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Relation of the structure of cytoplasmic polyhedrosis virus and the synthesis of its messenger RNA
VIROLOGY
105, 467-479 (1980)
Relation of the Structure of Cytoplasmic Polyhedrosis Virus and the Synthesis of Its Messenger RNA
KAZUMORI YAZAKI*
AND
KIN-ICHIRO MIURAt • 1
* Hepatitis Division, Tokyo Metropolitan Institute of Medical Science, Honkoneagome, Bunk yo-ku, Tokyo, and tNational Institute of Genetics, Mishiana, 411 Japan
11,3,
Accepted May 12, 1980 Cytoplasmic polyhedrosis virus (CPV) was observed under an electron microscope using a combination of staining and shadowing methods . Projections, not only in positions horizontal to the grid but also vertical, were clearly visualized . When the particle was mildly disrupted with EDTA, genome dsRNA was released from a projection . If the prticle was previously fixed with glutaraldehyde and then disrupted, dsRNA was released with a protein particle, which seemed to correspond to the base part of the projection . The protein particle was in most eases at the end of the strand, which sometimes takes a supercoiled structure . When CPV was incubated during mRNA synthesis, the protein particles appeared in various positions along the strands, and loop formations of dsRNA appeared . From these observations, we suggest that transcription in this virus particle proceeds as follows : genome dsRNA is transcribed by passing through the base part of the projection, where the enzymes for mRNA synthesis are located . A completed mRNA is released from the virion at the projection . INTRODUCTION
Cytoplasmic polyhedrosis virus (CPV) from the silkworm is an icosahedral particle with a projection or spike at each vertex (Asai et at ., 1972) . The virus contains 10 segments of double-stranded RNA (dsRNA) as its genome (Miura et al ., 1968 ; FujiiKawata et al ., 1970) . It contains RNA polymerase to synthesize its own mRNAs endogenously (Lewandowski et al ., 1969 ; Shimotohno and Miura, 1973a) . Shimotohno and Miura (1973b) showed that transcription of every segment starts at the same rate . After the transcription has been carried out for the whole length of each genome segment, single-stranded mRNAs are released from the particle . The molar ratios of the newly synthesized mRNA species are different as in the case of sheep blue tongue virus, which also contains 10 segments of double-stranded RNA as a genome and endogenous RNA polymerase (Huismans and Verwoerd, 1973) . Huismans and Verwoerd (1973) pointed out the presence of an en' Author to whom reprint requests should be addressed . 467
dogenous regulatory mechanism of mRNA synthesis in the virion . In reovirus, Bartlett et al. (1974) suggested that the nascent mRNA is released from the functioning core particle through the projections . If this is the case, some regulatory mechanism for mRNA synthesis may be involved in the structure of the spike or projection of the particle . We have investigated the structural relations between the genome and the organization of the virus particle, especially of the projection, by electron microscopic observation, comparing the states in the mature virus with those in the acting virus that is synthesizing mRNA . MATERIALS AND METHODS
Purification of virus . CPV was prepared from the midgut of diseased fifth-instar silkworms, Bombyx mori (L), as described by Miura et al . (1968) . Virus particles were purified by centrifugation in a CsCI density gradient and suspended in 10 mM Tris-HC1 buffer, pH 7.5, containing 0 .1 M NaCl . The final preparation was stored at 4° until use . Electron microscopy . Grids with parlodion 0042-6822/80/120467-12$02.00/0 Copyright ® 1'980 by Academic Presr, Inc . All rights of reproduction in any form reserved_
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film (Mallinckrodt) were prepared as described by Hirsh and Schleif (1976) . After evaporating carbon onto the parlodion surface, ion coating was performed with a Giko ion coater, IB-3, at 80 mTorr for 15 sec . To visualize the virus and nucleic acid, a combination of staining and shadowing was carried out as described by Bartlett et at . (1974) with slight modifications . Virus mounted onto the grid was stained with 0.25% uranyl acetate for 10 see, and shadowed with platinum-palladium on a rotary table at an angle 2to3°at2 x 10 6 TorrinaJEOL, JEE-4C evaporator . The grids were examined under an electron microscope, JEOL 1005, at 80 kV . Destruction of virus . The virus was destroyed on a grid as described by Yamagishi et al . (1973), with modifications . Virus was mounted on a grid by floating the grid on the surface of a drop of virus suspension for 5 min . Then, grids were floated on a drop of 0 .02M Tris-HCI buffer, pH 8.2, 0 .02 M EDTA solution, sample side down, and incubated at 37° for 20 min . To fix the virus before mounting onto the grid, virus was treated with 0 .125% glutaraldehyde in 10 mM Tris-HC1 buffer, pH 7 .5, 0.1 M NaCl for 5 min . mRNA synthesis . mRNA synthesis with the virus particle was carried out as described by Furuichi (1974) . After 15 min incubation, the virus was fixed with glutaraldehyde and mounted on a grid, followed by washing, three times, with 10 mM Tris-HC1 buffer, pH 7 .5 . The grids were stained and shadowed . Some grids were treated with EDTA solution to partially destroy the virus particles on the grid .
vertically were not seen so clearly by the usual negative staining of the virus . With negative staining and six-times rotation-photography, Miura et al . (1969) showed that the icosahedral head was 60 nm in diameter and the projection spikes were 20 nor in length, supporting the observation by Hosaka and Aizawa (1964) . Asai et al . (1972) observed that each projection carries a small spherical particle at the tip, when a fresh preparation of the virus was used . However, with the present method, consisting of staining and shadowing, each projection was observed to be a spherical blob rather than a long spike . The tip of a long spike may be easily taken off under the present conditions . The diameter of the icosahedral virion was found to be 68 nm and that of the projection to be 23 nor. There was a slight constriction at the attachment site of a spherical projection to the icosahedron . Partial Destruction
When the virus was immobilized on a grid, EDTA treatment caused mild disruption of virus particles . Then, genome dsRNA of the virus was released from the virion . When the disruption was mild, release of genome dsRNA from one of the projections was observed (Fig . 2). When the virus was disrupted more extensively by longer exposure to EDTA, genome dsRNA was released from the virion in a more compact form (Fig. 3) . When the virus was disrupted after glutaraldehyde fixation, the released genome dsRNA frequently carried a "knobshaped" protein particle (Fig . 4). The knob must be composed of proteins of a part of the virus particle, because the virus prepRESULTS aration used in this experiment was extremely purified by centrifugation in cesium Native Virus Particles chloride density gradient solution repeatCPV having projections at each end of edly. In most cases a protein particle was the 12 vertices of the icosahedral particles attached to the edge of a strand . According was visualized clearly with a combination to the stage of disruption, one (Figs. 4 a-c) of staining and shadowing as shown in Fig . or several strands (Figs . 4 d-f) were re1 . The shape is just like a 12-starred pyro- leased from a virion . The size of the knobtechnic mine . Not only were projections shaped protein particle was 23 nm in diamin positions horizontal to the surface of the eter on average, which just agrees with that grid seen, but also some in vertical posi- of a spherical projection of the virus . A tions . This is the first demonstration of a strand seems to be released from a vertical 12-starred pyrotechnic mine-shaped CPV projection of the icosahedron . Thus it is sugparticle, for the projections protruding gested that under the experimental condi-
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FIG . L A CPV particle visualized by a combination of staining and shadowing . Spherical projections are clearly seen . In (b), (c), and (d), three projections are in vertical positions, and in (e) one projection (center) is in the same state . In (a) a CPV particle with projections in both the positions, vertical and horizontal, is seen . The bar in (a) indicates 50 Ion . (b-e) are the same magnification as (a) .
Lions the proteins constructing the projection are released from the virion as they attach to dsRNA . In Fig . 5, the strand released from one virion (arrow) shows the supereoiled part of dsRNA and its loosened part . The appearance
of all other strands seen in Figs . 4 and 5 resembles just the supercoiled part, which is indicated by an arrow in Fig . 5. Therefore, the protein knob (projection) is attached to the end of two dsRNAs in the supercoiled state .
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FiG. 2. Mild disruption of CPV by EDTA . Genome dsRNA is released from one of the vertices of a particle . A bar indicates 50 nm. The disruption method is described in the text . A Particle Synthesizing mRNA
When CPV was incubated in medium adequate for mRNA synthesis, swelling or deformation of projections was observed as shown in Fig . 6. Figure 6 shows the case
min incubation . Incubation for as long as 6 hr, during which mRNA synthesis continues linearly (Shimotohno and Miura, 1973a ; Furuichi, 1974), gave similar features to Fig. 6, although a photograph is not shown here . After glutaraldehyde fixaof 15
VIRUS STRUCTURE AND MESSENGER RNA SYNTHESIS
FIG . 3 . Disruption of CPV particles . Genome dsRNA is released from a particle in the compact form (a) . During the release, a part of the compact form of the genome is loosened to show the dsRN A strand (h) . A bar indicates 5(1 run .
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FIG. 4. Release of dsRNA with a protein particle from a CPV . CPV was fixed with glutaraldehyde before disruption- dsRNAs seem to be released from vertices of the icosahedral particle. One (a-c) or several strands (d-f) are released . In most cases, a protein particle is attached to the edge of a strand . A bar indicates 50 nm .
VIRUS STRUCTURE AND MESSENGER RNA SYNTHESIS
FIG . 4 .-Continued .
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FIG . 5 . Released dsRNA with or without a protein particle . dsRNA is in the supercoiled form in some parts . The arrow indicates the switching place between the supercoiled form and its loosened form . The bar indicates 100 nm .
tion, the virus was disrupted by EDTA treatment . Genome dsRNA was released from a virion carrying a protein particle . In this case, protein particles were attached not only to the edge of a strand, but also at various positions along the strands (Fig. 7) . Loops of dsRNA were frequently seen (Figs. 7c-e) . DISCUSSION
Genome dsRNA of CPV is obtained as 10 segments, when the nucleic acid is extracted with phenol (Fujii-Kawata et al ., 1970) . On the other hand, Kavenoff et al . (1973) observed genome-sized open circular dsRNA under an electron microscope after Sarkosyl-Pronase treatment of the virus . By similar treatment or urea treatment of reovirus, which also contains 10 dsRNA segments as the genome, Kavenoff et al . (1975) and Dunnebacke and Kleinschmidt (1967) also extracted genome-sized RNAs
of compact spider-like shapes . But the organized structure of these genome dsRNAs in a virion still remains obscure, because with the treatments mentioned above, the structure of a virion was disrupted completely, and the extracted genomes were observed . In the present work, CPV was destructed mildly and directly on a grid . The structural changes of the genome with the experimental processes must be smaller than those observed in extracted genomes . As shown in Fig. 5, supercoillng of dsRNA carrying a protein particle was observed . This may be the first step in packaging of the genome into a CPV virion . Then, the genome would take the highly compact structure in a virion as seen in Fig . 3 . The supercoiled nature of double-stranded nucleic acid occurs naturally in closed circular DNAs such as various plasmids in Eseherichia coli . Worcel and Benyajati (1977) pointed out that the superhelix structure of doublestranded DNA was one of the primary struc-
VIRUS STRUCTURE AND MESSENGER RNA SYNTHESIS
4 75
FIG . 6. CPV particles in mRNA synthesis . CPV incubated in the mRNA synthesizing medium was fixed and visualized . Swelling or deformation of the projections was clearly seen . Bars indicate 50 nm.
tures of highly organized chromosomes of Drosophila melanogaster .
The following characteristic structures were shown for CPV with mild treatment : (1) genome dsRNA was released from a projection with mild disruption of the virus ; (2) dsRNA, carrying a protein particle which was thought to be a part of the projection, was seen under glutaraldehyde fixation conditions ; (3) the projection swelled, when the com-
petent virion was incubated in a medium for mRNA synthesis, and protein particles attached at various sites along the released strand, and loop structure of the strand were observed . From these features, the authors suggest the following mechanism for the primary step of mRNA synthesis of CPV. DsRNAs of CPV are thought to exist in a virion in intimate association with some part, probably the base part, of the projection, or with some proteins that are
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structural components of the projection . With mild disruption of the virus with EDTA, the association would cease, and a dsRNA strand is released from a projection as seen in case 1 . When the association is fixed by glutaraldehyde, dsRNA is probably released from a virion carrying a part of a projection as seen in case 2 . In mRNA synthesizing medium, the compact form of genome would be loosened to transcribe the genome. In this situation, the projection seems to swell as seen in case 3 . The projection must contain some elementary enzymes to transcript the genome of CPV as its structural component . Genome dsRNA exists in a virion keeping in close association with the special part of its core . In the initial stage of mRNA synthesis, loosening of the compact form of genome or unwinding of the supercoil of dsRNA would occur . As shown in Fig . 7, a protein particle attached to the genome that is believed to be a part of a projection was found at various positions along a dsRNA strand and loops of various lengths were observed . In the process of mRNA synthesis in CPV, the virus swells or its shape is loosened, but it does not change shape nor is the capsid decoated (Figs . 6, 7) . Based on this observation the authors imagine that the projection does not move on the surface of the particle, but genome dsRNA moves in the particle through a part (probably the base part) of the projection, just as a tape in a tape recorder moves across the head . The part, through which the dsRNA moves, would be constructed of some elementary enzymes that synthesize mRNA . The newly synthesized mRNA strand would be released through the top of the projection of a virion . Miura et al . (1969) pointed out the presence of a pore or "gate" at the attaching site of the projection to the icosahedron . The genome may move through the gate from the icosahedral part of a virion to the projection of vice versa . mRNA would be synthesized during this movement of genome dsRNA. Bartlett et al. (1974) pointed out, on electron microscopic observation, that the synthesized mRNA may be released from the projection of the reacting core particle of reovirus, although sufficient evidence was lacking proving that the observed
materials, which were released from the virus particle, were single-stranded mRNA . In the present experiments (see Figs . 6 and 7) a single-stranded RNA can not be visualized with an electron microscope under the given conditions . The ATP consumption in mRNA synthesis in CPV is larger than that calculated from ATP integrated into synthesized mRNA (Shimotohno, unpublished) . This overconsumption of ATP may be explained as an energy source required to move the genome in a virion . Shimotohno and Miura (1977) found a nucleoside triphosphate phosphohydrolase that releases the y-phosphate from nucleoside triphosphate in the CPV virion . Based on the fact that triphosphates at the 5'-terminus of externally added RNA are not used as substrates for the enzyme, the enzyme is thought to be located inside the particle . Although the primary role of this enzyme is probably the preparation of ADP from ATP for the cap component of the 5'-terminus of CPV mRNA (Shimotohno and Miura, 1977), this enzyme may also participate in the genome movement in a virion . In this paper, the authors were able to point out the importance of the base part of a projection of a CPV particle in its mRNA synthesis, and suggested the movement of genome dsRNA in a virion during mRNA synthesis, with the use of modified and newly developed techniques for electron microscopic study . REFERENCES ASAI, T., KAWAMOTO, F., and KAWASE, S. (1972) . On the structure of the cytoplasmic polyhedrosis virus of the silkworm, Bombyx mori . J . lnvertebr . Pathol . 19, 279-280 . BARTLETT, N. N., GILLIES, S. C ., BILLIVANT, S ., and BELLAMY, A. R . (1974) . Electron microscopy study of reovirus reaction cores . J. Virol . 14, 315326. DUNNEBACKE; T. H ., and KLEINSCHMIDT, A. K. (1967) . Ribonucleic acid from reovirus as seen in protein monolayers by electron microscopy . Z . Nor turforsch . 22b, 169-164 . FUJR-KAWATA, I ., MIURA, K ., and FUKE, M . (1970) . Segments of genome of viruses containing doublestranded ribonucleic acid . J- Mol. Biol . 51, 247253 . Fuaulcrl, Y . (1974). "Methylation coupled" transcrip-
VIRUS STRUCTURE AND MESSENGER RNA SYNTHESIS tion by virus-associated transcriptase of cytoplasmic polyhedrosis virus containing double-stranded RNA. Nucleic Acids Res . 1, 809-821 . HIRSCH, J ., and SCRLEIF, R . (1976) . High resolution electron microscopic studies of genetic regulation . J . Mol. Biol . 108, 471-490 . HOSAKA, Y ., and AIZAWA, K . (1964) . The fine structure of the cytoplasmic polyhedrosis virus of the silkworm, Bombyx mori (Linnaeus), J_ Insect Pathos . 6, 53-77 . HUISMANS, H ., and VERWOERD, D . W. (1973) . Control of transcription during the expression of the blue tongue virus genome . Virology 52, 81-88 . KAVENOFF, R ., KLOTZ, L . C ., and ZIMM, B . H . (1973) . On the nature of chromosome-sized DNA molecule . Cold Spring Harbor Syrup . Quant . Biol . 38, 1-8 . KAVENOFF, R ., TALCOVE, D ., and MUDD, J . A . (1975). Genome-sized RNA from reovirus particles . Proc . Nat . Acad . Sci . USA 72, 4317-4321 . LEWANDOWSKI, L . J ., KALMAKOFF, J ., and TANADA, Y . (1969) . Characterization of a ribonucleic acid polymerase activity associated with purified cytoplasmic polyhedrosis virus of the silkworm Boinhyx ram-i . J . Virol . 4, 857-865 . MIURA, K ., Fuju, I ., SAKAKI, T ., FUKE, M ., and KAWASE, S . (1968) . Double-stranded ribonucleic
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acid from cytoplasmic polyhedrosis virus of the silkworm. J . Virol . 2, 1211-1222 . MIURA, K ., FUJII-KAWATA, I ., IWATA, H ., and KAWASE, S . (1969) . Electron-microscopic observation of a cytoplasmic polyhedrosis virus from the silkworm . J . Invertebr. Pathol . 14, 262-265 . SHATKIN, A . J ., SIPE, J . D . . and LOS, P . (1968) . Separation of ten reovirus genome segments by polyacrylamide gel electrophoresis . J. Viral . 2, 986-991 . SHIMOTOHNO, K ., and M1URA, K . (l973a) . Singlestranded RNA synthesis in vitro by the RNA polymerase associated with cytoplasmic polyhedrosis virus containing double-stranded RNA . J. Biochem . 74, 117-125. SHIMOTOHNO, K ., and MIURA, K . (1973b) . Transcription of double-stranded RNA in cytoplasmic polyhedrosis virus in vitro . Virology 53, 283-286 . SHIMOTOHNO, K ., and MIURA, K. (1977) . Nucleoside triphosphate phosphohydrolase associated with cytoplasmic polyhedrosis virus . J . Biochem . . 81, 371-379 . WoRCEL, A ., and BENYAJATI, C . (1977) . Higher order coiling of DNA in chromatin . Cell 12, 83-100 . YAMAGISHI, H ., EGUCHI, G ., MAT5U0, H ., and OZEKT, H . (1973) . Visualization of thermal inactivation in phage lambda and (D80 . Virology 53, 277-280 .
105, 467-479 (1980)
Relation of the Structure of Cytoplasmic Polyhedrosis Virus and the Synthesis of Its Messenger RNA
KAZUMORI YAZAKI*
AND
KIN-ICHIRO MIURAt • 1
* Hepatitis Division, Tokyo Metropolitan Institute of Medical Science, Honkoneagome, Bunk yo-ku, Tokyo, and tNational Institute of Genetics, Mishiana, 411 Japan
11,3,
Accepted May 12, 1980 Cytoplasmic polyhedrosis virus (CPV) was observed under an electron microscope using a combination of staining and shadowing methods . Projections, not only in positions horizontal to the grid but also vertical, were clearly visualized . When the particle was mildly disrupted with EDTA, genome dsRNA was released from a projection . If the prticle was previously fixed with glutaraldehyde and then disrupted, dsRNA was released with a protein particle, which seemed to correspond to the base part of the projection . The protein particle was in most eases at the end of the strand, which sometimes takes a supercoiled structure . When CPV was incubated during mRNA synthesis, the protein particles appeared in various positions along the strands, and loop formations of dsRNA appeared . From these observations, we suggest that transcription in this virus particle proceeds as follows : genome dsRNA is transcribed by passing through the base part of the projection, where the enzymes for mRNA synthesis are located . A completed mRNA is released from the virion at the projection . INTRODUCTION
Cytoplasmic polyhedrosis virus (CPV) from the silkworm is an icosahedral particle with a projection or spike at each vertex (Asai et at ., 1972) . The virus contains 10 segments of double-stranded RNA (dsRNA) as its genome (Miura et al ., 1968 ; FujiiKawata et al ., 1970) . It contains RNA polymerase to synthesize its own mRNAs endogenously (Lewandowski et al ., 1969 ; Shimotohno and Miura, 1973a) . Shimotohno and Miura (1973b) showed that transcription of every segment starts at the same rate . After the transcription has been carried out for the whole length of each genome segment, single-stranded mRNAs are released from the particle . The molar ratios of the newly synthesized mRNA species are different as in the case of sheep blue tongue virus, which also contains 10 segments of double-stranded RNA as a genome and endogenous RNA polymerase (Huismans and Verwoerd, 1973) . Huismans and Verwoerd (1973) pointed out the presence of an en' Author to whom reprint requests should be addressed . 467
dogenous regulatory mechanism of mRNA synthesis in the virion . In reovirus, Bartlett et al. (1974) suggested that the nascent mRNA is released from the functioning core particle through the projections . If this is the case, some regulatory mechanism for mRNA synthesis may be involved in the structure of the spike or projection of the particle . We have investigated the structural relations between the genome and the organization of the virus particle, especially of the projection, by electron microscopic observation, comparing the states in the mature virus with those in the acting virus that is synthesizing mRNA . MATERIALS AND METHODS
Purification of virus . CPV was prepared from the midgut of diseased fifth-instar silkworms, Bombyx mori (L), as described by Miura et al . (1968) . Virus particles were purified by centrifugation in a CsCI density gradient and suspended in 10 mM Tris-HC1 buffer, pH 7.5, containing 0 .1 M NaCl . The final preparation was stored at 4° until use . Electron microscopy . Grids with parlodion 0042-6822/80/120467-12$02.00/0 Copyright ® 1'980 by Academic Presr, Inc . All rights of reproduction in any form reserved_
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film (Mallinckrodt) were prepared as described by Hirsh and Schleif (1976) . After evaporating carbon onto the parlodion surface, ion coating was performed with a Giko ion coater, IB-3, at 80 mTorr for 15 sec . To visualize the virus and nucleic acid, a combination of staining and shadowing was carried out as described by Bartlett et at . (1974) with slight modifications . Virus mounted onto the grid was stained with 0.25% uranyl acetate for 10 see, and shadowed with platinum-palladium on a rotary table at an angle 2to3°at2 x 10 6 TorrinaJEOL, JEE-4C evaporator . The grids were examined under an electron microscope, JEOL 1005, at 80 kV . Destruction of virus . The virus was destroyed on a grid as described by Yamagishi et al . (1973), with modifications . Virus was mounted on a grid by floating the grid on the surface of a drop of virus suspension for 5 min . Then, grids were floated on a drop of 0 .02M Tris-HCI buffer, pH 8.2, 0 .02 M EDTA solution, sample side down, and incubated at 37° for 20 min . To fix the virus before mounting onto the grid, virus was treated with 0 .125% glutaraldehyde in 10 mM Tris-HC1 buffer, pH 7 .5, 0.1 M NaCl for 5 min . mRNA synthesis . mRNA synthesis with the virus particle was carried out as described by Furuichi (1974) . After 15 min incubation, the virus was fixed with glutaraldehyde and mounted on a grid, followed by washing, three times, with 10 mM Tris-HC1 buffer, pH 7 .5 . The grids were stained and shadowed . Some grids were treated with EDTA solution to partially destroy the virus particles on the grid .
vertically were not seen so clearly by the usual negative staining of the virus . With negative staining and six-times rotation-photography, Miura et al . (1969) showed that the icosahedral head was 60 nm in diameter and the projection spikes were 20 nor in length, supporting the observation by Hosaka and Aizawa (1964) . Asai et al . (1972) observed that each projection carries a small spherical particle at the tip, when a fresh preparation of the virus was used . However, with the present method, consisting of staining and shadowing, each projection was observed to be a spherical blob rather than a long spike . The tip of a long spike may be easily taken off under the present conditions . The diameter of the icosahedral virion was found to be 68 nm and that of the projection to be 23 nor. There was a slight constriction at the attachment site of a spherical projection to the icosahedron . Partial Destruction
When the virus was immobilized on a grid, EDTA treatment caused mild disruption of virus particles . Then, genome dsRNA of the virus was released from the virion . When the disruption was mild, release of genome dsRNA from one of the projections was observed (Fig . 2). When the virus was disrupted more extensively by longer exposure to EDTA, genome dsRNA was released from the virion in a more compact form (Fig. 3) . When the virus was disrupted after glutaraldehyde fixation, the released genome dsRNA frequently carried a "knobshaped" protein particle (Fig . 4). The knob must be composed of proteins of a part of the virus particle, because the virus prepRESULTS aration used in this experiment was extremely purified by centrifugation in cesium Native Virus Particles chloride density gradient solution repeatCPV having projections at each end of edly. In most cases a protein particle was the 12 vertices of the icosahedral particles attached to the edge of a strand . According was visualized clearly with a combination to the stage of disruption, one (Figs. 4 a-c) of staining and shadowing as shown in Fig . or several strands (Figs . 4 d-f) were re1 . The shape is just like a 12-starred pyro- leased from a virion . The size of the knobtechnic mine . Not only were projections shaped protein particle was 23 nm in diamin positions horizontal to the surface of the eter on average, which just agrees with that grid seen, but also some in vertical posi- of a spherical projection of the virus . A tions . This is the first demonstration of a strand seems to be released from a vertical 12-starred pyrotechnic mine-shaped CPV projection of the icosahedron . Thus it is sugparticle, for the projections protruding gested that under the experimental condi-
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469
FIG . L A CPV particle visualized by a combination of staining and shadowing . Spherical projections are clearly seen . In (b), (c), and (d), three projections are in vertical positions, and in (e) one projection (center) is in the same state . In (a) a CPV particle with projections in both the positions, vertical and horizontal, is seen . The bar in (a) indicates 50 Ion . (b-e) are the same magnification as (a) .
Lions the proteins constructing the projection are released from the virion as they attach to dsRNA . In Fig . 5, the strand released from one virion (arrow) shows the supereoiled part of dsRNA and its loosened part . The appearance
of all other strands seen in Figs . 4 and 5 resembles just the supercoiled part, which is indicated by an arrow in Fig . 5. Therefore, the protein knob (projection) is attached to the end of two dsRNAs in the supercoiled state .
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FiG. 2. Mild disruption of CPV by EDTA . Genome dsRNA is released from one of the vertices of a particle . A bar indicates 50 nm. The disruption method is described in the text . A Particle Synthesizing mRNA
When CPV was incubated in medium adequate for mRNA synthesis, swelling or deformation of projections was observed as shown in Fig . 6. Figure 6 shows the case
min incubation . Incubation for as long as 6 hr, during which mRNA synthesis continues linearly (Shimotohno and Miura, 1973a ; Furuichi, 1974), gave similar features to Fig. 6, although a photograph is not shown here . After glutaraldehyde fixaof 15
VIRUS STRUCTURE AND MESSENGER RNA SYNTHESIS
FIG . 3 . Disruption of CPV particles . Genome dsRNA is released from a particle in the compact form (a) . During the release, a part of the compact form of the genome is loosened to show the dsRN A strand (h) . A bar indicates 5(1 run .
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FIG. 4. Release of dsRNA with a protein particle from a CPV . CPV was fixed with glutaraldehyde before disruption- dsRNAs seem to be released from vertices of the icosahedral particle. One (a-c) or several strands (d-f) are released . In most cases, a protein particle is attached to the edge of a strand . A bar indicates 50 nm .
VIRUS STRUCTURE AND MESSENGER RNA SYNTHESIS
FIG . 4 .-Continued .
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FIG . 5 . Released dsRNA with or without a protein particle . dsRNA is in the supercoiled form in some parts . The arrow indicates the switching place between the supercoiled form and its loosened form . The bar indicates 100 nm .
tion, the virus was disrupted by EDTA treatment . Genome dsRNA was released from a virion carrying a protein particle . In this case, protein particles were attached not only to the edge of a strand, but also at various positions along the strands (Fig. 7) . Loops of dsRNA were frequently seen (Figs. 7c-e) . DISCUSSION
Genome dsRNA of CPV is obtained as 10 segments, when the nucleic acid is extracted with phenol (Fujii-Kawata et al ., 1970) . On the other hand, Kavenoff et al . (1973) observed genome-sized open circular dsRNA under an electron microscope after Sarkosyl-Pronase treatment of the virus . By similar treatment or urea treatment of reovirus, which also contains 10 dsRNA segments as the genome, Kavenoff et al . (1975) and Dunnebacke and Kleinschmidt (1967) also extracted genome-sized RNAs
of compact spider-like shapes . But the organized structure of these genome dsRNAs in a virion still remains obscure, because with the treatments mentioned above, the structure of a virion was disrupted completely, and the extracted genomes were observed . In the present work, CPV was destructed mildly and directly on a grid . The structural changes of the genome with the experimental processes must be smaller than those observed in extracted genomes . As shown in Fig. 5, supercoillng of dsRNA carrying a protein particle was observed . This may be the first step in packaging of the genome into a CPV virion . Then, the genome would take the highly compact structure in a virion as seen in Fig . 3 . The supercoiled nature of double-stranded nucleic acid occurs naturally in closed circular DNAs such as various plasmids in Eseherichia coli . Worcel and Benyajati (1977) pointed out that the superhelix structure of doublestranded DNA was one of the primary struc-
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4 75
FIG . 6. CPV particles in mRNA synthesis . CPV incubated in the mRNA synthesizing medium was fixed and visualized . Swelling or deformation of the projections was clearly seen . Bars indicate 50 nm.
tures of highly organized chromosomes of Drosophila melanogaster .
The following characteristic structures were shown for CPV with mild treatment : (1) genome dsRNA was released from a projection with mild disruption of the virus ; (2) dsRNA, carrying a protein particle which was thought to be a part of the projection, was seen under glutaraldehyde fixation conditions ; (3) the projection swelled, when the com-
petent virion was incubated in a medium for mRNA synthesis, and protein particles attached at various sites along the released strand, and loop structure of the strand were observed . From these features, the authors suggest the following mechanism for the primary step of mRNA synthesis of CPV. DsRNAs of CPV are thought to exist in a virion in intimate association with some part, probably the base part, of the projection, or with some proteins that are
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structural components of the projection . With mild disruption of the virus with EDTA, the association would cease, and a dsRNA strand is released from a projection as seen in case 1 . When the association is fixed by glutaraldehyde, dsRNA is probably released from a virion carrying a part of a projection as seen in case 2 . In mRNA synthesizing medium, the compact form of genome would be loosened to transcribe the genome. In this situation, the projection seems to swell as seen in case 3 . The projection must contain some elementary enzymes to transcript the genome of CPV as its structural component . Genome dsRNA exists in a virion keeping in close association with the special part of its core . In the initial stage of mRNA synthesis, loosening of the compact form of genome or unwinding of the supercoil of dsRNA would occur . As shown in Fig . 7, a protein particle attached to the genome that is believed to be a part of a projection was found at various positions along a dsRNA strand and loops of various lengths were observed . In the process of mRNA synthesis in CPV, the virus swells or its shape is loosened, but it does not change shape nor is the capsid decoated (Figs . 6, 7) . Based on this observation the authors imagine that the projection does not move on the surface of the particle, but genome dsRNA moves in the particle through a part (probably the base part) of the projection, just as a tape in a tape recorder moves across the head . The part, through which the dsRNA moves, would be constructed of some elementary enzymes that synthesize mRNA . The newly synthesized mRNA strand would be released through the top of the projection of a virion . Miura et al . (1969) pointed out the presence of a pore or "gate" at the attaching site of the projection to the icosahedron . The genome may move through the gate from the icosahedral part of a virion to the projection of vice versa . mRNA would be synthesized during this movement of genome dsRNA. Bartlett et al. (1974) pointed out, on electron microscopic observation, that the synthesized mRNA may be released from the projection of the reacting core particle of reovirus, although sufficient evidence was lacking proving that the observed
materials, which were released from the virus particle, were single-stranded mRNA . In the present experiments (see Figs . 6 and 7) a single-stranded RNA can not be visualized with an electron microscope under the given conditions . The ATP consumption in mRNA synthesis in CPV is larger than that calculated from ATP integrated into synthesized mRNA (Shimotohno, unpublished) . This overconsumption of ATP may be explained as an energy source required to move the genome in a virion . Shimotohno and Miura (1977) found a nucleoside triphosphate phosphohydrolase that releases the y-phosphate from nucleoside triphosphate in the CPV virion . Based on the fact that triphosphates at the 5'-terminus of externally added RNA are not used as substrates for the enzyme, the enzyme is thought to be located inside the particle . Although the primary role of this enzyme is probably the preparation of ADP from ATP for the cap component of the 5'-terminus of CPV mRNA (Shimotohno and Miura, 1977), this enzyme may also participate in the genome movement in a virion . In this paper, the authors were able to point out the importance of the base part of a projection of a CPV particle in its mRNA synthesis, and suggested the movement of genome dsRNA in a virion during mRNA synthesis, with the use of modified and newly developed techniques for electron microscopic study . REFERENCES ASAI, T., KAWAMOTO, F., and KAWASE, S. (1972) . On the structure of the cytoplasmic polyhedrosis virus of the silkworm, Bombyx mori . J . lnvertebr . Pathol . 19, 279-280 . BARTLETT, N. N., GILLIES, S. C ., BILLIVANT, S ., and BELLAMY, A. R . (1974) . Electron microscopy study of reovirus reaction cores . J. Virol . 14, 315326. DUNNEBACKE; T. H ., and KLEINSCHMIDT, A. K. (1967) . Ribonucleic acid from reovirus as seen in protein monolayers by electron microscopy . Z . Nor turforsch . 22b, 169-164 . FUJR-KAWATA, I ., MIURA, K ., and FUKE, M . (1970) . Segments of genome of viruses containing doublestranded ribonucleic acid . J- Mol. Biol . 51, 247253 . Fuaulcrl, Y . (1974). "Methylation coupled" transcrip-
VIRUS STRUCTURE AND MESSENGER RNA SYNTHESIS tion by virus-associated transcriptase of cytoplasmic polyhedrosis virus containing double-stranded RNA. Nucleic Acids Res . 1, 809-821 . HIRSCH, J ., and SCRLEIF, R . (1976) . High resolution electron microscopic studies of genetic regulation . J . Mol. Biol . 108, 471-490 . HOSAKA, Y ., and AIZAWA, K . (1964) . The fine structure of the cytoplasmic polyhedrosis virus of the silkworm, Bombyx mori (Linnaeus), J_ Insect Pathos . 6, 53-77 . HUISMANS, H ., and VERWOERD, D . W. (1973) . Control of transcription during the expression of the blue tongue virus genome . Virology 52, 81-88 . KAVENOFF, R ., KLOTZ, L . C ., and ZIMM, B . H . (1973) . On the nature of chromosome-sized DNA molecule . Cold Spring Harbor Syrup . Quant . Biol . 38, 1-8 . KAVENOFF, R ., TALCOVE, D ., and MUDD, J . A . (1975). Genome-sized RNA from reovirus particles . Proc . Nat . Acad . Sci . USA 72, 4317-4321 . LEWANDOWSKI, L . J ., KALMAKOFF, J ., and TANADA, Y . (1969) . Characterization of a ribonucleic acid polymerase activity associated with purified cytoplasmic polyhedrosis virus of the silkworm Boinhyx ram-i . J . Virol . 4, 857-865 . MIURA, K ., Fuju, I ., SAKAKI, T ., FUKE, M ., and KAWASE, S . (1968) . Double-stranded ribonucleic
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acid from cytoplasmic polyhedrosis virus of the silkworm. J . Virol . 2, 1211-1222 . MIURA, K ., FUJII-KAWATA, I ., IWATA, H ., and KAWASE, S . (1969) . Electron-microscopic observation of a cytoplasmic polyhedrosis virus from the silkworm . J . Invertebr. Pathol . 14, 262-265 . SHATKIN, A . J ., SIPE, J . D . . and LOS, P . (1968) . Separation of ten reovirus genome segments by polyacrylamide gel electrophoresis . J. Viral . 2, 986-991 . SHIMOTOHNO, K ., and M1URA, K . (l973a) . Singlestranded RNA synthesis in vitro by the RNA polymerase associated with cytoplasmic polyhedrosis virus containing double-stranded RNA . J. Biochem . 74, 117-125. SHIMOTOHNO, K ., and MIURA, K . (1973b) . Transcription of double-stranded RNA in cytoplasmic polyhedrosis virus in vitro . Virology 53, 283-286 . SHIMOTOHNO, K ., and MIURA, K. (1977) . Nucleoside triphosphate phosphohydrolase associated with cytoplasmic polyhedrosis virus . J . Biochem . . 81, 371-379 . WoRCEL, A ., and BENYAJATI, C . (1977) . Higher order coiling of DNA in chromatin . Cell 12, 83-100 . YAMAGISHI, H ., EGUCHI, G ., MAT5U0, H ., and OZEKT, H . (1973) . Visualization of thermal inactivation in phage lambda and (D80 . Virology 53, 277-280 .