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The length of the helical nucleocapsid of Newcastle disease virus
VIROLOOY
33 (1967)
DISCUSSION AND PRELIMINARY The Length of the Helical Nucleocapsid Newcastle Disease Virus’
of
REPORTS
were disrupted, and the resulting extracts were fixed with 1% glutaraldehyde, applied Newcastle diseasevirus (NDV) is a mem- to Formvar-coated grids with heavy carbon ber of myxovirus subgoup II (1, g), which films, and stained with 2 % sodium phosphoalso includes mumps and the parainfluenza tungstate. Specimens were examined in a viruses. These viruses possess an outer, Hitachi HS-7S electron microscope. A large number of tightly coiled, long spike-covered lipoprotein envelope and an pieces of nucleocapsid were observed in the internal nucleoprotein component. This incell extracts (Fig. 1). The majority were disternal component, or nucleocapsid, is a tributed over the grid surface singly or in single-stranded helix 160-180 A in diameter, small groups. Occasionally, however, large which has many structural features in common with tobacco mosaic virus (3, 4). massesof intertwining nucleocapsid strands Although NDV nucleocapsid has been iso- were encountered. Such extracellular masses lated from virus particles and from infected probably result from the release by osmotic cells, it has usually been extensively frag- shock of the large cytoplasmic aggregates seen in thin sections of cells infected with mented, and the correct length of the intact myxoviruses of this subgroup (9, 11-13). A structure has not been determined (5-8). small number (
DISCUSSION
AND
PRELIMINARY
those of -2 /I length, could result from end-t,o-end a.ggregation of 1 y pieces. End-toend aggregation has been observed \vith tobacco mosaic virus (Id), and its occurrence with myxovirus nucleocapsids has been sug-
REPORTS
345
gested previously (IO, 15). The incorporation of the XDV nucleocapsid into the viral envelope occurs by a process of budding at the cell membrane. Sometimes large, pleomorphic virus particles are produced, and these
FIG. 1. NDV nucleocapsid released from infected cells by osmotic shock. (A) Tightly coiled pieces of nucleocapsid typical of those measured to determine the distribution of lengths. (B) Portion of a large aggregate of nucleocapsid which probably represents an inclusion after release from cells. (C) Circular and linear forms of nucleocapsid, both measuring approximately 1 p in length. (D) An apparently closed circle with a contour length of 1.07 p. Circular forms are thought to arise from end-to-end aggregation of linear nucleocapsid.
DISCUSSION
346
AND PRELIMINARY
REPORTS
capsids of this subgroup of viruses, a 1 ~1 length of nucleocapsid could reasonably be expected to contain RNA of approximately 7 million molecular weight. The agreement of this value with the size of RNA extracted from NDV (17) supports the conclusion that a 1 p length of nucleocapsid contains one NDV genome.
0:5 Nucleocopsid
115 length(p)
210
ACKNOWLEDGMENTS We are grateful to Dr. George E. Palade for extending to us the use of the electron microscopic facilities of the Cell Biology Laboratory. We thank Miss Cathleen O’Connell and Miss Linda Ferrer for excellent technical assistance.
REFERENCES A. P., Nature 193, 1163 (1962). capsid. W., in “Viral and Rickettsial Infections of Man” (F. L. Horsfall, Jr., and I. Tamm, eds.), p. 685. Lippincott, Philadelprobably contain multiples of the 1 p unit phia, Pennsylvania, 1965. length. 8. HORNE, R. W., and WATERSON, A. P., J. Mol. Since >99 % of the observed NDV nucleoBiol. 2, 75 (1960). capsids were linear, it is likely that this is 4. CHOPPIN, P. W., and STOECKENIUS, W. virthe usual form, rather than a closed circle. ology 23, 195 (1964). 6. SCHAFER, W., and ROTT, R., 2. Naturforsch. End-to-end aggregation, which presumably 14b, 629 (1959). gives rise to long pieces of nucleocapsid, 6. HORNE, R. W., and WATERSON, A. P., J. Mol. could join the two ends of a linear nucleoBiol. 2, 75 (1960). capsid to produce a circle of the type seen 7. WATERSON, A. P., in “Newcastle Disease Vihere. rus, an Evolving Pathogen” (R. P. Hanson, The present finding of an apparent unit ed.), p. 119. Univ. of Wisconsin Press, Madilength of approximately 1.06 cc for the son, 1964. nucleocapsid of NDV is in agreement with 8. ROTT, R., ibid, p. 133. the 1.02 CLlength reported for nucleocapsid 9. COMPANS, R. W., HOLMES, K. V., DALES, S., of the parainfluenza virus SV5 (IO), deterand CHOPPIN, P. W., Birology 30,411 (1966), mined by the method employed here. Previ10. COMPANS, R. W., and CHOPPIN, P. W., Proc. Natl. Acad. Sci. U.S. 57, 949 (1967). ously, Hosaka and co-workers (15) measurH., ing lengths of nucleocapsid within particles il. PROSE, P. H., BALK, S. D., LIEBHADER, and KRUGMAN, S., J. Esptl. Med. 122, 1151 of HVJ, another parainfluenza virus, found (1965). multiples of 1 p and concluded that 1 p was 12. HOWE, C., MORQAN, C., DE V-4ux ST. CYR, C., the minimum length associated with infecHsu, K. C., and ROSE, H. M., J. Virology 1, tive virus. On the basis of measurements on 215 (1967). 3 different members of the NDV-mumps1s. DUC-NGUYEN, H., and ROSENBLUM, E. N., J. parainfluenza subgroup of myxoviruses, it virology 1, 415 (1967). now seemslikely that a ~1 p unit length of 14. FRANCKI, R. I. B., Virology 30, 388 (1966). H., and IKEGUCHI, S., nucleocapsid may apply to all members of 16. HOSAKA, Y., KITANO, ViroZogy 29, 205 (1966). this subgroup. The length of the RNA genome would be 16. ADAMS, W. R., Federation Proc. 24, 159 (1965). expected to determine the unit length of the 17. DUESBERG, P. H., and ROBINSON, W. S., Proc. Natl. Acad. Sci. U.S. 54, 794 (1965). helical nucleocapsid of the virus. Recently, 18. KINQSBURY, D. W., J. Mol. Biol. 18,195 (1966). RNA of high molecular weight has been RICHARD W. COMP~NS FIG.
2.
Length
distribution
of NDV
nucleo-
isolated from NDV (I&18), and its size has been estimated to be 7.5 million daltons (17). We have suggested previously (10) that in view of the structure of the nucleo-
1. WATERSON, 2. HIRST, G.
The RockefellerUniversity New York, Accepted
New July
York 10021 6, 1967
PURNELL
W.
CHOPPIN
33 (1967)
DISCUSSION AND PRELIMINARY The Length of the Helical Nucleocapsid Newcastle Disease Virus’
of
REPORTS
were disrupted, and the resulting extracts were fixed with 1% glutaraldehyde, applied Newcastle diseasevirus (NDV) is a mem- to Formvar-coated grids with heavy carbon ber of myxovirus subgoup II (1, g), which films, and stained with 2 % sodium phosphoalso includes mumps and the parainfluenza tungstate. Specimens were examined in a viruses. These viruses possess an outer, Hitachi HS-7S electron microscope. A large number of tightly coiled, long spike-covered lipoprotein envelope and an pieces of nucleocapsid were observed in the internal nucleoprotein component. This incell extracts (Fig. 1). The majority were disternal component, or nucleocapsid, is a tributed over the grid surface singly or in single-stranded helix 160-180 A in diameter, small groups. Occasionally, however, large which has many structural features in common with tobacco mosaic virus (3, 4). massesof intertwining nucleocapsid strands Although NDV nucleocapsid has been iso- were encountered. Such extracellular masses lated from virus particles and from infected probably result from the release by osmotic cells, it has usually been extensively frag- shock of the large cytoplasmic aggregates seen in thin sections of cells infected with mented, and the correct length of the intact myxoviruses of this subgroup (9, 11-13). A structure has not been determined (5-8). small number (
DISCUSSION
AND
PRELIMINARY
those of -2 /I length, could result from end-t,o-end a.ggregation of 1 y pieces. End-toend aggregation has been observed \vith tobacco mosaic virus (Id), and its occurrence with myxovirus nucleocapsids has been sug-
REPORTS
345
gested previously (IO, 15). The incorporation of the XDV nucleocapsid into the viral envelope occurs by a process of budding at the cell membrane. Sometimes large, pleomorphic virus particles are produced, and these
FIG. 1. NDV nucleocapsid released from infected cells by osmotic shock. (A) Tightly coiled pieces of nucleocapsid typical of those measured to determine the distribution of lengths. (B) Portion of a large aggregate of nucleocapsid which probably represents an inclusion after release from cells. (C) Circular and linear forms of nucleocapsid, both measuring approximately 1 p in length. (D) An apparently closed circle with a contour length of 1.07 p. Circular forms are thought to arise from end-to-end aggregation of linear nucleocapsid.
DISCUSSION
346
AND PRELIMINARY
REPORTS
capsids of this subgroup of viruses, a 1 ~1 length of nucleocapsid could reasonably be expected to contain RNA of approximately 7 million molecular weight. The agreement of this value with the size of RNA extracted from NDV (17) supports the conclusion that a 1 p length of nucleocapsid contains one NDV genome.
0:5 Nucleocopsid
115 length(p)
210
ACKNOWLEDGMENTS We are grateful to Dr. George E. Palade for extending to us the use of the electron microscopic facilities of the Cell Biology Laboratory. We thank Miss Cathleen O’Connell and Miss Linda Ferrer for excellent technical assistance.
REFERENCES A. P., Nature 193, 1163 (1962). capsid. W., in “Viral and Rickettsial Infections of Man” (F. L. Horsfall, Jr., and I. Tamm, eds.), p. 685. Lippincott, Philadelprobably contain multiples of the 1 p unit phia, Pennsylvania, 1965. length. 8. HORNE, R. W., and WATERSON, A. P., J. Mol. Since >99 % of the observed NDV nucleoBiol. 2, 75 (1960). capsids were linear, it is likely that this is 4. CHOPPIN, P. W., and STOECKENIUS, W. virthe usual form, rather than a closed circle. ology 23, 195 (1964). 6. SCHAFER, W., and ROTT, R., 2. Naturforsch. End-to-end aggregation, which presumably 14b, 629 (1959). gives rise to long pieces of nucleocapsid, 6. HORNE, R. W., and WATERSON, A. P., J. Mol. could join the two ends of a linear nucleoBiol. 2, 75 (1960). capsid to produce a circle of the type seen 7. WATERSON, A. P., in “Newcastle Disease Vihere. rus, an Evolving Pathogen” (R. P. Hanson, The present finding of an apparent unit ed.), p. 119. Univ. of Wisconsin Press, Madilength of approximately 1.06 cc for the son, 1964. nucleocapsid of NDV is in agreement with 8. ROTT, R., ibid, p. 133. the 1.02 CLlength reported for nucleocapsid 9. COMPANS, R. W., HOLMES, K. V., DALES, S., of the parainfluenza virus SV5 (IO), deterand CHOPPIN, P. W., Birology 30,411 (1966), mined by the method employed here. Previ10. COMPANS, R. W., and CHOPPIN, P. W., Proc. Natl. Acad. Sci. U.S. 57, 949 (1967). ously, Hosaka and co-workers (15) measurH., ing lengths of nucleocapsid within particles il. PROSE, P. H., BALK, S. D., LIEBHADER, and KRUGMAN, S., J. Esptl. Med. 122, 1151 of HVJ, another parainfluenza virus, found (1965). multiples of 1 p and concluded that 1 p was 12. HOWE, C., MORQAN, C., DE V-4ux ST. CYR, C., the minimum length associated with infecHsu, K. C., and ROSE, H. M., J. Virology 1, tive virus. On the basis of measurements on 215 (1967). 3 different members of the NDV-mumps1s. DUC-NGUYEN, H., and ROSENBLUM, E. N., J. parainfluenza subgroup of myxoviruses, it virology 1, 415 (1967). now seemslikely that a ~1 p unit length of 14. FRANCKI, R. I. B., Virology 30, 388 (1966). H., and IKEGUCHI, S., nucleocapsid may apply to all members of 16. HOSAKA, Y., KITANO, ViroZogy 29, 205 (1966). this subgroup. The length of the RNA genome would be 16. ADAMS, W. R., Federation Proc. 24, 159 (1965). expected to determine the unit length of the 17. DUESBERG, P. H., and ROBINSON, W. S., Proc. Natl. Acad. Sci. U.S. 54, 794 (1965). helical nucleocapsid of the virus. Recently, 18. KINQSBURY, D. W., J. Mol. Biol. 18,195 (1966). RNA of high molecular weight has been RICHARD W. COMP~NS FIG.
2.
Length
distribution
of NDV
nucleo-
isolated from NDV (I&18), and its size has been estimated to be 7.5 million daltons (17). We have suggested previously (10) that in view of the structure of the nucleo-
1. WATERSON, 2. HIRST, G.
The RockefellerUniversity New York, Accepted
New July
York 10021 6, 1967
PURNELL
W.
CHOPPIN