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Intracellular vesicular stomatitis virus nucleocapsids and virions visualized by surface spreading
Journal of Virological Methods, 23 (1989) 1-12
Elsevier JVM 00809
Intracellular vesicular stomatitis virus nucleocapsids and virions visualized by surface spreading Kazumori Yazaki’, Toshihiko Sane’, Tomoko Okamoto2, Toshiyuki Urushibara2 and Chiaki Nishimura2 ITokyo Metropolitan Institute of Medical Science, Honkomagome, Bunkyo-ku, Tokyo and 2Department of Pharmaceutical Science, Kitasato University, Shirokane, Minato-ku, Tokyo, Japan
(Accepted4 July 1988)
Summary
Spreading of cells on a solution surface could visualize vesicular stomatitis virus nucleocapsids and virions in infected cells easily and clearly without the need for any purification. Characteristic structures observed by the spreading of the infected cells are described and discussed. VSV; Rhabdovirus;
Nucleocapsid;
Virus particle; Surface spreading
Introduction
In electron microscopic studies, ultra-thin sections and replicas of whole and fractured cells have relied on the studies of virus infection. By these methods, however, it is quite difficult to observe the intracellular viral genome, genomeprotein complex and virions as clearly as when observed in the purified state. The spreading of whole cells for electron microscopic observation has been used for the study of chromosomes and the synaptonemal complex (Gall, 1963; DuPraw, 1965; Moses, 1977). We used this method to examine viral nucleocapsids and virions in infected cells, and found it easy to carry out for the morphological study of virus maturation. In the present study, baby hamster kidney (BHK) cells infected with vesicular stomatitis virus (VSV) were examined. Correspondence to: Dr. K. Yazaki, Tokyo komagome, Bunkyo-ku, Tokyo 113, Japan. 0166-0934/89/$03.50
0
1989 Elsevier
Science
Metropolitan
Publishers
Institute
B.V.
of Medical
(Biomedical
Science,
Division)
3-18-22
Hon-
2
VW, a rhabdovirus, is enveloped and about 70 nm in diameter and 180 nm in length (Murphy and Harrison, 1980). It has a cone at one end of its cylindrical structure, thus giving it a bullet-shape appearance. The genome is single-stranded RNA of negative polarity with 11162 nucleotides (Schubert et al., 1984). The RNA forms a nucleocapsid with a major protein N and minor proteins NS and L. The N protein monomers are wedge-shaped, bilobed structures each about 9 nm in length and associate with RNA to form ribbon-shaped nucleocapsids 9 nm in width (Thomas et al., 1985). Each such nucleocapsid is a compact, builet-shaped coil about 50 to 53 nm in diameter with 34 to 35 turns in the virion and has the appearance of an internal skeleton (Simpson and Hauser, 1966; Nakai and Howatson, 1968; Cartwright et al., 1970a,b; Murphy and Harrison, 1980; Newcomb et al., 1982; Thomas et al., 1985). Newcomb and Brown (1981) and Newcomb et al. (1982) found that M protein must be present when an elongated nucIeocapsid coils to take on the bullet-like shape. A nucleocapsid forms a coil 20 nm in diameter in the absence of M at a high salt concentration (Heggeness et al., 1980). Neave et al. (1980) and Neave and Summers (1980) extracted nucleocapsids 20 nm in diameter from infected cells and found them to appear circular and helical and to serve as templates for virus replication. It has not been possible so far to observe differences in their structures which in all cases are 20 nm in diameter. A disc-like complex of N protein and VSV leader RNA has been extracted from cells at the late stage of viral infection (Blumberg et al., 1981; Blumberg and Kolakofsky, 1981). The structures of a nucleocapsid, the disc-like complex and virion mentioned above have been studied using purified and/or chemically treated samples. They were examined in this study by spreading infected cells on the surface of a solution.
Materials and Methods Virus and cells A monolayer culture of BHK cells grown in Eagle’s minimum essential medium supplemented 10% fetal calf serum was infected with VSV (wild-type, New Jersey serotype) at multiplicities of 5, and maintained at 35°C until harvest. At each sampling time, the cells were harvested by EDTA treatment. They were washed once by a hand-wind centrifuge and then resuspended in Dulbecco’s PBS(+) at a concentration adjusted to 2 x lo6 cells/ml. Surface spreading of cells Spreading was carried out according to the methods of Gall (1963), DuPraw (1965) and Moses (1977) with slight modification as follows: Fine talc powder was first sprinkled over the surface of a hypophase (PBS(+) supplemented with 10% sucrose) in a plastic Petri dish 5 or 9 cm in diameter. The cell suspension was taken in a platinum loop which was subsequently brought gently into contact with the hypophase surface and then removed from it. The spread cells appeared as a clear circular expansion free of talc powder. The grid was then brought into contact with
3
the clear surface of the circle. The samples were mounted on films of carbon-coated parlodion (Mallinckrodt, St Louis, MO, U.S.A.). The sample side of the grid was washed with PBS(+) and then distilled water. The samples were negatively stained with freshly prepared 2% uranyl acetate (TAAB), or shadowed with Pt-Pd on a rotary table. The grids were examined in a JEOL 1OOCelectron microscope, at 80 kv.
Results Outline of VSV infection
At 4 h following infection, nucleocapsids were present in abundance and could be seen as the stretched, coiled, or otherwise-shaped structures shown below. At 6 h, over 95% of the cells showed early cytopathic effects, nucleocapsids coiled as bullet-shaped forms and virions were abundantly present. At 8 h, many of the cells were round and easily removable. Attention was directed primarily to nucleocapsids and virions at 4 and 6 h after infection. Serpentine nucleocapsid
A serpentine nucleocapsid of VSV noted 4 h after infection is shown in Fig. la. Its length of 4.05 km was virtually the same as that of nucleocapsids obtained from infected cells (Neave and Summers, 1980), and calculated by Thomas et al. (1985). Ribbon-shaped nucleocapsids 9 nm in width were clearly visualized by the spreading of infected cells as evident in Fig. lb. Nucleocapsid
network
The nucleocapsid networks shown in Fig. 2a (Pt-Pd shadowing) and 2b (negative staining) were found in cells 4 h after infection and each contained a number
Fig. 1. Serpentine nucleocapsid of VSV (negative staining). pears serpentine along its length. (b) The nucleocapsid
(a) A nucleocapsid, 4.05 PITI in length is ribbon-shaped and 9 nm in width.
ap-
Fig. 2. Network of VSV nucleocapsids. (a) A number of serpentine nucleocapsids can be seen. Cytoskeletons with straight portions are indicated by arrows. A poiysome (P) can also be seen (Pt-Pd shadowing). (b) The nucleocapsids and cytoskeletons (arrows) clearly differ in their features. Membranous material (M), poiysomes (P) and disc-like structures (D) are evident (negative staining). (c) The nu~ieocapsids in the network appear situated along fine filaments, as indicated by arrows (Pt-Pd shadowing).
Fig. 3. A nucleocapsid showing the coils 20 nm in diameter and 7 to 9 nm pitch (negative staining).
of serpentine nucleocapsids. Cytoskeletons, distinguishable from nucleocapsids by their straightness (2a and b) and smoothness (2b) were found in all networks (see arrows). Polysomes (P) (2a and b), membranous material (M) and disc-like struc-
Fig. 4. “Double helical” VW nucleocapsids (Pt-Pd shadowing). (a) A nucleocapsid 4.1 urn in length has a branch (B). At D, the strands appear to be coiled around each other to form a double helix. (b) The nucleocapsid shows a double helix on its left half. The serpentine features indicated by arrows indicate the right half to possibly be a tightly coiled structure. (c) A nucleocapsid similar to (b), but note the virion (V) in the same field.
6
Fig. 5. Bullet- and cone-shaped toilings of VSV nucleocapsids (negative staining). (a) Bullet-shaped coiling is clearly evident. A nucleocapsid tail can be seen at the flat end. (b) The bullet-shaped coiling has 18 turns. At the flat end of the cylinder, N protein of the nucleocapsid enters the coil with its long axis directed radially (arrow A). A second coil appears at B. (c) Two bullet shapes, two cones and a circle are present in a nucleocapsid. (d) Seven cones can be seen. The inside of the cone appears empty (indicated by an arrow). (e) Twelve cones are arranged in succession. Al1 apices of the cones point in the same direction,
tures (D, discussed below) (2b) can also be seen. The nuc~eocapsids in the network were often situated along fine filaments (Fig. 2~). The thickness of such a filament was the same as that of one situated between neighbouring ribosomes in a polysome. The latter is certainly a single-stranded mRNA strand, noted in the sample (not shown). But whether the fine filament along which nucleocapsids was situated is a single-stranded RNA or some other cellular component has yet to be determined. Coils
20 nm in diameter
In cells 4 h after infection, some nucleocapsids appeared as partially coiled structures about 20 nm in diameter as shown in Fig. 3 (20 nm-coil) and had the same features as those indicated by Heggeness et al. (1980). Their pitch was from 7 to 9 nm.
7
“Double helical” nucleocapsids
In cells 4 and 6 h following infection, nucleocapsids with coils considerably different from both 20 nm-coils and bullet-shaped coils were observed, although less frequently than the latter two. Fig. 4a shows a nucleocapsid 11 to 12 nm in thickness with a branch (indicated as B). In each region indicated by an arrow (D), two strands appear coiled about each other. The length of the nucleocapsid without a branch was 4.1 pm. On the left half of the nucleocapsid shown in Fig. 4b, two strands each 11 to 12 nm in thickness are coiled around each other. This composite coil is referred to as a “double helix” in this paper to distinguish it from the 20 nm-coils and bullet-shaped coils in each of which a strand runs in one direction. The thickness of the strand comprising the right half of the nucleocapsid was 21 to 22 nm, twice that of the strand on the left. The thickness and serpentine features at the sites shown by arrows indicate the right half to possibly be a tightly coiled version of the left. The length of the nucleocapsid was 2.02 km, one half the full length of a nucleocapsid of VSV (Thomas et al., 1985). It thus appears that the structure in 4b is a full length nucleocapsid folded as a double helix and that in 4a is partially unfolded nucleocapsid with a branch. The double helix and 20 nm-coil in Fig. 3 are about the same in thickness, but differ very much in other respects. Fig. 4c shows a structure similar to those in 4a and b. Note the virion (V) present in the same field. Bullet- and cone-shaped
nucleocapsid
toilings
In cells 6 h after infection, networks of the nucleocapsids could hardly be observed but nucleocapsids and virions could be seen in abundance. Most of the nu-
Fig. 6. Disc-like
structures (negative staining). The structure is 13 nm in diameter with a central 3 to 4 nm in diameter. The same structures are also shown in Fig. 2b.
hole
8
cleocapsids were situated away from each other and variously shaped as shown in Fig. 5. The bullet coil in Fig. 5a possesses 30 turns. Note the tail at the flat end, indicating the entire length of the nucleocapsid to be still uncoiled. The structure in 5b has 18 turns. It is clearly evident that N proteins enter the coil with their long axis directed radially at the end of the cylinder as described by Thomas et al. (1985) (indicated by arrow A). The nucleocapsid extending from the flat end shows a second coil indicated by arrow B. The features indicated by arrows A and B clearly demonstrate coiling of the nucleocapsid to proceed from left to right. A central hole 3 to 4 nm in diameter is evident at the top of the cone which has 9 turns. The nucleocapsids in Fig. 5c-e each possess more than one bullet-shaped coil and cone. In 5c, two bullet-shaped coils, two cones and a circle can be seen. In 5d, seven cones are present in one nucleocapsid. A cone indicated by an arrow is clearly visualized inside of the coiled structure. In 5e, twelve cones are arranged in succes-
Fig. 7. be seen ent. (b) the
VSV particles in cells 6 h after infection (negative staining). (a) Internal skeletons can clearly in the virions. Within the inner cavities of the skeletons, an amorphous structure appears presA virion with a terminal hemisphere at its flat end. The hemisphere appears continuous with inner substance. (c) Radial view of a virion. Three concentric circles are clearly apparent.
9
sion. Note that their apices and bullet-coils all point in the same direction. inner cavities of the coils in Fig. 5 (especially evident in d) appear empty.
The
Disc-like structures
The disc-like structures in Fig. 6 (and Fig. 2b) first became apparent 4 h following infection and were abundant by 6 h. Each disc was about 13 nm in outer diameter and had a central hole 3 to 4 nm in diameter. In each, rod-like molecules were arranged in such a way as to form about twelve to fifteen circular petals. Virus particles
By the present method of spreading, VSV virions were observed to be variously shaped. In Fig. 7a, the internal skeletons of virions are clearly apparent. Each skeleton has as many as 38 turns, this being more than those observed in previous studies (see Introduction). The inner cavities of virion skeletons do not appear empty. An amorphous structure appears to be present. A limited number of virions had round membranous hemispheres at their flat ends (7b). These are the “terminal hemisphere” as described by Orenstein et al. (1976). Each membranous hemisphere appears to extend to and become continuous with the inner cavity of the skeleton. A radial view of the virus is shown in 7c. One striking feature is the three concentric circles within a circular membrane of 10.5 to 12 nm in thickness. The distance between the outer and inner circle is about 10 nm, about the same as the length of the N protein.
Discussion
After cells had been spread over a hypotonic solution or one without Mg++ as in the case of PBS(-), polysomes tended to undergo degradation. The present study was conducted under conditions that would not allow this. By using a Petri dish of smaller size, the extent to which each cell would spread was limited and polysomes, cytoskeletons and membranes could be observed. This condition favored negative staining. In a large dish, each cell would spread more extensively than when using a smaller dish. Cytoskeleton and membranes were scattered about, making observation difficult. However, polysome and helical structures of nucleocapsids could be clearly seen by metal shadowing (Figs. 2a,c, 4). What is seen in Fig. 2 may actually be extended structures of the “virus factory” region where nucleocapsids are crowded together. In this study, it was possible for the first time to observe the structures of this region at high resolution. The networks of nucleocapsids and cytoskeletons may prevent the destruction of the “virus factory” during spreading. Heggeness et al. (1980) found the nucleocapsid to change reversibly from an unwound state to the 20 nm-coil at low and high salt concentrations without the occurrence of M protein binding. Each coil seen in Fig. 3 may have been formed as a result of the highly salty microenvironment surrounding the nucleocapsid. The pitch of the coil strongly implies that the long axis of N protein monomers is not
10
oriented radially in the coil, but runs parallel or nearly so to its length. At 4 h after infection, hardly any bullet-shaped coils could be seen. Thus, the 20 nm-coils observed at 4 h after infection are quite possibly nucleocapsids not yet bound to M protein. This binding occurred by 6 h to give rise to bullet and cone shapes. At the sites of bullet-shaped coils in the cells, M protein has been detected only on nucleocapsids, not on membranes (Odenwald et al., 1986). Spreading may allow the coils to stretch again to various degrees. The cytoskeletons and membranes surrounding and suspending the nucleocapsids were scattered, thus possibly freeing the nucleocapsids. Autonomous coiling may start from various points in a nucleocapsid to produce the cone and bullet shape in association with bound M protein. The coiled structures shown in Fig. 5 may arise in this manner. But various characteristics of nucleocapsids can be discerned from the toilings, such as: (i) there is a central hole about 3 to 4 nm in diameter at the top of the cone; (ii) each cone has 9 turns, and coiling becomes cylindrical from the tenth turn from the top and (iii) coil direction shows polarity. The apices of the cones and bullets in a nucleocapsid were noted to always point in the same direction. In no case were two cones connected at their apices or base planes, nor did any cone point in a direction opposite to that of the others. Most of the nucleocapsids at 6 h had the structures shown in Fig. 5, or were in virions as internal skeletons. But double helical nucleocapsids were also found, although in limited number, as is especially evident from Fig. 4c. A replicative nucleocapsid of VSV is a circular helical structure (Neave and Summers, 1980), and nascent RNA also has the form of a nucleocapsid (Patton et al., 1983, 1984). The nucleocapsids in Fig. 4 are quite possibly circular, and the structure in Fig. 4a is a replicative intermediate of the virus, while those in Fig. 4b and c are compact forms of the template. No double helical nucleocapsids showed bullet- or cone-shaped coiling, possibly being unable to do so even after M protein binding or possibly because this binding failed to occur. In no case did a nucleocapsid coil into a bullet shape. If such bullet-shaped coiling were a pre-requisite for a nucleocapsid to form a mature virion, it would be impossible for a double helical nucleocapsid to form a virion. Although full length nucleocapsids containing RNAs of positive and negative polarity have been found in infected cells, only those with negative polarity have been detected in the virus (Roy et al., 1973; Wagner, 1975). The nucleocapsid having RNA of plus polarity, the template for replication, may be sorted out from the viral maturation process by differences in secondary structure. The disc-like structure in Figs. 2b and 6 appeared identical to the 18s disc-like complex (18s complex) extracted from cells at a late stage of infection (Blumberg and Kolakofsky, 1981), and was also found in abundance in cells at late stages of infection. It is thus quite likely that this structure is also the 18s complex of which the function may be regulation of transcription and translation of the virus (Blumberg and Kolakofsky, 1981). Prosomes, of which the function may be post-transcriptional regulation, have been isolated from various animal and plant cells (&humid et al., 1984; Schuldt and Kloetzel, 1985; Arrigo et al., 1985; Horsch et al., 1985; Martins de Sa et al., 1986; Kremp et al., 1986). A prosome consists of single-stranded RNA of 40 to 110 nucleotides and about ten kinds of protein. RNA
11
and proteins differ in the particular prosomes they possess, depending on the cells from which they are isolated. Purified prosomes, however, all have in common a circular disc 13 nm in outer diameter and a central hole 3 to 4 nm in diameter, and sediment at 18 or 19s. The morphology of a prosome appears exactly the same as that of the 18s complex and the structures in Fig. 6. From the close correlation of function with morphology, the 18s complex may be considered a prosome characteristically appearing in cells at a late stage of infection. Its constituents come from the virus. There is also the possibility that some of the disc-like structures in Fig. 6 (and Fig. 2b) are prosomes comprised of cellular derivatives, or are hybrids of the viral and cellular derivatives. According to the previous papers, nucleocapsids must interact with M protein and membranes to take on the compact complete bullet shape (Dubovi and Wagner, 1977; Zakowski and Wagner, 1980; Newcomb and Brown, 1981; Newcomb et al., 1982). What appears to be an amorphous structure observed at the inner cavity of the skeleton in the virus (Fig. 7), possibly part of a membrane, may be essential for preventing the formation of irregularly-shaped coils such as those in Fig. 5. Of the three concentric circles observed in a radial view of the virus (Fig. 7c), the outer and inner circles possibly derive from circularly arranged bilobed N protein monomers. The central region of the nucleocapsid becomes clearly visible as the central circle. The central regions of the nucleocapsids in Figs. 1, 3 and 5 do not appear as the lines shown in Fig. 7d. Some conformational change may occur in the region. The present method of spreading infected cells on a solution surface holds promise as a simple means for morphologically studying virus infection, making diagnosis and detecting new viruses. This method is now being applied in conjugation with immunological techniques.
Acknowledgement
The authors are grateful to Dr. Kin-ichiro Miura, University of Tokyo, for his helpful discussion. References Arrigo, A.-P., Darlix, J.-L., Khandjian, E.W., Simon, M. and Spahr, P.-F. (1985) Characterization of the prosome from Drosophila and its similarity to the cytoplasmic structures formed by the low molecular weight heat-shock proteins. Eur. Mol. Biol. Organ. J. 4, 399-406. Blumberg, B.M., Leppert, M. and Kolakofsky, D. (1981) Interaction of vsv leader RNA and nucleocapsid protein may control vsv genome replication. Cell 23, 837-845. Blumberg, B.M. and Kolakofsky, D. (1981) Intracellular vesicular stomatitis virus leader RNAs are found in nucleocapsid structures. J. Virol. 40, 568-576. Cartwright, B., Smale, C.J. and Brown, F. (1970a) Dissection of vesicular stomatitis virus into the infective ribonucleoprotein and immunizing components. J. Gen. Viral. 7, 19-32. Cartwright, B., Talbot, P. and Brown, F. (1970b) The proteins of biochemically active sub-units of vesicular stomatitis virus. J. Gen. Virol. 7, 267-272. Dubovi, E.J. and Wagner, R.R. (1977) Spatial relationships of the proteins of vesicular stomatitis vi-
12 FUS:induction of reversible oligomers by cleavable protein cross-linkers and oxidation. J. Viral. 22, 500-509. DuPraw, E.J. (1965) The organization of nuclei and chromosomes in honeybee embryonic cells. Proc. Natl. Acad. Sci. USA 53, 161-168. Gall, J. (1963) Chromosome fibers from an interphase nucleus. Science 139, 120-121. Heggeness, M.H., Scheid, A. and Choppin, P.W. (1980) Conformation of the helical nucleocapsids of paramyxoviruses and vesicular stomatitis virus: reversible coiling and uncoiling induced by changing in salt concentration. Proc. Natl. Acad. Sci. USA 77, 2631-2635. Hotsch, A., Kohler, K. and Schmid, H.-P. (2985) Prosomes are involved in the repression of viral mRNA. Z. Naturforsch. 4Oc, 449-450. Kremp, A., Schliephacke, M.. Kull, U. and Schmid, H.-P. (1986) Prosome exists in plant cells too. Exp. Cell Res. 166. 553-557. Martins de Sa, C., Grossi de Sa, M.-F.. Akhayat, O., Broders, F., Scherrer. K.. Horsch, A. and Schmid. H.-P. (1986) Prosome ubiquity and inter-species structural variations. J. Mol. Biol. 187, 479-493. Moses, M.J. (197’7) Synaptonemal complex karyotyping in spermato~ytes of the Chinese hamster (Cricetulus griseus). Chromosoma 60, 99-125. Murphy, F.A. and Harrison, A.K. (1980) Electron microscopy of the rhabdoviruses of animals. In: D.H.L. Bishop (Ed.), Rhabdoviruses. Vol. 1. pp. 65-106. CRC Press Inc., Boca Raton, FL, USA. Nakai, T. and Howatson, A.F. (1968) The fine structure of vesicular stomatitis virus. Virology 35. 268-281. Neave, C.W. and Summers, D.F. (1980) Electron microscopy of vesicular stomatitis virus ribonucleoprotein. J. Virol. 34, 764-771. Neave, C.W., Kolakofsky, CM. and Summers, D.F. (1980) Comparison of vesicular stomatitis virus intracellular and virion ribonucleoproteins. J. Virol. 33, 856-865. Newcomb, W.W. and Brown, J.C. (1981) Role of the vesicular stomatitis virus matrix protein in maintaining the viral nucleocapsid in the condensed form found in native virions. J. Virol. 39, 295-299. Newcomb, W.W.. Tobin, G.J.. McGowan, J.J. and Brown, J.C. (1982) In vitro reassembly of vesicular stomatitis virus skeletons. J. Viroi. 41, 1055-1062. Odenwald. W.F., Amheiter, H., Dubois-Dalcq, M. and Lazzarini, R.A. (1986) Stereo images of vesicular stomatitis virus assembly. J. Virol. 57. 922-932. Orenstein, J., Johnson, L., Shelton, E. and Larzarini, R.A. (1976) The shape of vesicular stomatit~s virus. Virology 71, 291-301. Patton, J.T., Davis, N.L. and Wertz. G.W. (1983) Cell-free synthesis and assembly of vesicular stomatitis virus nucleocapsid. J. Viral. 45, 155-164. Patton, J.T., Davis, N.L. and Wertz. G.W. (1984) N protein alone satisfies the requirement for protein synthesis during RNA replication of vesicular stoma&is virus. J. Virol. 49, 303-309. Roy, P., Repik, P., Hefti, E. and Bishop. B.H.L. (1973) Complementary RNA species isalated from vesicular stomatitis virus (HR) strain defective virions. J. Viral. 11, 915-925. Schmid, H.-P., Akhayat, O., Martins de Sa, C.. Puvion, F.. Koehler, K. and Scherrer, K. (1984) The prosome: an ubiquitous morphologically distinct RNP particle associated with repressed mRNPs and containing specific &RNA and a characteristic set of proteins. EMBO J. 3, 29-34. Schubert, M., Harmisson, G.G. and Meier. E. (1984) Primary structure of the vesicular stomatitis virus polymerase (L) gene: evidence for a high frequency of mutation. J. Viral. 51. 505-514. Schuldt, C. and KIoetzeI, P.-M. (1985) Analysis of cytoplasmic 19s ring-type particles in Drosophih which contain hsp 23 at normal growth temperature. Develop. Biol. 110, 65-74. Simpson, R.W. and Hauser, R.E. (1966) Structural components of vesicular stomatitis virus. Virology 29. 654-667. Thomas, D., Newcomb, W.W., Brown, J.C., Wall, J.S.. Hainfeld, L.F., Trus, B.L. and Steven, A.C. (1985) Mass and molecular composition of vesicular stomatitis virus: a scanning transmission eiectron microscopy analysis. J. Virol. 54, 598-607. Wagner, R.R. (19’75)Reproduction of rhabdoviruses. In: H. Fraenkel-Conrad and R.R. Wagner (Eds.), Comprehensive Virology, Vol. 4. pp. l-95. Plenum Publishing Corp., New York. Zabowski, J.J. and Wagner, R.R. (1980) Localization of membrane-associated proteins in vesicular stomatitis virus by use of hydrophobic membrane probes and cross-linking reagents. J. Virol. 36. 93-102.
Elsevier JVM 00809
Intracellular vesicular stomatitis virus nucleocapsids and virions visualized by surface spreading Kazumori Yazaki’, Toshihiko Sane’, Tomoko Okamoto2, Toshiyuki Urushibara2 and Chiaki Nishimura2 ITokyo Metropolitan Institute of Medical Science, Honkomagome, Bunkyo-ku, Tokyo and 2Department of Pharmaceutical Science, Kitasato University, Shirokane, Minato-ku, Tokyo, Japan
(Accepted4 July 1988)
Summary
Spreading of cells on a solution surface could visualize vesicular stomatitis virus nucleocapsids and virions in infected cells easily and clearly without the need for any purification. Characteristic structures observed by the spreading of the infected cells are described and discussed. VSV; Rhabdovirus;
Nucleocapsid;
Virus particle; Surface spreading
Introduction
In electron microscopic studies, ultra-thin sections and replicas of whole and fractured cells have relied on the studies of virus infection. By these methods, however, it is quite difficult to observe the intracellular viral genome, genomeprotein complex and virions as clearly as when observed in the purified state. The spreading of whole cells for electron microscopic observation has been used for the study of chromosomes and the synaptonemal complex (Gall, 1963; DuPraw, 1965; Moses, 1977). We used this method to examine viral nucleocapsids and virions in infected cells, and found it easy to carry out for the morphological study of virus maturation. In the present study, baby hamster kidney (BHK) cells infected with vesicular stomatitis virus (VSV) were examined. Correspondence to: Dr. K. Yazaki, Tokyo komagome, Bunkyo-ku, Tokyo 113, Japan. 0166-0934/89/$03.50
0
1989 Elsevier
Science
Metropolitan
Publishers
Institute
B.V.
of Medical
(Biomedical
Science,
Division)
3-18-22
Hon-
2
VW, a rhabdovirus, is enveloped and about 70 nm in diameter and 180 nm in length (Murphy and Harrison, 1980). It has a cone at one end of its cylindrical structure, thus giving it a bullet-shape appearance. The genome is single-stranded RNA of negative polarity with 11162 nucleotides (Schubert et al., 1984). The RNA forms a nucleocapsid with a major protein N and minor proteins NS and L. The N protein monomers are wedge-shaped, bilobed structures each about 9 nm in length and associate with RNA to form ribbon-shaped nucleocapsids 9 nm in width (Thomas et al., 1985). Each such nucleocapsid is a compact, builet-shaped coil about 50 to 53 nm in diameter with 34 to 35 turns in the virion and has the appearance of an internal skeleton (Simpson and Hauser, 1966; Nakai and Howatson, 1968; Cartwright et al., 1970a,b; Murphy and Harrison, 1980; Newcomb et al., 1982; Thomas et al., 1985). Newcomb and Brown (1981) and Newcomb et al. (1982) found that M protein must be present when an elongated nucIeocapsid coils to take on the bullet-like shape. A nucleocapsid forms a coil 20 nm in diameter in the absence of M at a high salt concentration (Heggeness et al., 1980). Neave et al. (1980) and Neave and Summers (1980) extracted nucleocapsids 20 nm in diameter from infected cells and found them to appear circular and helical and to serve as templates for virus replication. It has not been possible so far to observe differences in their structures which in all cases are 20 nm in diameter. A disc-like complex of N protein and VSV leader RNA has been extracted from cells at the late stage of viral infection (Blumberg et al., 1981; Blumberg and Kolakofsky, 1981). The structures of a nucleocapsid, the disc-like complex and virion mentioned above have been studied using purified and/or chemically treated samples. They were examined in this study by spreading infected cells on the surface of a solution.
Materials and Methods Virus and cells A monolayer culture of BHK cells grown in Eagle’s minimum essential medium supplemented 10% fetal calf serum was infected with VSV (wild-type, New Jersey serotype) at multiplicities of 5, and maintained at 35°C until harvest. At each sampling time, the cells were harvested by EDTA treatment. They were washed once by a hand-wind centrifuge and then resuspended in Dulbecco’s PBS(+) at a concentration adjusted to 2 x lo6 cells/ml. Surface spreading of cells Spreading was carried out according to the methods of Gall (1963), DuPraw (1965) and Moses (1977) with slight modification as follows: Fine talc powder was first sprinkled over the surface of a hypophase (PBS(+) supplemented with 10% sucrose) in a plastic Petri dish 5 or 9 cm in diameter. The cell suspension was taken in a platinum loop which was subsequently brought gently into contact with the hypophase surface and then removed from it. The spread cells appeared as a clear circular expansion free of talc powder. The grid was then brought into contact with
3
the clear surface of the circle. The samples were mounted on films of carbon-coated parlodion (Mallinckrodt, St Louis, MO, U.S.A.). The sample side of the grid was washed with PBS(+) and then distilled water. The samples were negatively stained with freshly prepared 2% uranyl acetate (TAAB), or shadowed with Pt-Pd on a rotary table. The grids were examined in a JEOL 1OOCelectron microscope, at 80 kv.
Results Outline of VSV infection
At 4 h following infection, nucleocapsids were present in abundance and could be seen as the stretched, coiled, or otherwise-shaped structures shown below. At 6 h, over 95% of the cells showed early cytopathic effects, nucleocapsids coiled as bullet-shaped forms and virions were abundantly present. At 8 h, many of the cells were round and easily removable. Attention was directed primarily to nucleocapsids and virions at 4 and 6 h after infection. Serpentine nucleocapsid
A serpentine nucleocapsid of VSV noted 4 h after infection is shown in Fig. la. Its length of 4.05 km was virtually the same as that of nucleocapsids obtained from infected cells (Neave and Summers, 1980), and calculated by Thomas et al. (1985). Ribbon-shaped nucleocapsids 9 nm in width were clearly visualized by the spreading of infected cells as evident in Fig. lb. Nucleocapsid
network
The nucleocapsid networks shown in Fig. 2a (Pt-Pd shadowing) and 2b (negative staining) were found in cells 4 h after infection and each contained a number
Fig. 1. Serpentine nucleocapsid of VSV (negative staining). pears serpentine along its length. (b) The nucleocapsid
(a) A nucleocapsid, 4.05 PITI in length is ribbon-shaped and 9 nm in width.
ap-
Fig. 2. Network of VSV nucleocapsids. (a) A number of serpentine nucleocapsids can be seen. Cytoskeletons with straight portions are indicated by arrows. A poiysome (P) can also be seen (Pt-Pd shadowing). (b) The nucleocapsids and cytoskeletons (arrows) clearly differ in their features. Membranous material (M), poiysomes (P) and disc-like structures (D) are evident (negative staining). (c) The nu~ieocapsids in the network appear situated along fine filaments, as indicated by arrows (Pt-Pd shadowing).
Fig. 3. A nucleocapsid showing the coils 20 nm in diameter and 7 to 9 nm pitch (negative staining).
of serpentine nucleocapsids. Cytoskeletons, distinguishable from nucleocapsids by their straightness (2a and b) and smoothness (2b) were found in all networks (see arrows). Polysomes (P) (2a and b), membranous material (M) and disc-like struc-
Fig. 4. “Double helical” VW nucleocapsids (Pt-Pd shadowing). (a) A nucleocapsid 4.1 urn in length has a branch (B). At D, the strands appear to be coiled around each other to form a double helix. (b) The nucleocapsid shows a double helix on its left half. The serpentine features indicated by arrows indicate the right half to possibly be a tightly coiled structure. (c) A nucleocapsid similar to (b), but note the virion (V) in the same field.
6
Fig. 5. Bullet- and cone-shaped toilings of VSV nucleocapsids (negative staining). (a) Bullet-shaped coiling is clearly evident. A nucleocapsid tail can be seen at the flat end. (b) The bullet-shaped coiling has 18 turns. At the flat end of the cylinder, N protein of the nucleocapsid enters the coil with its long axis directed radially (arrow A). A second coil appears at B. (c) Two bullet shapes, two cones and a circle are present in a nucleocapsid. (d) Seven cones can be seen. The inside of the cone appears empty (indicated by an arrow). (e) Twelve cones are arranged in succession. Al1 apices of the cones point in the same direction,
tures (D, discussed below) (2b) can also be seen. The nuc~eocapsids in the network were often situated along fine filaments (Fig. 2~). The thickness of such a filament was the same as that of one situated between neighbouring ribosomes in a polysome. The latter is certainly a single-stranded mRNA strand, noted in the sample (not shown). But whether the fine filament along which nucleocapsids was situated is a single-stranded RNA or some other cellular component has yet to be determined. Coils
20 nm in diameter
In cells 4 h after infection, some nucleocapsids appeared as partially coiled structures about 20 nm in diameter as shown in Fig. 3 (20 nm-coil) and had the same features as those indicated by Heggeness et al. (1980). Their pitch was from 7 to 9 nm.
7
“Double helical” nucleocapsids
In cells 4 and 6 h following infection, nucleocapsids with coils considerably different from both 20 nm-coils and bullet-shaped coils were observed, although less frequently than the latter two. Fig. 4a shows a nucleocapsid 11 to 12 nm in thickness with a branch (indicated as B). In each region indicated by an arrow (D), two strands appear coiled about each other. The length of the nucleocapsid without a branch was 4.1 pm. On the left half of the nucleocapsid shown in Fig. 4b, two strands each 11 to 12 nm in thickness are coiled around each other. This composite coil is referred to as a “double helix” in this paper to distinguish it from the 20 nm-coils and bullet-shaped coils in each of which a strand runs in one direction. The thickness of the strand comprising the right half of the nucleocapsid was 21 to 22 nm, twice that of the strand on the left. The thickness and serpentine features at the sites shown by arrows indicate the right half to possibly be a tightly coiled version of the left. The length of the nucleocapsid was 2.02 km, one half the full length of a nucleocapsid of VSV (Thomas et al., 1985). It thus appears that the structure in 4b is a full length nucleocapsid folded as a double helix and that in 4a is partially unfolded nucleocapsid with a branch. The double helix and 20 nm-coil in Fig. 3 are about the same in thickness, but differ very much in other respects. Fig. 4c shows a structure similar to those in 4a and b. Note the virion (V) present in the same field. Bullet- and cone-shaped
nucleocapsid
toilings
In cells 6 h after infection, networks of the nucleocapsids could hardly be observed but nucleocapsids and virions could be seen in abundance. Most of the nu-
Fig. 6. Disc-like
structures (negative staining). The structure is 13 nm in diameter with a central 3 to 4 nm in diameter. The same structures are also shown in Fig. 2b.
hole
8
cleocapsids were situated away from each other and variously shaped as shown in Fig. 5. The bullet coil in Fig. 5a possesses 30 turns. Note the tail at the flat end, indicating the entire length of the nucleocapsid to be still uncoiled. The structure in 5b has 18 turns. It is clearly evident that N proteins enter the coil with their long axis directed radially at the end of the cylinder as described by Thomas et al. (1985) (indicated by arrow A). The nucleocapsid extending from the flat end shows a second coil indicated by arrow B. The features indicated by arrows A and B clearly demonstrate coiling of the nucleocapsid to proceed from left to right. A central hole 3 to 4 nm in diameter is evident at the top of the cone which has 9 turns. The nucleocapsids in Fig. 5c-e each possess more than one bullet-shaped coil and cone. In 5c, two bullet-shaped coils, two cones and a circle can be seen. In 5d, seven cones are present in one nucleocapsid. A cone indicated by an arrow is clearly visualized inside of the coiled structure. In 5e, twelve cones are arranged in succes-
Fig. 7. be seen ent. (b) the
VSV particles in cells 6 h after infection (negative staining). (a) Internal skeletons can clearly in the virions. Within the inner cavities of the skeletons, an amorphous structure appears presA virion with a terminal hemisphere at its flat end. The hemisphere appears continuous with inner substance. (c) Radial view of a virion. Three concentric circles are clearly apparent.
9
sion. Note that their apices and bullet-coils all point in the same direction. inner cavities of the coils in Fig. 5 (especially evident in d) appear empty.
The
Disc-like structures
The disc-like structures in Fig. 6 (and Fig. 2b) first became apparent 4 h following infection and were abundant by 6 h. Each disc was about 13 nm in outer diameter and had a central hole 3 to 4 nm in diameter. In each, rod-like molecules were arranged in such a way as to form about twelve to fifteen circular petals. Virus particles
By the present method of spreading, VSV virions were observed to be variously shaped. In Fig. 7a, the internal skeletons of virions are clearly apparent. Each skeleton has as many as 38 turns, this being more than those observed in previous studies (see Introduction). The inner cavities of virion skeletons do not appear empty. An amorphous structure appears to be present. A limited number of virions had round membranous hemispheres at their flat ends (7b). These are the “terminal hemisphere” as described by Orenstein et al. (1976). Each membranous hemisphere appears to extend to and become continuous with the inner cavity of the skeleton. A radial view of the virus is shown in 7c. One striking feature is the three concentric circles within a circular membrane of 10.5 to 12 nm in thickness. The distance between the outer and inner circle is about 10 nm, about the same as the length of the N protein.
Discussion
After cells had been spread over a hypotonic solution or one without Mg++ as in the case of PBS(-), polysomes tended to undergo degradation. The present study was conducted under conditions that would not allow this. By using a Petri dish of smaller size, the extent to which each cell would spread was limited and polysomes, cytoskeletons and membranes could be observed. This condition favored negative staining. In a large dish, each cell would spread more extensively than when using a smaller dish. Cytoskeleton and membranes were scattered about, making observation difficult. However, polysome and helical structures of nucleocapsids could be clearly seen by metal shadowing (Figs. 2a,c, 4). What is seen in Fig. 2 may actually be extended structures of the “virus factory” region where nucleocapsids are crowded together. In this study, it was possible for the first time to observe the structures of this region at high resolution. The networks of nucleocapsids and cytoskeletons may prevent the destruction of the “virus factory” during spreading. Heggeness et al. (1980) found the nucleocapsid to change reversibly from an unwound state to the 20 nm-coil at low and high salt concentrations without the occurrence of M protein binding. Each coil seen in Fig. 3 may have been formed as a result of the highly salty microenvironment surrounding the nucleocapsid. The pitch of the coil strongly implies that the long axis of N protein monomers is not
10
oriented radially in the coil, but runs parallel or nearly so to its length. At 4 h after infection, hardly any bullet-shaped coils could be seen. Thus, the 20 nm-coils observed at 4 h after infection are quite possibly nucleocapsids not yet bound to M protein. This binding occurred by 6 h to give rise to bullet and cone shapes. At the sites of bullet-shaped coils in the cells, M protein has been detected only on nucleocapsids, not on membranes (Odenwald et al., 1986). Spreading may allow the coils to stretch again to various degrees. The cytoskeletons and membranes surrounding and suspending the nucleocapsids were scattered, thus possibly freeing the nucleocapsids. Autonomous coiling may start from various points in a nucleocapsid to produce the cone and bullet shape in association with bound M protein. The coiled structures shown in Fig. 5 may arise in this manner. But various characteristics of nucleocapsids can be discerned from the toilings, such as: (i) there is a central hole about 3 to 4 nm in diameter at the top of the cone; (ii) each cone has 9 turns, and coiling becomes cylindrical from the tenth turn from the top and (iii) coil direction shows polarity. The apices of the cones and bullets in a nucleocapsid were noted to always point in the same direction. In no case were two cones connected at their apices or base planes, nor did any cone point in a direction opposite to that of the others. Most of the nucleocapsids at 6 h had the structures shown in Fig. 5, or were in virions as internal skeletons. But double helical nucleocapsids were also found, although in limited number, as is especially evident from Fig. 4c. A replicative nucleocapsid of VSV is a circular helical structure (Neave and Summers, 1980), and nascent RNA also has the form of a nucleocapsid (Patton et al., 1983, 1984). The nucleocapsids in Fig. 4 are quite possibly circular, and the structure in Fig. 4a is a replicative intermediate of the virus, while those in Fig. 4b and c are compact forms of the template. No double helical nucleocapsids showed bullet- or cone-shaped coiling, possibly being unable to do so even after M protein binding or possibly because this binding failed to occur. In no case did a nucleocapsid coil into a bullet shape. If such bullet-shaped coiling were a pre-requisite for a nucleocapsid to form a mature virion, it would be impossible for a double helical nucleocapsid to form a virion. Although full length nucleocapsids containing RNAs of positive and negative polarity have been found in infected cells, only those with negative polarity have been detected in the virus (Roy et al., 1973; Wagner, 1975). The nucleocapsid having RNA of plus polarity, the template for replication, may be sorted out from the viral maturation process by differences in secondary structure. The disc-like structure in Figs. 2b and 6 appeared identical to the 18s disc-like complex (18s complex) extracted from cells at a late stage of infection (Blumberg and Kolakofsky, 1981), and was also found in abundance in cells at late stages of infection. It is thus quite likely that this structure is also the 18s complex of which the function may be regulation of transcription and translation of the virus (Blumberg and Kolakofsky, 1981). Prosomes, of which the function may be post-transcriptional regulation, have been isolated from various animal and plant cells (&humid et al., 1984; Schuldt and Kloetzel, 1985; Arrigo et al., 1985; Horsch et al., 1985; Martins de Sa et al., 1986; Kremp et al., 1986). A prosome consists of single-stranded RNA of 40 to 110 nucleotides and about ten kinds of protein. RNA
11
and proteins differ in the particular prosomes they possess, depending on the cells from which they are isolated. Purified prosomes, however, all have in common a circular disc 13 nm in outer diameter and a central hole 3 to 4 nm in diameter, and sediment at 18 or 19s. The morphology of a prosome appears exactly the same as that of the 18s complex and the structures in Fig. 6. From the close correlation of function with morphology, the 18s complex may be considered a prosome characteristically appearing in cells at a late stage of infection. Its constituents come from the virus. There is also the possibility that some of the disc-like structures in Fig. 6 (and Fig. 2b) are prosomes comprised of cellular derivatives, or are hybrids of the viral and cellular derivatives. According to the previous papers, nucleocapsids must interact with M protein and membranes to take on the compact complete bullet shape (Dubovi and Wagner, 1977; Zakowski and Wagner, 1980; Newcomb and Brown, 1981; Newcomb et al., 1982). What appears to be an amorphous structure observed at the inner cavity of the skeleton in the virus (Fig. 7), possibly part of a membrane, may be essential for preventing the formation of irregularly-shaped coils such as those in Fig. 5. Of the three concentric circles observed in a radial view of the virus (Fig. 7c), the outer and inner circles possibly derive from circularly arranged bilobed N protein monomers. The central region of the nucleocapsid becomes clearly visible as the central circle. The central regions of the nucleocapsids in Figs. 1, 3 and 5 do not appear as the lines shown in Fig. 7d. Some conformational change may occur in the region. The present method of spreading infected cells on a solution surface holds promise as a simple means for morphologically studying virus infection, making diagnosis and detecting new viruses. This method is now being applied in conjugation with immunological techniques.
Acknowledgement
The authors are grateful to Dr. Kin-ichiro Miura, University of Tokyo, for his helpful discussion. References Arrigo, A.-P., Darlix, J.-L., Khandjian, E.W., Simon, M. and Spahr, P.-F. (1985) Characterization of the prosome from Drosophila and its similarity to the cytoplasmic structures formed by the low molecular weight heat-shock proteins. Eur. Mol. Biol. Organ. J. 4, 399-406. Blumberg, B.M., Leppert, M. and Kolakofsky, D. (1981) Interaction of vsv leader RNA and nucleocapsid protein may control vsv genome replication. Cell 23, 837-845. Blumberg, B.M. and Kolakofsky, D. (1981) Intracellular vesicular stomatitis virus leader RNAs are found in nucleocapsid structures. J. Virol. 40, 568-576. Cartwright, B., Smale, C.J. and Brown, F. (1970a) Dissection of vesicular stomatitis virus into the infective ribonucleoprotein and immunizing components. J. Gen. Viral. 7, 19-32. Cartwright, B., Talbot, P. and Brown, F. (1970b) The proteins of biochemically active sub-units of vesicular stomatitis virus. J. Gen. Virol. 7, 267-272. Dubovi, E.J. and Wagner, R.R. (1977) Spatial relationships of the proteins of vesicular stomatitis vi-
12 FUS:induction of reversible oligomers by cleavable protein cross-linkers and oxidation. J. Viral. 22, 500-509. DuPraw, E.J. (1965) The organization of nuclei and chromosomes in honeybee embryonic cells. Proc. Natl. Acad. Sci. USA 53, 161-168. Gall, J. (1963) Chromosome fibers from an interphase nucleus. Science 139, 120-121. Heggeness, M.H., Scheid, A. and Choppin, P.W. (1980) Conformation of the helical nucleocapsids of paramyxoviruses and vesicular stomatitis virus: reversible coiling and uncoiling induced by changing in salt concentration. Proc. Natl. Acad. Sci. USA 77, 2631-2635. Hotsch, A., Kohler, K. and Schmid, H.-P. (2985) Prosomes are involved in the repression of viral mRNA. Z. Naturforsch. 4Oc, 449-450. Kremp, A., Schliephacke, M.. Kull, U. and Schmid, H.-P. (1986) Prosome exists in plant cells too. Exp. Cell Res. 166. 553-557. Martins de Sa, C., Grossi de Sa, M.-F.. Akhayat, O., Broders, F., Scherrer. K.. Horsch, A. and Schmid. H.-P. (1986) Prosome ubiquity and inter-species structural variations. J. Mol. Biol. 187, 479-493. Moses, M.J. (197’7) Synaptonemal complex karyotyping in spermato~ytes of the Chinese hamster (Cricetulus griseus). Chromosoma 60, 99-125. Murphy, F.A. and Harrison, A.K. (1980) Electron microscopy of the rhabdoviruses of animals. In: D.H.L. Bishop (Ed.), Rhabdoviruses. Vol. 1. pp. 65-106. CRC Press Inc., Boca Raton, FL, USA. Nakai, T. and Howatson, A.F. (1968) The fine structure of vesicular stomatitis virus. Virology 35. 268-281. Neave, C.W. and Summers, D.F. (1980) Electron microscopy of vesicular stomatitis virus ribonucleoprotein. J. Virol. 34, 764-771. Neave, C.W., Kolakofsky, CM. and Summers, D.F. (1980) Comparison of vesicular stomatitis virus intracellular and virion ribonucleoproteins. J. Virol. 33, 856-865. Newcomb, W.W. and Brown, J.C. (1981) Role of the vesicular stomatitis virus matrix protein in maintaining the viral nucleocapsid in the condensed form found in native virions. J. Virol. 39, 295-299. Newcomb, W.W.. Tobin, G.J.. McGowan, J.J. and Brown, J.C. (1982) In vitro reassembly of vesicular stomatitis virus skeletons. J. Viroi. 41, 1055-1062. Odenwald. W.F., Amheiter, H., Dubois-Dalcq, M. and Lazzarini, R.A. (1986) Stereo images of vesicular stomatitis virus assembly. J. Virol. 57. 922-932. Orenstein, J., Johnson, L., Shelton, E. and Larzarini, R.A. (1976) The shape of vesicular stomatit~s virus. Virology 71, 291-301. Patton, J.T., Davis, N.L. and Wertz. G.W. (1983) Cell-free synthesis and assembly of vesicular stomatitis virus nucleocapsid. J. Viral. 45, 155-164. Patton, J.T., Davis, N.L. and Wertz. G.W. (1984) N protein alone satisfies the requirement for protein synthesis during RNA replication of vesicular stoma&is virus. J. Virol. 49, 303-309. Roy, P., Repik, P., Hefti, E. and Bishop. B.H.L. (1973) Complementary RNA species isalated from vesicular stomatitis virus (HR) strain defective virions. J. Viral. 11, 915-925. Schmid, H.-P., Akhayat, O., Martins de Sa, C.. Puvion, F.. Koehler, K. and Scherrer, K. (1984) The prosome: an ubiquitous morphologically distinct RNP particle associated with repressed mRNPs and containing specific &RNA and a characteristic set of proteins. EMBO J. 3, 29-34. Schubert, M., Harmisson, G.G. and Meier. E. (1984) Primary structure of the vesicular stomatitis virus polymerase (L) gene: evidence for a high frequency of mutation. J. Viral. 51. 505-514. Schuldt, C. and KIoetzeI, P.-M. (1985) Analysis of cytoplasmic 19s ring-type particles in Drosophih which contain hsp 23 at normal growth temperature. Develop. Biol. 110, 65-74. Simpson, R.W. and Hauser, R.E. (1966) Structural components of vesicular stomatitis virus. Virology 29. 654-667. Thomas, D., Newcomb, W.W., Brown, J.C., Wall, J.S.. Hainfeld, L.F., Trus, B.L. and Steven, A.C. (1985) Mass and molecular composition of vesicular stomatitis virus: a scanning transmission eiectron microscopy analysis. J. Virol. 54, 598-607. Wagner, R.R. (19’75)Reproduction of rhabdoviruses. In: H. Fraenkel-Conrad and R.R. Wagner (Eds.), Comprehensive Virology, Vol. 4. pp. l-95. Plenum Publishing Corp., New York. Zabowski, J.J. and Wagner, R.R. (1980) Localization of membrane-associated proteins in vesicular stomatitis virus by use of hydrophobic membrane probes and cross-linking reagents. J. Virol. 36. 93-102.