Envelope and nucleoid ultrastructure of Molluscum contagiosum virus

Envelope and nucleoid ultrastructure of Molluscum contagiosum virus

VIROLOGY 83, 120-130 (1977) Envelope and Nucleoid Ultrastructure Virus of Molhscum contagiosum JOH. VREESWIJK,‘,2 G. L. KALSBEEK,’ AND N. NANNI...

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VIROLOGY

83, 120-130 (1977)

Envelope

and Nucleoid

Ultrastructure Virus

of Molhscum

contagiosum

JOH. VREESWIJK,‘,2 G. L. KALSBEEK,’ AND N. NANNINGA With the assistance of J. H. D. Leutscher Laboratory

of Electron

Microscopy,

University

of Amsterdam,

Amsterdam

Accepted June 28,1977 The ultrastructure of the mature and immature poxvirus Molluscum contagiosum has been examined by electron microscopy. With freeze-fracturing one fracture plane was observed in the viral envelope. During the conversion of immature into mature virus, particles inside the envelope become partly rearranged into parallel bands. Depressions in freeze-fractured cores of mature particles suggest the presence of water-rich regions. The organization of the nucleoid was investigated by thin sections analyzed in a tilting rotating specimen stage. The nucleoid consists of fibers with a diameter of approximately 10 nm which are probably connected to each other, thus forming one long fiber. In thin sections four profiles of the nucleoid were observed which were transformed into one another by tilting. Serial sections together with tilting revealed a twist of about 180” between the fibers of the two lobules of the nucleoid. A model for the conformation of the nucleoid in the mature brickstone particles is presented. INTRODUCTION

The Molluscum contagiosum virus (MCV) belongs to the group of poxviruses (Joklik, 1966; Postlethwaite, 1970). So far, the virus has been found only in human epidermal cells. The information available concerning the morphology of MCV points to a close similarity between MCV and other poxviruses such as vaccinia (Postlethwaite, 1970; Dourmashkin and Bernhard, 1959; Vreeswijk et al., 1976, 1977). In contrast to other enveloped viruses, poxviruses assemble their own membranes in so-called “factories” lying in the cytoplasm (Dales and Mosbach, 1968; Stern and Dales, 1974). After the membranes have enclosed part of the viroplasm, virus maturation proceeds in discrete steps. Before the shape of the virus is finally changed into a brickstone, the DNA is folded into a complex structure denoted as core (Morgan, 1976). Detailed ’ Present address: Department of Dermatology, Academic Hospital, Free University, 1117 De Boelelaan, Amsterdam, The Netherlands. 2 Send reprint request to Dr. Joh. Vreeswijk at the address given in footnote 1.

information on the core and on the packing of the DNA are lacking because of the small dimensions of the virus, and because different fixation procedures have different effects on the internal conformation of the virus. Hyde and Peters (1970,1971), for instance, observed tubular structures in the core after various fixation procedures. They suggested that the nucleoid is folded into an S-shaped rod. The visualization of DNA architecture in several other viruses has also been reported (for example, Brown et al., 1975, Mattern et al., 1974). So far one main purpose has been to elucidate the process of DNA packing. In particular the proteins involved in the condensation process have drawn attention (Cremisi et al., 1976). During viral maturation, alteration of the viral envelope coincides with alteration of DNA packing (Morgan, 1976). Therefore, the analysis of the architecture of poxvirus DNA, the largest viral DNA with a molecular weight of approximately 1.4 x lOa (Geshelin and Berns, 19741, might contribute to understanding the process of viral maturation.

120 Copyright 0 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.

ISSN 0042-6622

ULTRASTRUCTURE

In this report we describe the ultrastructure of the freeze-fractured envelope of immature and mature MCV. Furthermore the conformation of the DNA structure in the core has been studied. The use of serial sections in combination with a tilting rotating stage (goniometer) led to a model of the nucleoid which might also apply to other poxviruses. Finally the possibility is discussed that the DNA itself is the driving force in the final maturation step. MATERIALS

AND

METHODS

Molluscum contagiosum lesions in various stages were obtained by curettage of children’s skin. (i) Thin sectioning. Lesions were quickly brought into a buffer (phosphatebuffered saline) containing 30% glycerol and then incubated for 1 hr. Thereafter glycerol was substituted by a series of decreasing glycerol concentrations, each step containing l’% acrolein. Postfixation was carried out in 1% 0~0, and in 1% aqueous uranyl acetate. Dehydration in ethanol and embedding in Epon were carried out by means of standard techniques. Series of 40-70 nm sections were cut with an LKB ultrotome and mounted on one-hole grids as described before (Vreeswijk et al., 1977). The sections were stained with 1% aqueous uranyl acetate for 20 min followed by lead citrate according to Venable and Coggeshall (1.965). They were examined in a Philips EM 300 electron microscope equipped with a tilting rotating stage (goniometer) at an accelerating voltage of 80 kV. (ii) Freeze-fracturing. Skin lesions of MCV incubated in 30 or 40% glycerol (1 hr at 4”) were frozen in Freon 22. Fracturing was carried out in a Balzers apparatus (BA 360 M) at -110” and 2~10-~ torr. (iii) Negative and positive staining. Particles were fixed for 1 min in 2% glutaraldehyde in 10 n&f Tris-HCl, pH 7.4, and 0.001 M EDTA, and were stained with aqueous 1% many1 acetate. RESULTS

Organization

of the Viral Envelope

The viroplasmic region in human epidermal cells infected with MCV was stud-

OF MCV

121

ied by freeze-fracturing. It is easily recognized by the presence of arc-shaped viral membranes (Fig. 1). The viroplasm and cross-fractured content of the immature particles have no substructure visible as compared with mature ones (see below). In the cross-fractured envelope evidence is sometimes found for a regular substructure (arrows, Fig. lb). This regularity can also be seen in thin sections (Fig. la). A regular arrangement is not observed in the concave and convex fracture faces of the envelope. This would mean that the envelope fracture is located more internally. This interpretation is also supported by the fact that the convex fracture faces are surrounded by a rather thick ridge (Fig. 1). The convex and concave fracture faces of the immature virus particles are strikingly different from each other. The convex face is closely packed with particles with a diameter of the order of 15 nm (Fig. 1). The concave face carries only few particles with similar dimensions (Fig. 1). It thus appeared that freeze-fractured immature MCV particles are structurally indisvaccinia tinguishable from immature (Easterbrook, 1971, 1972). Mature Virus Mature MCV could be identified by the approximately rectangular outline of certain cross-fractured profiles and by the internal substructure (see below). Freezefracturing produces three types of fracture faces, i.e., in the viral envelope a convex face and its concave counterpart and the face exposed by cross-fracturing the viral content. (i) The convex fracture face. This face (Fig. 2a) closely resembles that of the immature viral structure (Fig. 1). However, at certain orientations of the mature virus in the frozen tissue the particles on the convex face appear to be arranged in parallel bands (Figs. 2f and 2g). (ii) The concave fracture face. This face (Fig. 2d) also resembles that of immature viruses (Fig. 1). Some additional features occur, however, in the concave fracture faces of the mature viral envelope. The particles on the concave face have a filamentous appearance, whereas sometimes

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FIG. 1. Freeze-fractured viroplasmic region (VP) with “growing” membranes and immature virus particles. Some immature particles are cross-fractured (CF); some show the inner fracture face (IFF) and others the outer fracture face (OFF) of the viral envelope. Viroplasm and content of immature virus display a granular appearance. ~75,000. (la) The viroplasm has a fibrillar appearance afier mixed glutaraldehydeosmium fixation. Growing membranes appear to be covered with particles (arrows). ~75,000. (lb) Crossfractured immature virus particles showing repeating units (spicules) on the outer part of the envelope (arrows). x 75,000.

the filaments occur in parallel bands (Fig. 2e). In the latter case the orientation is same as in some convex fracture faces (compare Figs. 2e and 0. It is not known whether the filamentous appearance has been caused by plastic deformation (Dunlop and Robards, 1972). So far, we found evidence for only one fracture plane in the viral envelope. From the viruses with convex fracture faces it can be deduced that the fracture plane is somewhat below the viral exterior, because they are surrounded by a thick ridge.

(iii) Cross-fractured mature MC virus. More information can be obtained from this virus than the immature counterpart (Fig. 1). In the differentiated virus one observes the biconcave core clearly (Figs. 2b and c). It can be seen that the core protrudes from the background, presumably because it is embedded in a waterrich environment. After freezing, the ice would have been sublimated during etching in the freeze-fracture apparatus. If this reasoning is correct, the core itself also contains water-rich regions because of the local depressions that are visible (Figs. 2b

ULTRASTRUCTURE

OF MCV

123

FIG. 2. Freeze-fractured epidermal tissue infected with MCV. (CF) cross fractures; (OFF) outer fracture face; (IFF) inner fracture face. ~75,000. (2a) The inner fracture face (IFF) is covered with particles and surrounded by a thick ridge. (2b and 2~) Cross fractures of mature particles showing the outline of the core. Holes are visible (arrows) in the “figure-eight” structure of the core and between the core and the envelope. For better contrast, (2~) was printed in reverse. (2d and 2e) The filaments of the outer fracture face lie parallel to each. other, presumably perpendicular to the length axis of the virus. (2f and 2g) The particles of the IFF (2a) are often arranged in parallel bands (compare 2e and 0.

and c, arrows). In certain orientations of the core, th.e depressions transform the structure core into a figure-eight-like (Figs. 2b and c). The Organization

of the Viral Nucleoid

The use of‘ acrolein in combination with glycerol resulted in clearly visible electron-dense fibers in the core. Figure 3 represents an example obtained by this procedure. The nucleoid seems to be a long thread which is folded inside the core. Depending on the orientation of the particles, four different profiles of the nucleoid can be distinguished. (i) Profile A. This profile (Fig. 3a),

which has an oval outline, is supposed to represent a vertical plane along the longitudinal axis of the particles as indicated by A in Fig. 3e. Electron-dense fibers run along this axis in a rather narrow core. (ii) Profile B. This profile (Fig. 3b), which has a brickstone shape, is indicated by the horizontal plane B in Fig. 3e. The outline of the core closely resembles that of the whole particle. The electrondense fibers run approximately parallel to each other and along the longitudinal axis of the core. Profiles A and B suggest that the DNA structure in the core forms a single long fiber. (iii) Profile C. This profile (Fig. 3~) has an oval, almost round shape with a figure-

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eight-like structure of the core. Instead of fibers, electron-dense dots are observed which are supposed to represent the DNA structure in cross-sectional profile. Profile C, which lies in a plane perpendicular to the longitudinal axis, is indicated in Fig. 3e as plane C. (iv) Profile D. Although the shape resembles that of profile C (Fig. 3d), the fibers of the nucleoid look like a herring bone. This profile is not shown in Fig. 3e,

AND

NANNINGA

but is believed to be about half-way between profiles B and C. In Fig. 3e a provisional model of the viral nucleoid is presented. This provisional model is meant as an aid for the interpretation of the electron micrographs (see below). Tilting

Experiments

To check further the spatial relation of the four projections indicated in Figs. 3a-

FIG. 3. Thin section of an MCV inclusion body in human epidermal cells. The four types of profiles shown are indicated by the letters A-D. (3a) Plane A, along the longitudinal axis of the virus (A in 3e). (3b) The horizontal plane B with parallel orientation of the nucleoprotein fibres (B in 3e). (3~) Plane C with crosssectional profile of the nucleoprotein fibers. Electron-dense dots are arranged in a figure-eight-like pattern. Long axis perpendicular to the longitudinal axis (C in 3e). (3d) Herringbone appearance ofthe nucleoprotein fibers CD). Long axis perpendicular to the longitudinal axis in (3e). Plane D is not indicated in (3e) (text). (3eI A provisional model of the arrangement of the nucleoprotein fibers in the viral core. The localization of profiles A, B, and C are indicated in the lower drawing. The fibers in the model represent tightly packed coils of DNA, located inside or just below the core shell.

ULTRASTRUCTURE

cl, we have carried out experiments with the tilting rotating specimen stage. Figures 4a-d show the conversion of plane A (A in Fig. 4;~) into plane B (B in Fig. 4a) and vice versa. When starting from plane B (not shown), little change is seen from tilting along the longitudinal axis in either direction. This indicates that the outline of the core in this area is rather constant. In Figs. 5a-d we show the conversion of profile C into D (cf. Fig. 3d) and vice versa. More information can be obtained when tilting is combined with rotation. In Fig. 6, the central particle (projection 2) has been tilted 40” in both directions: along the longitudinal axis (conversion of projection 4 into projection 5, and vice versa) and along the shortest axis (conversion of projection 1 into 3, an.d vice versa). Projection 2 is supposed to ‘be slightly above plane B (Fig. 3e). By tilting along the longitudinal axis two different projections are visible. Projection 4 in Fig. 6 is presumed to be close to plane A (Fig. 3e). Note the S shape of the DNA structure (cf. Fig. la). After tilting in the other direction (projection 5 in Fig. 6) we see an indication for striations in an oblique orientation with respect to the longitudinal axis. The plane of projection 5 is presumed to be slightly below plane B (Fig. 3e). By tilting along the shortest axis we also observed two different projections. Projection 1 (Fig. 6) bears some resemblance to projection 5. Projection 3 has a X shape. The results already suggest that the orientation of the fibers is more complex than was indicated in the provisional model of Fig. 3e. It seems that the DNA structure palssesses a twisted conformation with a crossover point in the center of the particle. Serial Sectioning

and Tilting

To find further evidence for a crossover point, which is thought to be caused by twisting of the fibers, we have made serial sections and we have combined serial sectioning with tilting. To facilitate the interpretation we present with each electron micrograph a rough sketch of the orienta-

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OF MCV

tion of the DNA fibers. (i) Serial sectioning. Sections were cut with a thickness ranging from 40 to 70 nm. An A-type profile (Figs. 3a and e) is shown in Fig. 7. In the center of the nucleoid a constriction is visible (Figs. 7b and c), whereas in the lateral directions a concave (Fig. 7a) or convex structure (Fig. 7d) can be observed. This suggests the presence of an axis of rotation along the intersection of the A and B planes. Serial sections of Ctype profiles (cf. Figs. 3c and e) show a constriction of the figure-eight in the central section (not shown). When moved backwards or forwards the central part of the nucleoid widens again. The central section of Fig. 8 shows a herringbone appearance (cf. Fig. 3d), whereas in Fig. 8b the nucleoid is more flattened. The complicated appearance of the herringbone structure can be better understood if serial sectioning is combined with tilting. (ii) Tilting of serial sections. Figure 9 shows four consecutive sections running approximately parallel to the C plane (cf. Fig. 3e). Tilting of the section in Figs. 9b and c produces a herringbone-like structure (Figs. 9b’ and c’). The structure is supposed to reflect in part the parallel orientation of the nucleoid fibers in the B plane and in part the twisting of the nucleoid (Fig. 9b”). The fibers of the outermost sections of tilted series often appear to converge in opposite directions, which indicates that the nucleoprotein is folded into a spool. We have attempted to integrate the various observations into a model (Figs. 10a and b) which will be discused below. DISCUSSION

The Viral Envelope Immature MCV in epidermal cells reveals a convex inner fracture face (IFF) with many membrane particles and a concave outer fracture face (OFF) with few particles. This particular distribution of particles between the two fracture faces is also typical for many cellular membranes (Branton, 1969). Mature freeze-fractured MCV envelopes are different from those of immature

FIGS. 4-6. (4) Tilting experiments for the localization of profile A. (4a) A schematic presentation of the observations in (4b-d). Tilting was carried out over + or - 36”. For the series in (4b-d), the zero position is indicated in the scheme. (5) Tilting experiments for the localization of the profiles C and D. The zero (5~) and -36” (5d) positions show parallel striations with an oblique orientation. Tilting over +36” produces crosssectioned fibers of profile C. (6) Tilting along the longitudinal axis (4, 5) and along the axis perpendicular to the longitudinal axis (1, 3). The results suggest the occurrence of a twist in the centre of the nucleoid (see text). 126

ULTRASTRUCTURE

OF MCV

127

e

FIGS. 7-8. Serial sections (with schematic interpretations) Sections of a virus particle with profile A in the plane of sectioning. The crossover point of the twisted fibers can be observed in (7~). (8) sections of a virus particle with projection D in the plane of sectioning. The crossover point is observed in (8~) (herringbone).

MCV. The surface layer of regularly arranged spicules visible in cross-fractured immature M:CV has disappeared. Furthermore, in some orientations of the viruses, parallel bands are observed in the convex as well as in the concave fracture faces (Figs. 2e and 0. MCV maturation thus involves not only a change in the overall shape of the virus but also structural alterations in the viral envelope. It is plausible to assume that for the transition from a spherical to an approximately brickstone shape it is a prerequisite that the regular arrangement of the spicules has to be disrupted. On the other hand, the curvature of the developing envelopes in the viroplasm will depend on the occurrence of regularly arranged units (spicules). This is implied in the work of Dales and Mosbach (1968). They found that the specific curvature of the growing envelope does not arise if vaccinia-infected cells are treated with rifampitin. In such1 cells, abberrant viral mem-

branes are formed which lack spicules. Shape and bonding properties of such units can be supposed to generate the curvature of the developing immature virus envelope by self-assembly. The Viral

Nucleoid

In the course of viral maturation the DNA becomes condensed into a biconcave structure in the center of the brickstoneshaped particle (Morgan, 1976). In immature round particles, parallel orientation of the DNA precedes the formation of the biconcave core. During maturation the DNA conformation thus proceeds from a less ordered to a more ordered structure. Nucleoid maturation proceeds concomitantly with alterations in the immature viral envelope. These alterations, however, occur with loss of a regular substructure (the spicules). It therefore seems that an increase of order in the nucleoid is accompanied by a decrease of order in the envelope. Once the core has been formed it

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FIG. 9. Serial sections (with schematic interpretations) combined with tilting. Tilting of profile C (9b) over -36” results in a herringbone structure (9b’l while tilting over +36” renders the crossover point more easily visible in (9b”).

is a very stable structure. This was clearly observed after Brij-DOC treatment followed by Nonidet-P40 degradation of the envelope. Vaccinia and MCV cores prepared in this way (Figs. 1Oc and d) possess a dumbbell shape with an electron-lucent area located in either lobule. They closely resemble cores of negatively stained BrijDOC-treated particles (Fig. 10e) not subjected to degradation. From freeze-fracture photographs it was deduced that the inner compartment of the core and the lateral parts represent areas with high water content (Figs. 2b and c). This might imply that the well-defined translucent spheres seen in Fig. 10e are

water-rich structures enveloped by a barrier which prevents penetration of the negative stain. The barrier could be partly composed of tightly packed nucleoprotein. In the present study the use of glycerol and acrolein followed by postfixation in osmium tetroxide and uranyl acetate resulted in the preservation of regularly arranged fibers in the core. Although fixation is likely to induce a variable fiber thickness, we believe that the arrangement observed in the profiles shown in Fig. 3 is related to the original conformation and represents a basic feature. Our results confirm and extend the studies carried out on vaccinia (Peters and Muller,

FIG. 10. (lOa) A final model of the architecture of the nucleoid. The fiber was folded into a spool with eight turns. The spool was fixed on two points and twisted over 160”. In this way a dumbbell was generated. (lob) A side view of the spool. Note the “holes” in both lobules of the dumbbell. (10~ and d) Isolated cores as observed after incubation with Brij-DOC followed by NP40 and glycerol. Stained with uranyl acetate. In both lobules of the core translucent areas are visible. ~160,000. (lOe1 Negatively stained Brij-DOC-treated particle. In the core translucent spheres are present and probably represent areas in which the stain cannot penetrate. ~160,000.

19631, fowlpox (Hyde and Peters, 1970, 19711 and Amsacta poxvirus (Bergoin and Dales, 1971) which suggested the presence of a substructure in the core. The existence of a substructure in the core of MCV was recognized by Peters and Kiiper (1970) who employed different fixation procedures. The observations presented in Fig. 3 have led us to compose a provisional model (Fig. 3e) of the fiber arrangement; 16 fibers were counted in type C profiles (by counting electron-dense dots), which might correspond to a fiber length of about 2-4.5 pm. The fiber is therefore supposed to represent tightly coiled DNA. It is clear, however, that the nucleoid

has a more complex architecture. The observations obtained from tilting experiments combined with rotation (Fig. 6) and from serial sectioning combined with tilting (Figs. 7-9) suggest that the nucleoid is twisted (Figs. 10a and b). In the model a spool was twisted over 180”. The spool is composed of one continuous fiber with a length 16 times the length of the core (see above). A side view photograph of the model is shown in Fig. lob. We feel that this model explains the parallel striations seen in Fig. 6, projection 5 (Fig. lOa, small arrows), as well as the Sshaped structure (Fig. lOa, large arrow). It also explains the depressions of the dumbbell and the electronlucent areas observed

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in negatively stained cores (compare Figs. lob. with 1Oc and d). Hyde and Peters (1971) showed that the immature nucleoid contains fiber sheets which might represent spooled DNA fibers. Modeling of these fibers seems to impose further maturation events. The architecture of the nucleoid, as presented in this paper, suggests that the dumbbell has been generated by twisting of spooled fibers. As has been remarked before (Morgan, 19761, the gradual transition into the brickstone mature particle was never observed in thin sections. With the present results in hand, it it tempting to speculate about this transition. In the final maturation step the DNA fibers could have been collapsed into a tighter conformation, possibly comparable to extensive supercoiling of circular duplex DNA. An excess of water is probably withdrawn from the DNA during this process (as has been inferred from freeze-fracturing). The collapse of the DNA is accompanied by a change of the immature round particle into the mature brickstone particle. A prerequisite for this process seems to be the disappearance of spicules. At present it is not clear which factor(s) cause their disappearance. ACKNOWLEDGMENTS The fiber model of the virus was designed by Mr. J. H. D. Leutscher who also made the drawings of the tilting diagrams. We thank Mrs. A. R. Wierdsma for help with the English text and Mrs. J. Raphael-Snyer for the photographic work. Thanks are also extended to Mr. P. J. Barends for technical assistance. REFERENCES M., and DALES, S. (1971). Comparative observations on poxviruses of invertebrates and vertebrates, In Comparative Virology’ (K. Maramorosch and E. Kurstak, eds.), pp. 169-205. Academic Press, New York. BRANTON, D. (1969). Membrane structure. Annu. Rev. Plant Physiol. 20, 209-233. BROWN, D. T., WESTPHAL, M., BURLINGHAM, B. T., WINTERHOFF, U., and DOERFLER, W. (1975). Structure and composition of the adenovirus type 2 core. J. Virol. 16, 366-387. CREMISI, C., PIGNATTI, P. F., CROISSANT, O., and YANIV, M. (1976). Chromatin-like structures in polyoma virus and simian virus 40 lytic cycle. J. Virol. 17, 204-211. BERGOIN,

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S., and MOSBACH, E. H. (1968). Vaccinia as a model of membrane biogenesis. Virology 35, 564583. DOIJRMASHKIN, R., and BERNHARD, W. J. (1959). A study with the electron microscope of the skin tumour ofMolluscum contagiosum. J. Ultrastruct. Res. 3, 11-38. DUNLOP, F. W., and ROBARDS, A. W. (1972). Some artifacts of the freeze-etching technique. J. Ultrastruct. Res. 40, 391-400. EASTERBROOK, K. B., and ROZEE, K. R. (1971). The intracellular development of vaccinia as observed in freeze-etched preparations. Canad. J. Microbiol. 17, 753-757. EASTERBROOK, K. B. (1972). Stereomicrography of freeze-etched poxvirus. J. Microsc. 15, 13-20. GESHELIN, P., and BERNS, K. I. (1974). Characterization and localization of the naturally occurring cross-links in vaccinia virus DNA. J. Mol. Biol. 88, 785-796. HYDE, J. M., and PETERS, D. (1970). Organization of the fowlpox virus nucleoid. In ‘Septieme Congrees International de Microscopic Electronique, Grenoble,’ pp. 291-292. HYDE, J. M., and PETERS, D. (1971). The organization of nucleoprotein within fowlpox virus. J. Ultrastruct. Res. 35, 626-641. JOKLIK, W. K. (1966). The poxviruses. Bacterial Reu. 30, 33-66. MATTERN, C. F. T., HRUSKA, J. G., and DIAMOND, L. S. (1974). Viruses of Entumoeba histolytica; V. Ultrastructure of the polyhedral virus V,,,. J. Virol. 13, 247-249. MORGAN, C. (1976). Vaccinia virus reexamined: Development and release. Virology 73, 43-58. PETERS, D., and MILLER, G. (1963). The fine structure of the DNA-containing core of vaccinia virus. Virology 21, 266-269. PETERS, D., and K~PER, H. (19701. Die Nucleoidstructur des Molluscum contagiosum-Virus wahrend der Reifung. Arch. Gesamte Virusforsch. 31, 137-151. POSTLETHWAITE, R. (1970). Molluscum contagiosum: A review. Arch. Environ. Health 21, 432-452. STERN, W., and DALES, S. (1974). Biogenesis of vaccinia: Concerning the origin of the envelope phospholipids. Virology 62, 293-306. VENABLE, J. H., and COGGESHALL, R. (1965). A simplified lead citrate stain for use in electron microscopy. J. Cell Biol. 25, 407-408. VREESWIJK, JoH., LEENE, W., and KALSBEEK, G. L. (1976). Early interactions of the virus Molluscum contugiosum with its host cell: virus induced alterations in the basal and supra-basal layers of the epidermis. J. Vltrastruct. Res. 54, 37-52. VREESWIJK, JoH., LEENE W., and KAL~BEEK G. L. (1977). Early host-cell-viral interactions: MolZuscum contagiosum interactions with the basal epidermal cells. J. Inuest. Dermatol. 69, in press. DALES,