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A morphological study on the ultrastructure and assembly of murine leukemia virus using a temperature-sensitive mutant restricted in assembly
VmOLOOY 80, 260-274 (1977)
A Morphological Study on the Ultrastructure and Assembly of Murine Leukemia Virus Using a Temperature-Sensitive Mutant Restricted in Assembly P. H. Y U E N AND P. K. Y. WONG ~ Department of Microbiology and School of Basic Medical Sciences, University of Illinois, Urbana, Illinois 61801 Accepted March 2, 1977 Ts3, a temperature-sensitive mutant of Moloney murine leukemia virus, was observed to produce large numbers of both normal particles and multiploids, (i.e., virions with more than one ribonucleoprotein component [RNP]) in all stages of assembly at the nonpermissive temperature. In thin sections, budding particles and extracellular immature particles show the general structural components characteristic of other RNA tumor viruses. Examination of the core shell confirms that it consists of polygonal subunits. The double striated tracks seen in negatively stained virions appear to be the edges of these subunits and are not likely to be a ~core membrane." An additional substructure was observed in the core shell which extends from the center of each polygonal subunit to the outer periphery of the RNP. The presence of substructures in the form of rings or short tubular substructures in the RNP is consistent with the hypothesis that the RNP is a hollow sphere formed by supercoiling of a single- or doublestranded helix. Multiploids were also observed in ts3-infected cells grown at the permissive temperature and in MuLV infected cells. We have demonstrated that the individual RNPs of a multiploid remain discrete entities. The volume of a spherical duplex is about twice that of a normal particle which suggests that it contains two complete genomes. The assembly of both normal particles and multiploids are essentially similar. Multiploids are apparently produced when two or more normal virions assemble in close proximity or assemble in succession at about the same site. The final steps in the assembly of both types of particles are preceded by the resealing of the cellular membrane. The laying down of the remaining section of the viral envelope occurs as a separate event. Elongation of the cytoplasmic strands linking the virions to the cell seems to occur before virion release. The significance of multiploids in mixed infections is discussed. INTRODUCTION
The ultrastructure of RNA tumor viruses has been investigated using different preparatory methods. These include negative staining of extracellular virions or virions that have been disrupted by various physical and chemical means and positive staining of sections of cell-associated virions or viral pellets. From these studies, several basically similar models of the arrangement of the viral components have been suggested (de Th~ and O'Connor, 1966; Kakefuda and Bader, 1969; Sarkar et Author to w h o m r e p r i n t requests should be addressed.
al., 1971; Nermut et al., 1972). However, none of them adequately account for all the known viral-specific protein components revealed by biochemical and immunological analysis. No matter which preparatory method is used, one of the factors contributing to the ease and success in elucidating the fine structure of viruses under the electron microscope is the availability of large populations of particles with similar morphology. Perhaps for this reason most investigators have tended to use concentrated purified viral preparations even though in most cases 99% of the virions from such harvests are mature virions which have lost the ordered arrange260
Copyright © 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.
ISSN 0042-6822
ULTRASTRUCTURE AND ASSEMBLY OF MuLV ment of structural components seen in budding or in immature extracellular virions. Although the immature and cell-associated virions are more ideally suited for such investigations, their infrequent occurrence (about five budding virions/10 cell sections) makes the task of studying them arduous and often prohibitively difficult. Recently, a temperature-sensitive (ts) m u t a n t of Moloney murine leukemia virus (Mo-MuLV), designated ts3, was isolated (Wong et al., 1973) which alleviates some of these problems. Scanning electron microscopic studies on ts3-infected cells (Wong and MacLeod, 1975) showed that at the nonpermissive temperature (39°) virions in a late stage of assembly accumulate on the cell surface. Maximum density was reached by 48 hr postinfection. Within 1 hr after temperature shift-down, more than 90% of these virions disappeared from the cell surface. The large number of budding virions in ts3-infected cells grown at 39° (about 22/cell section) and the availability of many immature virions trapped in the intercellular spaces simply by shifting the infected culture from 39 to 34°, facilitated a detailed examination of the fine structure of the budding and extracellular immature virions. This investigation revealed not only viral structures hitherto unreported but also the occurrence of multiploids. The implications of the occurrence of multiploids in mixed infections is discussed. MATERIALS AND METHODS
Cells Detailed procedures for the propagation of TB cells, a fibroblastic cell line established from mixed cultures of bone marrow and thymus CFW/D mouse cells (Ball et al., 1964) have been previously described (Wong and McCarter, 1974). Virus TB cells were infected in suspension at a m.o.i, of 5 with wild-type Moloney murine leukemia virus (Mo-MuLV) or ts3, a temperature-sensitive m u t a n t of MuLV. Infected cells (3 x 105) were plated on each 60-mm plastic petri dish and incubated at the permissive (34°) or nonpermissive tem-
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perature (39°) for 48 hr. Ts3-infected cells were also incubated at the nonpermissive temperature for 48 hr then shifted to the permissive temperature for 30 or 60 min.
Concentration of Viral Harvests The viral harvests were filtered through 0.45-t~m Millipore filters and pelleted at 25,000 rpm for 2 hr in an ultracentrifuge with a SW 40 rotor. The supernatant was aspirated and the viral pellet was either resuspended in TNE for negative staining or fixed for study in thin sections. Electron Microscopy Thin section. Viral pellets, and wildtype MuLV or ts3-infected TB cells scraped off with a rubber policeman were fixed in 2% glutaraldehyde in sodium cacodylate buffer or phosphate-buffered saline (PBS), then postfixed in 2% osmic acid overnight or in a freshly prepared mixture of 0.12 g of potassium ferrocyanide in 6 ml of 1% osmic acid. After dehydration through graded ethyl alcohol, the cells were embedded in Spurr's plastic. Sections were mounted directly on 300-mesh grids and stained with uranyl acetate at 70° in a water bath and with Reynold's lead citrate. Specimens were viewed in a Siemens 102 at 80 kv. Negative staining. Formvar carboncoated grids were lowered onto a drop of the virus suspension and allowed to adsorb for 1-3 min. The excess fluid was then drained off and the grids were stained with 1% uranyl acetate or 1% ammonium molybdate. RESULTS
Ts3 Virions Thin sections of ts3-infected cells grown at the nonpermissive temperature (39°) show many, essentially spherical, C-type particles, and atypically shaped virions in different stages of assembly at the cell membrane. Extracellular immature virions with ordered arrangement of the viral structural components and electron lucent centers were only very rarely encountered. Mature virions with dense cores centrally located were not observed. However, by shifting a ts3-infected culture grown at the nonpermissive to the permissive tempera-
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ture for 30 min, rapid assembly and release of the virions occurred.
Fine Structure and Arrangement of the Viral Components of Normal C-Type Particles Normal extracellular immature ts3 particles measure about 108 nm in diameter. They are morphologically similar to the particles of wild-type MuLV. In thin sections, budding particles and extracellular immature particles show the general structural components characteristic of other murine RNA tumor viruses (Fig. 1F). There appears to be selective preservation of the viral components depending on the fixatives used. In the glutaraldehydeosmium tetroxide-fixed specimens, the envelope usually appears as a single dense ring (Figs. 1C and F) surrounded by a fuzzy substance which, on rare occasions, appears to consist of structures resembling knobs and spikes (Fig. 1C). The typical bilayer was revealed when ts3-infected cells were postfixed in an osmium tetroxide-potassium ferrocyanide mixture (Fig. 1D). Within the viral envelope, two concentric rings, measuring about 58 and 77 nm in diameter, can be distinguished (Fig. 1E). The region between the outer ring and the envelope is less electron dense and substructures have not been resolved in this region. The outer of the two concentric rings is the core shell. It is not as electron dense as the inner ring and measures about 10 nm in width. Sections sliced through the core shell but outside the inner concentric ring reveal a central structurally uniform region (Figs. 1A and B), consisting of closely packed polygons each
of which has an electron dense dot at the center (Figs. 1A and B). In some of the above sections, a chain of rings was sometimes observed at the outer edge of the core shell. However, in some sections through the center of the virion, elongated substructures arranged periodically were observed to extend across the core shell (Fig. 1G), while in other parts of the same sections or in other sections dense dots were observed (Fig. 1F). The inner concentric ring is the ribonucleoprotein component (RNP) which consists of the viral RNA and attendant protein. It is more electron dense and measures about 3 nm in width. In some sections, the RNP appears to consist of open rings of varying sizes (Fig. 1F), while in others it seems to consist of short tubular substructures (Fig. 1E). Fragments of electron dense material (Fig. 1G) and dense spherical masses resembling polyribosomes and measuring about 22 nm in diameter (Figs. 4E and 7A) were frequently observed in the center of the virus.
Negatively Stained Extracellular ts3 Virions Ammonium molybdate (AM)-stained virions are of varying sizes. They may be essentially spherical particles (Fig. 2A) or particles with characteristic tails (Fig. 2B). Where the stain has penetrated particularly well, as shown in Fig. 2A, several substructures were observed. Although the outline of the virion was distinct, the detail of the viral envelope was not resolved. In some virions, the viral surface appears to consist of closely packed polygonal subunits. These substructures are not unlike those seen in thin sections through
FIo. 1. Electron micrographs ofvirions from ts3-infected TB cells. Magnification: A-B and E-G, bar = 40 nm; C-D, bar = 50 nm. A and B, Sections through the core shells of budding virions: A, polygonal subunit (white arrow); B, polygonal subunit with a dense center (black arrow). C, Section of an extracellular immature virion showing a spike and knob on the cell surface (arrow); D, section of an extracellular virion showing the lipoprotein bilayer of the viral envelope; E, section of an extracellular immature virion showing the core shell (~) and a chain of short tubular substructures in the RNP (between arrows); F, section of an extracellular immature virion showing a chain of open rings in the RNP (between arrows); G, Section of a budding multiploid virion showing the structural components characteristic of C-type virus: viral envelope (short black arrow), core shell (long black arrow), RNP (white arrow), elongate substructures extending from RNP to the outer edge of the core shell (between bars). Electron dense material in the center of the virion could be unpackaged RNP or polyribosomal fragments.
ULTRASTRUCTURE AND ASSEMBLY OF MuLV
FIGURE 1
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FIGURE 2
ULTRASTRUCTURE AND ASSEMBLY OF MuLV tl~e core shell of the virions (Figs. 1A and B). W h e t h e r these s u b s t r u c t u r e s occur on the surface of the envelope or w h e t h e r t h e y are part of the core shell c a n n o t be d e t e r m i n e d with certainty. Short filamentous strands, probably the ribonucleoprorein component, m e a s u r i n g about 9 n m in width were also observed inside the virion (Fig. 2A, double black arrows) and protruding from an a p p a r e n t l y disrupted particle (Fig. 2A, double white arrows). Figure 2A (single black arrow) also shows a double striated t r a c k just b e n e a t h the viral envelope. A u r a n y l acetate (UA)-stained virion is shown in Fig. 2C. The viral envelopes of UA-stained viruses a p p e a r indistinct and difficult to distinguish from t h e i r surroundings. Surface substructures, resembling the polygonal subunits found in AMstained preparations, were also observed. Again it is not clear w h e t h e r these polygonal subunits occur on the surface of the viral envelope or w h e t h e r t h e y are p a r t of the core shell. Within the viral envelope two distinct concentric rings can be distinguished. The outer ring (Fig. 2C, double arrows) measures about 80 n m in diameter, while the i n n e r ring (Fig. 2C, single arrow) m e a s u r e s about 60 n m in diameter. The sizes of these rings are similar to those obtained for the core shell and R N P of sectioned i m m a t u r e virions, suggesting t h a t t h e y comprise the outer edges of the core shell and R N P of an i m m a t u r e virion, respectively. Some dense staining material was also observed in the interior of the virus.
Sequence of Events in the Assembly of a Normal C-type ts3 Virion The sequence of events in the assembly of a n o r m a l C-type ts3 virion is shown in Fig. 3. The earliest stage detected in the
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assembly of the virion is a slight evagination on the cell m e m b r a n e which is easily recognized by the dense ribonucleoprotein component (RNP). Successive stages in the assembly of the virion involve the orderly a n d progressive packaging of the viral s t r u c t u r a l components. The RNP first appears as a slight arc (Figs. 3A and B), t h e n a semicircle (Figs. 3C and D), t h e n a horseshoe (Figs. 3E and F), and finally a ring (Fig. 3G and H). F i g u r e 3H shows t h a t even after the virion is completely assembled and the cellular memb r a n e is resealed, it is still cytoplasmically linked to the cell. An extracellular immat u r e and a m a t u r e virion are shown in Figs. 3I and J, respectively. It is unclear at present w h e t h e r the virions are e v e n t u a l l y released by a mechanical sloughing off of the cytoplasmic s t r a n d or by some kind of enzymatic action.
Structure and Assembly of Multipolid Virions The fine s t r u c t u r e of the budding atypically shaped and a b n o r m a l l y large spherical particles is essentially similar to t h a t of the n o r m a l particles (Fig. 1G). Cross sections of these particles show t h a t each of t h e m consists of two or more distinct RNPs, which indicates t h a t each is a composite of two or more particles. Hence, t h e y will subsequently be referred to as multiploids. Figure 4 shows the probable sequence of events in the assembly of the simplest form of multiploid, an essentially spherical particle containing two RNPs enclosed in a single envelope. The formation of such a particle appears to be initiated w h e n two n o r m a l C-type particles assemble in juxtaposition. T h a t the two RNPs r e m a i n discrete entities after assembly is indicated by the distinct b r e a k s in the ribonucleo-
FIG. 2. Negatively stained virions from culture medium ofts3-infected TB cells grown at the nonpermissive temperature for 48 hr then shifted to the permissive temperature for 1 hr. A-B, bar = 100 nm; C, bar = 40 nm. A, Ammonium molybdate-stained virions showing the polygonal subunits of the envelope or core shell (single white arrow), double striated track located just beneath the envelope (single black arrow), and the RNP within a virion (double black arrow) and protruding from a disrupted virion (double white arrow); B, ammonium molybdate-stained virions with the characteristic tails; C, a uranyl acetate-stained virion showing the viral envelope ~short black arrow), the core shell (double arrows), and the RNP (long black arrow).
FIc. 3. A - H , Different stages in the a s s e m b l y of n o r m a l virions of ts3; I, an e x t r a c e l l u l a r i m m a t u r e virion with ordered a r r a n g e m e n t of the s t r u c t u r a l components; J, an extracellular m a t u r e virion with centrally located dense core. Bar = 100 nm. 266
U L T R A S T R U C T U R E A N D A S S E M B L Y OF MuLV
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FIG. 4. The probable sequence of e v e n t s in the a s s e m b l y of a spherical duplex. B a r = 100 nm. Black a r r o w s indicate b r e a k s in the R N P of the virions.
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protein component (Fig. 4, arrows). Whether or not each RNP contains a complete genome cannot be determined until more is known about the packaging of the viral RNA. The fact that a spherical duplex is larger (averaging about 125 nm in diameter) than a normal particle and its volume is about twice that of a normal particle suggests that it may consist of two complete RNPs. The structure and size of the normal ts3 particles and the spherical duplexes in different stages of development are compared in Fig. 5. In addition to these duplexes, thin section electron microscopy also revealed larger, almost spherical particles and atypically shaped particles containing three or more discrete RNPs. These multiploid particles appear to be assembled either in the same manner as suggested for the formation of duplexes or they are assembled into rod-shaped virions by the successive assembly of two or more particles at about the same site. Figure 6 shows representatives of multiploids in the early stages of assembly in ts3 infected cells grown at the nonpermissive temperature. That these virions are still in the process of assembly is indicated by the virions still being broadly linked to the cell cytoplasm. Those multiploids shown in Figs. 6B and E may eventually dissociate as single, essentially spherical, virions. By contrast, the first particle assembled in the rod-shaped virions shown in Figs. 6C and F may dissociate independently from the rest. Figure 7 shows some of the almost completely assembled multiploids in ts3-infected cells grown at the nonpermissive temperature and in ts3-infected cells grown at the nonpermissive temperature then shifted to the permissive temperature for 30 min. That these multiploids are in the final stages of assembly is indicated by the complete assembly of viral components, the resealing of the cell membrane (Figs. 7A, C, G, and I), and elongation of the cytoplasmic linkages. These multiploids may be divided into two broad groups: those that are linked to the cell by a common cytoplasmic strand but may eventually dissociate as individual particles each with its own complete genome
(Figs. 7D, G, and H), and those that will most likely remain as multiploids after dissociation from the cell membrane (Figs. 7A-C, E, F, and I). The heterogeneity of virion size in the viral pellet from a ts3infected culture grown at the nonpermissive temperature then shifted to the permissive temperature for 1 hr (Fig. 8) and in the negatively stained preparations (Figs. 2A and B) indicates that these multiploids were released into the culture medium. Their infectivity has not as yet been determined. DISCUSSION Our present findings on the structure of the budding and immature extracellular virions agree remarkably well with the gross structural components that have been previously reported (de Th6 and O'Connor, 1966; Sarkar et al., 1971; Nermut et al., 1972; Kakefuda and Bader, 1969). We have, however, observed substructures in the core shell and ribonucleoprotein component (RNP) which consists of the viral RNA and attendant protein hitherto unreported. Nermut et al. (1972) reported that the core shell of the Rauscher and Friend leukemia viruses consists of two structural elements: (i) a ~core membrane" and (ii) a layer of globular or ring-like morphological subunits forming a close hexagonal pattern. (The arrangement of these two layers of the core shell was reversed in a later report [Schafer et al., 1975]) We agree with Nermut et al. (1972) that the outer layer of the core shell consists of polygonal subunits. However, we cannot agree with their interpretation that the double striated tracks in negatively stained preparations represent a separate substructure of the core shell, viz, the "core membrane." The double tracks observed in ammonium molybdate-stained whole viral preparations appear to be the outer and inner edges of the polygonal subunits of the core shell and the striae to be the walls between the subunits. In uranyl acetate-stained whole viral preparations, the double tracks were not observed. Instead, two concentric rings with dense outer edges were resolved. The diameters
FIG. 5. A comparison of the size differences between normal ts3 virions (A, C, E) and spherical duplexes (B, D, F). A and B, Late budding virions; C and D, extracellular i m m a t u r e virions; E and F, m a t u r e virions. Bar = 50 nm. 269
FIG. 6. Early stages in the assembly of multiploids showing the broad cytoplasmic linkages of the virions to the cell. Bar = 100 nm. A, A budding patch consisting of at least six budding virions in close proximity to one another; B and E, essentially spherical multiploids; C, the simultaneous assembly of a normal virion and multiploids in juxtaposition; D and F, rod-shaped virions with two or more virions assembling in tandem. 270
FIG. 7. Multiploids in the final stages of assembly showing complete or near complete assembly of the structural components and elongate cytoplasmic linkages of the virion to the cell. Bar = 100 nm. A, A spherical multiploid with probably three pieces of genomic RNA; B, a n essentially spherical multiploid with four or five pieces of genomic RNA; C, a clover-shaped multiploid with three pieces of genomic RNA; D, A dumbbell-shaped duplex; E, a n egg-shaped duplex; F, a rod-shaped virion with at least three pieces or genomic RNA; G, a rod-shaped virion consisting of three virions assembling in tandem. H, three normal virions probably dissociating as a cluster; I, a large multiploid with five or more pieces of genomic RNA. 271
FIG. 8. Section of a viral pellet from a ts3-infected TB cell c u l t u r e g r o w n at t h e n o n p e r m i s s i v e t e m p e r a t u r e t h e n s h i f t e d to t h e p e r m i s s i v e t e m p e r a t u r e for 1 h r s h o w i n g i m m a t u r e d u p l e x e s (double arrows) a n d i m m a t u r e n o r m a l v i r i o n s (single arrow). B a r = 200 n m . 272
ULTRASTRUCTURE AND ASSEMBLY OF MuLV of the two concentric rings in the UAstained specimens could best be interpreted as the outer edges of the core shell and RNP of an immature virion. In the budding and immature virions, there is an elongated substructure in the core shell which extends from the center of each polygonal subunit to the periphery of the RNP. We tentatively suggest that these substructures serve as binding sites for the RNP during assembly and are probably destroyed on condensation of the RNP in the extracellular virion. We further propose that the layer of polygonal subunits is the most rigid component of the virus, and this rigidity determines the shape of the virus during assembly. This proposed role for the polygonal subunits of the core shell is consistent with the observation t h a t the uniformly packaged RNP in budding and immature virions follows the contour of this shell. In the budding and immature virions, the core shell is separated from the envelope by a uniform space. This ordered arrangement is lost on condensation of the RNP. Although no structural component has as yet been identified between the core shell and the envelope, we believe that some structure must be present to enable viral structural protein-protein interactions during assembly. However, subsequent to assembly, fracturing along this region is conceivable. The presence of rings and short tubular substructures in the RNP is consistent with the suggestion that the configuration of the RNP is a hollow sphere formed by supercoiling of a single-stranded or double-stranded helix (Kakefuda and Bader, 1969; Nowinski et al., 1970; Sarkar et al., 1971). The formation of multiploids is not peculiar to ts3 grown at the nonpermissive temperature. Multiploids, mostly duplexes, were also observed consistently, although in relatively lower numbers, in ts3infected cells grown at the permissive temperature, in MuLV-infected cells and also in Moloney murine sarcoma virus (MoMuSV) infected cells. Two virions assembling in juxtaposition have been reported in Rauscher leukemia virus (de Th~ and
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O'Connor, 1966) and particles with two cores have also been observed in Friend leukemia virus (Nermut et al., 1972). The latter type of particles is probably similar to the dumbbell-shaped duplexes shown in Fig. 7D, rather t h a n the essentially spherical particle shown in Fig. 4E. From published micrographs, for example, those of extracellular whole virus preparations (deHarven et al., 1973) or sections of viral pellets (de Th~ and O'Connor, 1966), variation in the size of the virions can be observed. Recently (Dmochowski et al., 1976), morphological differences in the budding, immature, and mature C-type viruses from mice were demonstrated. The viruses shown in the micrographs certainly included multiploids. Unfortunately, no mention was made as to whether these were observed in one or more virus populations. These observations suggest that the occurrence of multiploids, especially duplexes, is a more general phenomenon than has hitherto been appreciated. The occurrence of multiploid particles would be of special significance in mixed infection studies, since they may be capable of introducing into the same cell genomes from both parents. Heterozygosis has been postulated as a possible step in the formation of recombinants in Rous sarcoma viruses (Weiss et al., 1973). Multiploids could at least in part explain the phenotypic heterogeneity observed in the viral progeny of mixed infections such as those reported by Fischinger and O'Connor (1970), Ball et al. (1973), and Hopkins et al. (1976). Multiploids are also of importance in recombination studies. Recombination frequencies obtained could be artificially enhanced due to the presence of heterozygotes, as was found to be the case in crosses between three ts mutants of MoMuLV (Wong and McCarter, 1973) where a recombination frequency of about 8% was obtained. However, subsequent recloning of the apparent wild-type progenies from the 34° harvest of one of the crosses, tsl × ts3, showed t h a t 6/14 (i.e., nearly 40%) of the clones segregated with ts phenotype (unpublished data). Since the possibility exists that the frequency in the
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formation of heterozygotes may vary from o n e m i x e d i n f e c t i o n to a n o t h e r , a t t e m p t s at mapping of the RNA tumor viral genome by recombination frequencies will entail at least recloning of the progeny of the first filial generation. ACKNOWLEDGMENTS We wish to thank Ms. H. C. Chen, Ms. K. Hedges, Ms. L. Jones, and Ms. K. O'Connor for their skillful assistance. This investigation is supported by Public Health Service Research Grant No. CA17695 awarded by the National Cancer Institute to PKYW. REFERENCES BALL, J. K., HUH, T. Y., and MCCARTER, J. A. (1964). On the statistical distribution of epidermal papillomata in mice. Brit. J. Cancer 18, 120-123. BALL,J. K., MCCARTER,J. A., and SUNDERLAND,S. M. (1973). Evidence for helper independent murine sarcoma virus. Virology 56, 268-284. DMOCHOWSKI,L., BOWEN, J. M., MYERS, B., PRIORI, E. S., MILLER,M. F., SEMAN, G., CHAN, J. C., DODSON, M. L., SCANLON, M., OHTSUKI, Y., and YOSHIDA, H. (1976). Comparative studies of type C, type B and M-PM oncornaviruses. In "Comparative Leukemia Research 1975", (J. Clemmesen and V. S. Yohn, eds.), Bibl. Haernatol., No. 43, pp. 417-424. Karger, Basel. FISCHINGER,P. J., and O'CONNOR,T. E. (1970). Replication of defective and competent forms of murine sarcoma virus in mouse cells. Virology 41, 233-243. DEHARVEN,E., BEJU, D., EVENSON,D. P., BAsu, S., and SCHIDLOVSKY,G. (1973). Structure of critical point dried oncornaviruses. Virology 55, 535-540. HOPKINS, N., TRAKTMAN, P., and WHALEN, K. (1976). N-Tropic variants obtained after co-infection with N- and B-tropic murine leukemia viruses. J. Virol. 18, 324-331. KAKEFUDA, T., and BADER, J. P. (1969). Electron microscopic observations of the ribonucleic acid of murine leukemia virus. J. Virol. 4, 460-474.
NERMUT, M. V., FRANK, H., and SCHAFER, W. (1972). Properties of mouse leukemia viruses. III. Electron microscopic appearance as revealed after conventional preparation techniques as well as freeze-drying and freeze-etching. Virology 49, 345-358. NOWINSKI,R. C., OLD, L. J., SARKAR,N. H., and MOORE, D. H. (1970). Common properties of the oncogenic RNA viruses (oncornaviruses). Virology 42, 1152-1157. SARKAR, N. H., NOWINSKI,R. C., and MOORE, D. H. (1971). Helical neocapsid structure of the oncogenic ribonucleic acid viruses (oncornaviruses). J. Virol. 8, 564-572. SCHAFER, W., DEMSEY, A., FRANK, H., HUNSMAN, G., LANGE, J., MOENNIG, V., PISTER, L., BOLOGNESI, D. P., GREEN,R. W., LUFTIG,R. B., SHAPER, J., and HUPER, G. (1975). Mammalian C-type oncorna viruses. In "Comparative Leukemia Research 1973" (Y. Ito and R. M. Dutcher, eds.), Leukemogenesis, Bibl. Haemat., No. 40, Karger, Basel. DE THe, G., and O'CONNOR, T. E. (1966). Structure of a murine leukemia virus after disruption with Tween-ether and comparison with two myxoviruses. Virology 28, 713-728. WEISS, R. A., MASON, W. S., and VOGT, P. K. (1973). Genetic recombinants and heterozygotes derived from endogenous and exogenous avian RNA tumor viruses. Virology 52, 535-552. WONG, P. K. Y., and MCCARTER,J. A. (1973). Genetic studies of temperature sensitive mutants of Moloney murine leukemia virus. Virology 53, 319326. WONG,P. K. Y., and MCCARTER,J. A. (1974). Studies of two temperature-sensitive mutants of Molohey murine leukemia virus. Virology 58, 396-408. WONG, P. K. Y., and MACLEOD,R. (]975). Studies on the budding process of a temperature-sensitive mutant of murine leukemia virus with a scanning electron microscope. J. Virol. 16, 434-442. WONG, P. K. Y., Russ, L. J., and MCCARTER, J. A. (1973). Rapid, selective procedure for isolation of spontaneous temperature-sensitive mutants of Moloney leukemia virus. Virology 51, 424-431.
A Morphological Study on the Ultrastructure and Assembly of Murine Leukemia Virus Using a Temperature-Sensitive Mutant Restricted in Assembly P. H. Y U E N AND P. K. Y. WONG ~ Department of Microbiology and School of Basic Medical Sciences, University of Illinois, Urbana, Illinois 61801 Accepted March 2, 1977 Ts3, a temperature-sensitive mutant of Moloney murine leukemia virus, was observed to produce large numbers of both normal particles and multiploids, (i.e., virions with more than one ribonucleoprotein component [RNP]) in all stages of assembly at the nonpermissive temperature. In thin sections, budding particles and extracellular immature particles show the general structural components characteristic of other RNA tumor viruses. Examination of the core shell confirms that it consists of polygonal subunits. The double striated tracks seen in negatively stained virions appear to be the edges of these subunits and are not likely to be a ~core membrane." An additional substructure was observed in the core shell which extends from the center of each polygonal subunit to the outer periphery of the RNP. The presence of substructures in the form of rings or short tubular substructures in the RNP is consistent with the hypothesis that the RNP is a hollow sphere formed by supercoiling of a single- or doublestranded helix. Multiploids were also observed in ts3-infected cells grown at the permissive temperature and in MuLV infected cells. We have demonstrated that the individual RNPs of a multiploid remain discrete entities. The volume of a spherical duplex is about twice that of a normal particle which suggests that it contains two complete genomes. The assembly of both normal particles and multiploids are essentially similar. Multiploids are apparently produced when two or more normal virions assemble in close proximity or assemble in succession at about the same site. The final steps in the assembly of both types of particles are preceded by the resealing of the cellular membrane. The laying down of the remaining section of the viral envelope occurs as a separate event. Elongation of the cytoplasmic strands linking the virions to the cell seems to occur before virion release. The significance of multiploids in mixed infections is discussed. INTRODUCTION
The ultrastructure of RNA tumor viruses has been investigated using different preparatory methods. These include negative staining of extracellular virions or virions that have been disrupted by various physical and chemical means and positive staining of sections of cell-associated virions or viral pellets. From these studies, several basically similar models of the arrangement of the viral components have been suggested (de Th~ and O'Connor, 1966; Kakefuda and Bader, 1969; Sarkar et Author to w h o m r e p r i n t requests should be addressed.
al., 1971; Nermut et al., 1972). However, none of them adequately account for all the known viral-specific protein components revealed by biochemical and immunological analysis. No matter which preparatory method is used, one of the factors contributing to the ease and success in elucidating the fine structure of viruses under the electron microscope is the availability of large populations of particles with similar morphology. Perhaps for this reason most investigators have tended to use concentrated purified viral preparations even though in most cases 99% of the virions from such harvests are mature virions which have lost the ordered arrange260
Copyright © 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.
ISSN 0042-6822
ULTRASTRUCTURE AND ASSEMBLY OF MuLV ment of structural components seen in budding or in immature extracellular virions. Although the immature and cell-associated virions are more ideally suited for such investigations, their infrequent occurrence (about five budding virions/10 cell sections) makes the task of studying them arduous and often prohibitively difficult. Recently, a temperature-sensitive (ts) m u t a n t of Moloney murine leukemia virus (Mo-MuLV), designated ts3, was isolated (Wong et al., 1973) which alleviates some of these problems. Scanning electron microscopic studies on ts3-infected cells (Wong and MacLeod, 1975) showed that at the nonpermissive temperature (39°) virions in a late stage of assembly accumulate on the cell surface. Maximum density was reached by 48 hr postinfection. Within 1 hr after temperature shift-down, more than 90% of these virions disappeared from the cell surface. The large number of budding virions in ts3-infected cells grown at 39° (about 22/cell section) and the availability of many immature virions trapped in the intercellular spaces simply by shifting the infected culture from 39 to 34°, facilitated a detailed examination of the fine structure of the budding and extracellular immature virions. This investigation revealed not only viral structures hitherto unreported but also the occurrence of multiploids. The implications of the occurrence of multiploids in mixed infections is discussed. MATERIALS AND METHODS
Cells Detailed procedures for the propagation of TB cells, a fibroblastic cell line established from mixed cultures of bone marrow and thymus CFW/D mouse cells (Ball et al., 1964) have been previously described (Wong and McCarter, 1974). Virus TB cells were infected in suspension at a m.o.i, of 5 with wild-type Moloney murine leukemia virus (Mo-MuLV) or ts3, a temperature-sensitive m u t a n t of MuLV. Infected cells (3 x 105) were plated on each 60-mm plastic petri dish and incubated at the permissive (34°) or nonpermissive tem-
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perature (39°) for 48 hr. Ts3-infected cells were also incubated at the nonpermissive temperature for 48 hr then shifted to the permissive temperature for 30 or 60 min.
Concentration of Viral Harvests The viral harvests were filtered through 0.45-t~m Millipore filters and pelleted at 25,000 rpm for 2 hr in an ultracentrifuge with a SW 40 rotor. The supernatant was aspirated and the viral pellet was either resuspended in TNE for negative staining or fixed for study in thin sections. Electron Microscopy Thin section. Viral pellets, and wildtype MuLV or ts3-infected TB cells scraped off with a rubber policeman were fixed in 2% glutaraldehyde in sodium cacodylate buffer or phosphate-buffered saline (PBS), then postfixed in 2% osmic acid overnight or in a freshly prepared mixture of 0.12 g of potassium ferrocyanide in 6 ml of 1% osmic acid. After dehydration through graded ethyl alcohol, the cells were embedded in Spurr's plastic. Sections were mounted directly on 300-mesh grids and stained with uranyl acetate at 70° in a water bath and with Reynold's lead citrate. Specimens were viewed in a Siemens 102 at 80 kv. Negative staining. Formvar carboncoated grids were lowered onto a drop of the virus suspension and allowed to adsorb for 1-3 min. The excess fluid was then drained off and the grids were stained with 1% uranyl acetate or 1% ammonium molybdate. RESULTS
Ts3 Virions Thin sections of ts3-infected cells grown at the nonpermissive temperature (39°) show many, essentially spherical, C-type particles, and atypically shaped virions in different stages of assembly at the cell membrane. Extracellular immature virions with ordered arrangement of the viral structural components and electron lucent centers were only very rarely encountered. Mature virions with dense cores centrally located were not observed. However, by shifting a ts3-infected culture grown at the nonpermissive to the permissive tempera-
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ture for 30 min, rapid assembly and release of the virions occurred.
Fine Structure and Arrangement of the Viral Components of Normal C-Type Particles Normal extracellular immature ts3 particles measure about 108 nm in diameter. They are morphologically similar to the particles of wild-type MuLV. In thin sections, budding particles and extracellular immature particles show the general structural components characteristic of other murine RNA tumor viruses (Fig. 1F). There appears to be selective preservation of the viral components depending on the fixatives used. In the glutaraldehydeosmium tetroxide-fixed specimens, the envelope usually appears as a single dense ring (Figs. 1C and F) surrounded by a fuzzy substance which, on rare occasions, appears to consist of structures resembling knobs and spikes (Fig. 1C). The typical bilayer was revealed when ts3-infected cells were postfixed in an osmium tetroxide-potassium ferrocyanide mixture (Fig. 1D). Within the viral envelope, two concentric rings, measuring about 58 and 77 nm in diameter, can be distinguished (Fig. 1E). The region between the outer ring and the envelope is less electron dense and substructures have not been resolved in this region. The outer of the two concentric rings is the core shell. It is not as electron dense as the inner ring and measures about 10 nm in width. Sections sliced through the core shell but outside the inner concentric ring reveal a central structurally uniform region (Figs. 1A and B), consisting of closely packed polygons each
of which has an electron dense dot at the center (Figs. 1A and B). In some of the above sections, a chain of rings was sometimes observed at the outer edge of the core shell. However, in some sections through the center of the virion, elongated substructures arranged periodically were observed to extend across the core shell (Fig. 1G), while in other parts of the same sections or in other sections dense dots were observed (Fig. 1F). The inner concentric ring is the ribonucleoprotein component (RNP) which consists of the viral RNA and attendant protein. It is more electron dense and measures about 3 nm in width. In some sections, the RNP appears to consist of open rings of varying sizes (Fig. 1F), while in others it seems to consist of short tubular substructures (Fig. 1E). Fragments of electron dense material (Fig. 1G) and dense spherical masses resembling polyribosomes and measuring about 22 nm in diameter (Figs. 4E and 7A) were frequently observed in the center of the virus.
Negatively Stained Extracellular ts3 Virions Ammonium molybdate (AM)-stained virions are of varying sizes. They may be essentially spherical particles (Fig. 2A) or particles with characteristic tails (Fig. 2B). Where the stain has penetrated particularly well, as shown in Fig. 2A, several substructures were observed. Although the outline of the virion was distinct, the detail of the viral envelope was not resolved. In some virions, the viral surface appears to consist of closely packed polygonal subunits. These substructures are not unlike those seen in thin sections through
FIo. 1. Electron micrographs ofvirions from ts3-infected TB cells. Magnification: A-B and E-G, bar = 40 nm; C-D, bar = 50 nm. A and B, Sections through the core shells of budding virions: A, polygonal subunit (white arrow); B, polygonal subunit with a dense center (black arrow). C, Section of an extracellular immature virion showing a spike and knob on the cell surface (arrow); D, section of an extracellular virion showing the lipoprotein bilayer of the viral envelope; E, section of an extracellular immature virion showing the core shell (~) and a chain of short tubular substructures in the RNP (between arrows); F, section of an extracellular immature virion showing a chain of open rings in the RNP (between arrows); G, Section of a budding multiploid virion showing the structural components characteristic of C-type virus: viral envelope (short black arrow), core shell (long black arrow), RNP (white arrow), elongate substructures extending from RNP to the outer edge of the core shell (between bars). Electron dense material in the center of the virion could be unpackaged RNP or polyribosomal fragments.
ULTRASTRUCTURE AND ASSEMBLY OF MuLV
FIGURE 1
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FIGURE 2
ULTRASTRUCTURE AND ASSEMBLY OF MuLV tl~e core shell of the virions (Figs. 1A and B). W h e t h e r these s u b s t r u c t u r e s occur on the surface of the envelope or w h e t h e r t h e y are part of the core shell c a n n o t be d e t e r m i n e d with certainty. Short filamentous strands, probably the ribonucleoprorein component, m e a s u r i n g about 9 n m in width were also observed inside the virion (Fig. 2A, double black arrows) and protruding from an a p p a r e n t l y disrupted particle (Fig. 2A, double white arrows). Figure 2A (single black arrow) also shows a double striated t r a c k just b e n e a t h the viral envelope. A u r a n y l acetate (UA)-stained virion is shown in Fig. 2C. The viral envelopes of UA-stained viruses a p p e a r indistinct and difficult to distinguish from t h e i r surroundings. Surface substructures, resembling the polygonal subunits found in AMstained preparations, were also observed. Again it is not clear w h e t h e r these polygonal subunits occur on the surface of the viral envelope or w h e t h e r t h e y are p a r t of the core shell. Within the viral envelope two distinct concentric rings can be distinguished. The outer ring (Fig. 2C, double arrows) measures about 80 n m in diameter, while the i n n e r ring (Fig. 2C, single arrow) m e a s u r e s about 60 n m in diameter. The sizes of these rings are similar to those obtained for the core shell and R N P of sectioned i m m a t u r e virions, suggesting t h a t t h e y comprise the outer edges of the core shell and R N P of an i m m a t u r e virion, respectively. Some dense staining material was also observed in the interior of the virus.
Sequence of Events in the Assembly of a Normal C-type ts3 Virion The sequence of events in the assembly of a n o r m a l C-type ts3 virion is shown in Fig. 3. The earliest stage detected in the
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assembly of the virion is a slight evagination on the cell m e m b r a n e which is easily recognized by the dense ribonucleoprotein component (RNP). Successive stages in the assembly of the virion involve the orderly a n d progressive packaging of the viral s t r u c t u r a l components. The RNP first appears as a slight arc (Figs. 3A and B), t h e n a semicircle (Figs. 3C and D), t h e n a horseshoe (Figs. 3E and F), and finally a ring (Fig. 3G and H). F i g u r e 3H shows t h a t even after the virion is completely assembled and the cellular memb r a n e is resealed, it is still cytoplasmically linked to the cell. An extracellular immat u r e and a m a t u r e virion are shown in Figs. 3I and J, respectively. It is unclear at present w h e t h e r the virions are e v e n t u a l l y released by a mechanical sloughing off of the cytoplasmic s t r a n d or by some kind of enzymatic action.
Structure and Assembly of Multipolid Virions The fine s t r u c t u r e of the budding atypically shaped and a b n o r m a l l y large spherical particles is essentially similar to t h a t of the n o r m a l particles (Fig. 1G). Cross sections of these particles show t h a t each of t h e m consists of two or more distinct RNPs, which indicates t h a t each is a composite of two or more particles. Hence, t h e y will subsequently be referred to as multiploids. Figure 4 shows the probable sequence of events in the assembly of the simplest form of multiploid, an essentially spherical particle containing two RNPs enclosed in a single envelope. The formation of such a particle appears to be initiated w h e n two n o r m a l C-type particles assemble in juxtaposition. T h a t the two RNPs r e m a i n discrete entities after assembly is indicated by the distinct b r e a k s in the ribonucleo-
FIG. 2. Negatively stained virions from culture medium ofts3-infected TB cells grown at the nonpermissive temperature for 48 hr then shifted to the permissive temperature for 1 hr. A-B, bar = 100 nm; C, bar = 40 nm. A, Ammonium molybdate-stained virions showing the polygonal subunits of the envelope or core shell (single white arrow), double striated track located just beneath the envelope (single black arrow), and the RNP within a virion (double black arrow) and protruding from a disrupted virion (double white arrow); B, ammonium molybdate-stained virions with the characteristic tails; C, a uranyl acetate-stained virion showing the viral envelope ~short black arrow), the core shell (double arrows), and the RNP (long black arrow).
FIc. 3. A - H , Different stages in the a s s e m b l y of n o r m a l virions of ts3; I, an e x t r a c e l l u l a r i m m a t u r e virion with ordered a r r a n g e m e n t of the s t r u c t u r a l components; J, an extracellular m a t u r e virion with centrally located dense core. Bar = 100 nm. 266
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FIG. 4. The probable sequence of e v e n t s in the a s s e m b l y of a spherical duplex. B a r = 100 nm. Black a r r o w s indicate b r e a k s in the R N P of the virions.
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protein component (Fig. 4, arrows). Whether or not each RNP contains a complete genome cannot be determined until more is known about the packaging of the viral RNA. The fact that a spherical duplex is larger (averaging about 125 nm in diameter) than a normal particle and its volume is about twice that of a normal particle suggests that it may consist of two complete RNPs. The structure and size of the normal ts3 particles and the spherical duplexes in different stages of development are compared in Fig. 5. In addition to these duplexes, thin section electron microscopy also revealed larger, almost spherical particles and atypically shaped particles containing three or more discrete RNPs. These multiploid particles appear to be assembled either in the same manner as suggested for the formation of duplexes or they are assembled into rod-shaped virions by the successive assembly of two or more particles at about the same site. Figure 6 shows representatives of multiploids in the early stages of assembly in ts3 infected cells grown at the nonpermissive temperature. That these virions are still in the process of assembly is indicated by the virions still being broadly linked to the cell cytoplasm. Those multiploids shown in Figs. 6B and E may eventually dissociate as single, essentially spherical, virions. By contrast, the first particle assembled in the rod-shaped virions shown in Figs. 6C and F may dissociate independently from the rest. Figure 7 shows some of the almost completely assembled multiploids in ts3-infected cells grown at the nonpermissive temperature and in ts3-infected cells grown at the nonpermissive temperature then shifted to the permissive temperature for 30 min. That these multiploids are in the final stages of assembly is indicated by the complete assembly of viral components, the resealing of the cell membrane (Figs. 7A, C, G, and I), and elongation of the cytoplasmic linkages. These multiploids may be divided into two broad groups: those that are linked to the cell by a common cytoplasmic strand but may eventually dissociate as individual particles each with its own complete genome
(Figs. 7D, G, and H), and those that will most likely remain as multiploids after dissociation from the cell membrane (Figs. 7A-C, E, F, and I). The heterogeneity of virion size in the viral pellet from a ts3infected culture grown at the nonpermissive temperature then shifted to the permissive temperature for 1 hr (Fig. 8) and in the negatively stained preparations (Figs. 2A and B) indicates that these multiploids were released into the culture medium. Their infectivity has not as yet been determined. DISCUSSION Our present findings on the structure of the budding and immature extracellular virions agree remarkably well with the gross structural components that have been previously reported (de Th6 and O'Connor, 1966; Sarkar et al., 1971; Nermut et al., 1972; Kakefuda and Bader, 1969). We have, however, observed substructures in the core shell and ribonucleoprotein component (RNP) which consists of the viral RNA and attendant protein hitherto unreported. Nermut et al. (1972) reported that the core shell of the Rauscher and Friend leukemia viruses consists of two structural elements: (i) a ~core membrane" and (ii) a layer of globular or ring-like morphological subunits forming a close hexagonal pattern. (The arrangement of these two layers of the core shell was reversed in a later report [Schafer et al., 1975]) We agree with Nermut et al. (1972) that the outer layer of the core shell consists of polygonal subunits. However, we cannot agree with their interpretation that the double striated tracks in negatively stained preparations represent a separate substructure of the core shell, viz, the "core membrane." The double tracks observed in ammonium molybdate-stained whole viral preparations appear to be the outer and inner edges of the polygonal subunits of the core shell and the striae to be the walls between the subunits. In uranyl acetate-stained whole viral preparations, the double tracks were not observed. Instead, two concentric rings with dense outer edges were resolved. The diameters
FIG. 5. A comparison of the size differences between normal ts3 virions (A, C, E) and spherical duplexes (B, D, F). A and B, Late budding virions; C and D, extracellular i m m a t u r e virions; E and F, m a t u r e virions. Bar = 50 nm. 269
FIG. 6. Early stages in the assembly of multiploids showing the broad cytoplasmic linkages of the virions to the cell. Bar = 100 nm. A, A budding patch consisting of at least six budding virions in close proximity to one another; B and E, essentially spherical multiploids; C, the simultaneous assembly of a normal virion and multiploids in juxtaposition; D and F, rod-shaped virions with two or more virions assembling in tandem. 270
FIG. 7. Multiploids in the final stages of assembly showing complete or near complete assembly of the structural components and elongate cytoplasmic linkages of the virion to the cell. Bar = 100 nm. A, A spherical multiploid with probably three pieces of genomic RNA; B, a n essentially spherical multiploid with four or five pieces of genomic RNA; C, a clover-shaped multiploid with three pieces of genomic RNA; D, A dumbbell-shaped duplex; E, a n egg-shaped duplex; F, a rod-shaped virion with at least three pieces or genomic RNA; G, a rod-shaped virion consisting of three virions assembling in tandem. H, three normal virions probably dissociating as a cluster; I, a large multiploid with five or more pieces of genomic RNA. 271
FIG. 8. Section of a viral pellet from a ts3-infected TB cell c u l t u r e g r o w n at t h e n o n p e r m i s s i v e t e m p e r a t u r e t h e n s h i f t e d to t h e p e r m i s s i v e t e m p e r a t u r e for 1 h r s h o w i n g i m m a t u r e d u p l e x e s (double arrows) a n d i m m a t u r e n o r m a l v i r i o n s (single arrow). B a r = 200 n m . 272
ULTRASTRUCTURE AND ASSEMBLY OF MuLV of the two concentric rings in the UAstained specimens could best be interpreted as the outer edges of the core shell and RNP of an immature virion. In the budding and immature virions, there is an elongated substructure in the core shell which extends from the center of each polygonal subunit to the periphery of the RNP. We tentatively suggest that these substructures serve as binding sites for the RNP during assembly and are probably destroyed on condensation of the RNP in the extracellular virion. We further propose that the layer of polygonal subunits is the most rigid component of the virus, and this rigidity determines the shape of the virus during assembly. This proposed role for the polygonal subunits of the core shell is consistent with the observation t h a t the uniformly packaged RNP in budding and immature virions follows the contour of this shell. In the budding and immature virions, the core shell is separated from the envelope by a uniform space. This ordered arrangement is lost on condensation of the RNP. Although no structural component has as yet been identified between the core shell and the envelope, we believe that some structure must be present to enable viral structural protein-protein interactions during assembly. However, subsequent to assembly, fracturing along this region is conceivable. The presence of rings and short tubular substructures in the RNP is consistent with the suggestion that the configuration of the RNP is a hollow sphere formed by supercoiling of a single-stranded or double-stranded helix (Kakefuda and Bader, 1969; Nowinski et al., 1970; Sarkar et al., 1971). The formation of multiploids is not peculiar to ts3 grown at the nonpermissive temperature. Multiploids, mostly duplexes, were also observed consistently, although in relatively lower numbers, in ts3infected cells grown at the permissive temperature, in MuLV-infected cells and also in Moloney murine sarcoma virus (MoMuSV) infected cells. Two virions assembling in juxtaposition have been reported in Rauscher leukemia virus (de Th~ and
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O'Connor, 1966) and particles with two cores have also been observed in Friend leukemia virus (Nermut et al., 1972). The latter type of particles is probably similar to the dumbbell-shaped duplexes shown in Fig. 7D, rather t h a n the essentially spherical particle shown in Fig. 4E. From published micrographs, for example, those of extracellular whole virus preparations (deHarven et al., 1973) or sections of viral pellets (de Th~ and O'Connor, 1966), variation in the size of the virions can be observed. Recently (Dmochowski et al., 1976), morphological differences in the budding, immature, and mature C-type viruses from mice were demonstrated. The viruses shown in the micrographs certainly included multiploids. Unfortunately, no mention was made as to whether these were observed in one or more virus populations. These observations suggest that the occurrence of multiploids, especially duplexes, is a more general phenomenon than has hitherto been appreciated. The occurrence of multiploid particles would be of special significance in mixed infection studies, since they may be capable of introducing into the same cell genomes from both parents. Heterozygosis has been postulated as a possible step in the formation of recombinants in Rous sarcoma viruses (Weiss et al., 1973). Multiploids could at least in part explain the phenotypic heterogeneity observed in the viral progeny of mixed infections such as those reported by Fischinger and O'Connor (1970), Ball et al. (1973), and Hopkins et al. (1976). Multiploids are also of importance in recombination studies. Recombination frequencies obtained could be artificially enhanced due to the presence of heterozygotes, as was found to be the case in crosses between three ts mutants of MoMuLV (Wong and McCarter, 1973) where a recombination frequency of about 8% was obtained. However, subsequent recloning of the apparent wild-type progenies from the 34° harvest of one of the crosses, tsl × ts3, showed t h a t 6/14 (i.e., nearly 40%) of the clones segregated with ts phenotype (unpublished data). Since the possibility exists that the frequency in the
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formation of heterozygotes may vary from o n e m i x e d i n f e c t i o n to a n o t h e r , a t t e m p t s at mapping of the RNA tumor viral genome by recombination frequencies will entail at least recloning of the progeny of the first filial generation. ACKNOWLEDGMENTS We wish to thank Ms. H. C. Chen, Ms. K. Hedges, Ms. L. Jones, and Ms. K. O'Connor for their skillful assistance. This investigation is supported by Public Health Service Research Grant No. CA17695 awarded by the National Cancer Institute to PKYW. REFERENCES BALL, J. K., HUH, T. Y., and MCCARTER, J. A. (1964). On the statistical distribution of epidermal papillomata in mice. Brit. J. Cancer 18, 120-123. BALL,J. K., MCCARTER,J. A., and SUNDERLAND,S. M. (1973). Evidence for helper independent murine sarcoma virus. Virology 56, 268-284. DMOCHOWSKI,L., BOWEN, J. M., MYERS, B., PRIORI, E. S., MILLER,M. F., SEMAN, G., CHAN, J. C., DODSON, M. L., SCANLON, M., OHTSUKI, Y., and YOSHIDA, H. (1976). Comparative studies of type C, type B and M-PM oncornaviruses. In "Comparative Leukemia Research 1975", (J. Clemmesen and V. S. Yohn, eds.), Bibl. Haernatol., No. 43, pp. 417-424. Karger, Basel. FISCHINGER,P. J., and O'CONNOR,T. E. (1970). Replication of defective and competent forms of murine sarcoma virus in mouse cells. Virology 41, 233-243. DEHARVEN,E., BEJU, D., EVENSON,D. P., BAsu, S., and SCHIDLOVSKY,G. (1973). Structure of critical point dried oncornaviruses. Virology 55, 535-540. HOPKINS, N., TRAKTMAN, P., and WHALEN, K. (1976). N-Tropic variants obtained after co-infection with N- and B-tropic murine leukemia viruses. J. Virol. 18, 324-331. KAKEFUDA, T., and BADER, J. P. (1969). Electron microscopic observations of the ribonucleic acid of murine leukemia virus. J. Virol. 4, 460-474.
NERMUT, M. V., FRANK, H., and SCHAFER, W. (1972). Properties of mouse leukemia viruses. III. Electron microscopic appearance as revealed after conventional preparation techniques as well as freeze-drying and freeze-etching. Virology 49, 345-358. NOWINSKI,R. C., OLD, L. J., SARKAR,N. H., and MOORE, D. H. (1970). Common properties of the oncogenic RNA viruses (oncornaviruses). Virology 42, 1152-1157. SARKAR, N. H., NOWINSKI,R. C., and MOORE, D. H. (1971). Helical neocapsid structure of the oncogenic ribonucleic acid viruses (oncornaviruses). J. Virol. 8, 564-572. SCHAFER, W., DEMSEY, A., FRANK, H., HUNSMAN, G., LANGE, J., MOENNIG, V., PISTER, L., BOLOGNESI, D. P., GREEN,R. W., LUFTIG,R. B., SHAPER, J., and HUPER, G. (1975). Mammalian C-type oncorna viruses. In "Comparative Leukemia Research 1973" (Y. Ito and R. M. Dutcher, eds.), Leukemogenesis, Bibl. Haemat., No. 40, Karger, Basel. DE THe, G., and O'CONNOR, T. E. (1966). Structure of a murine leukemia virus after disruption with Tween-ether and comparison with two myxoviruses. Virology 28, 713-728. WEISS, R. A., MASON, W. S., and VOGT, P. K. (1973). Genetic recombinants and heterozygotes derived from endogenous and exogenous avian RNA tumor viruses. Virology 52, 535-552. WONG, P. K. Y., and MCCARTER,J. A. (1973). Genetic studies of temperature sensitive mutants of Moloney murine leukemia virus. Virology 53, 319326. WONG,P. K. Y., and MCCARTER,J. A. (1974). Studies of two temperature-sensitive mutants of Molohey murine leukemia virus. Virology 58, 396-408. WONG, P. K. Y., and MACLEOD,R. (]975). Studies on the budding process of a temperature-sensitive mutant of murine leukemia virus with a scanning electron microscope. J. Virol. 16, 434-442. WONG, P. K. Y., Russ, L. J., and MCCARTER, J. A. (1973). Rapid, selective procedure for isolation of spontaneous temperature-sensitive mutants of Moloney leukemia virus. Virology 51, 424-431.