.; MÜLLER, G. W. & COSTA, A. S. Electron microscope investigations on tristeza. In: Proc. 3rd. Conf. Int. Org. Citrus Virologists, 1965. p. 1-9.

[23] 23. KLIMENTKO, S. M.; UVAROV, V. N. & GUAJDAMOVICH, S. J. Spatial arrangement of ribonucleoprotein strand of vesicular stomatitis virus. In: Proc. 6th Int. Conf. Electron Microscopy (Kyoto), 1966. v. 2. p. 183-184.

[24] 24. KÖHLER, E. Versuch einer Deutung der Partikellángen pflanzlicher Virusarten. Naturwissenschaften 43:230-231, 1956.

[25] 25. LISTER, R. M. Possible relationship of virus-specific products of tobacco rattle virus infection. Virology 28:350-353, 1966.

[26] 26. LUFT, J. H. Improvements in epoxy resin embedding methods. J. Biophys. Biochem. Cytol. 9:409-414, 1961.

[27] 27. MATTERN, C. F. T. Electron microscopic observations of tobacco mosaic virus structure. Virology 17:76-83, 1962.

[28] 28. MILLONIG, G. Studio sui fattori che determinano la preservazione della ultrastruttura. In: From molecule to cell-Symposium on electron microscopy. Buff a, P., ed. Roma, C.N.R., 1964. p. 347-362.

[29] 29. NIXON, H. L. & HARRISON, B. D. Electron microscopic evidence on the structure of the particles of tobacco rattle virus. J. Gen. Microbiol. 21:582-590, 1959.

[30] 30. OFFORD, R. E. Electron microscopic investigations on the structure of the particles of tobacco rattle virus. J. Mol. Biol. 17:370-375, 1966.

[31] 31. OLIVEIRA, A. R. Serologia aplicada ao estudo do virus do anel do pimentăo. (Tese para doutoramento, E.S.A. "Luiz de Queiroz", Piracicaba, 1967) 40fls.

[32] 32. REYNOLDS, E. S. The use of lead citrate at high pH as an electron opaque stain in electron microscopy. J. Cell Biol. 17:208-211, 1963.

[33] 33. SÄNGER, H. L. & BRANDENBURG, E. über die Gewinnung von infektiősen Pressaft aus "Wintertype" Pflanzen des Tabak-Rattle-Virus durch Phenol Extraktionen. Naturwissenschften 48:391, 1961.

[34] 34. SILVA, D. M. Estudos sôbre purificaçăo, composiçăo e algumas propriedades do vírus do anel do pimentăo. (Tese para livre docęncia, E.S.A. "Luiz de Queiroz", Piracicaba, 1965) 106fls.

[35] 35. SIMPSON, R. W. & HAUSER, R. W. Structural components of vesicular stomatitis virus. Virology 29:654-667, 1966.

[36] 36. TAKAHASHI, W.N. The effect of the high pH on the linear aggregation of the tobacco mosaic virus. Am. J. Bot. 36:642-645, 1949.

[37] 37. WATSON, M. L. Straining of tissue sections for electron microscopy with heavy metals. J. Biophys. Biochem. Cytol. 4:475-478, 1958.