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Study of poliovirus infection of human and monkey cells by indirect immunoferritin technique
VIROLOGY
39, 211-223 (1969)
Study
of Poliovirus Cells
by Indirect
J. D. LEVINTHAL, Department
Infection
of Human
Immunoferritin
T. H. DIJNNEBACKE,
of Molecular Biology
and
Monkey
Technique’ AND
and Virus Laboratory, Berkeley, California 94720
R. C. WILLIAMS University
of California,
Accepted May $4, 1969
Primary human amnion, human chorion, and human embryonic kidney, continuous lines of human amnion, human HEp-2, and HeLa, and continuous line monkey BS-C-1 cells were infected with poliovirus type 1, Brunhilde strain at a multiplicity of 2@200 PFU/cell. After aldehyde fixation and digitonin permeabilisation, cells were reacted with rabbit antipoliovirus serum followed by ferritin-labeled goat antirabbit globulin. Full and empty particles, ferritin coated by this technique and presumed to represent complete and incomplete progeny virus, were found 3 or more hours after infection: (a) free in the extracellular medium, (b) emerging at the plasma membrane by way of a tubulovacuolar excretory system, (c) free in the cytoplasm near the plasma membrane and among clusters of vesicles, and (d) aligned on fibrils, in the cytoplasm and in cytoplasmic blebs. Fibrils, vesicles, and the smooth endoplasmic reticulum were progressively more heavily coated with ferritin over the 3-6period of poliovirus urotein nroduction. an observation interpreted as demonstrating iocal accumulations*of free viral protein. INTRODUCTION
Studies by electron microscopy (Kallman et al., 1958; Dales and Franklin, 1962; Dales et al., 1965) indicate that the features com-
mon to cytolytic picornavirus infection include disaggregation of host polyribosomes followed by appearance of viral polyribosomes, the formation of aggregates of irregularly dense, coarse, granular material which have been called viroplasm (Dales et al., 1965), and the proliferation of multiple, full or empty, single-walled vesicles in the cytoplasm. Membrane-bound bodies are formed that contain material resembling cytoplasmic matrix and ribosomes; the nucleus shrinks and is displaced; and loss of plasma membrane integrity is shown by gradual leakage of cytoplasmic matrix. The 1 This investigation was supported in part by U.S. Public Health Service Research Grant CA 02245 and Training Grant CA 05628 from the National Cancer Institute; and National Science Foundation Research Grant GB 6918.
identification of single viral particles is complicated by the fact that they are about the same size as ribosomes. Nonetheless, some of the progeny virus are recognized when (a) aggregated into intracytoplasmic crystals, (b) lying as free particles deep in the cytoplasm among membranous vesicles; (c) enmeshed in a network of membrane-bounded channels, and (d) aligned along fib& in the cytopIasm (Rifkind et cd., 1961). No obvious sites of viral assembly appear, however, nor is it evident what structural changes occur secondary to virus-induced interference with cellular RNA, protein, and lipid metabolism (Penman and Summers, 1965), or secondary to “toxic” viral products (Bablanian et at., 1956a,b; Amako and Dales, 1967). In order to clarify the mechanisms of the initial and final stages of the viral cycle, Dunnebacke, Levinthal, and Williams have studied~byelectrcm microscopy the adsorption and penetration of poliovirus and the 211
‘11’) - -
LEVINTHAL,
DUNNEBACKE,
(below), gave similar findings when stained by immunofluorescence, although the staining after the latter method was less brilliant. Ferritin and fluorescein double-labeled antirabbit globulin was prepared as previously described (Levinthal et ab., 1967a) from goat antirabbit globulin purchased from Antibodies, Inc., Davis, California, and used at a concentration between 0.6 and 1.8 % proteins (as measured by Perkin-EImer Hitachi hand refractometer). Cells. Cells from human amniotic membranes, human chorion, and human embryonic kidney (HEK), kindly made available to us by Dr. A. W. Xakepeace and Mrs. Sylvia Mcleod (Kaiser Foundation Hospital, Oakland, California), were trypsinized and cultured in medium 199 containing 10% human serum and 10 % calf serum. After the cells became established, the human serum was omitted from the medium. HeLa, FL, and Lad-Al (a strain of human amnion developed in this laboratory) were cultured in Medium 199 with 10% calf serum, KB in Eagle’s ME!M with 2% fetal bovine serum, and HEp-2 and BS-C-1 (African green monkey) in L15 (Leibovitz) with 10 % fetal bovine serum, and antibiotics. The KB, HEp-2, Lad-Al, and BS-C-1 lines with which most of the immunoferritin work was done were contaminated by PPLO, but this did not interfere with the course of the infection by poliovirus or with the reaction with antibody. The basic media were prepared from powered mixtures (Grand Island Biological Company). Cells were plated in plastic petri dishes and incubated in a moistened 5 % CO2 atmosphere, at 37”. Virus. Poliovirus, type 1, strain Brunhilde, 20-200 PFU/cell, was added to cultures after 2 washes of Dulbecco buffer. After 45 min at
releahc of progeny virus (1969). In brief, they found that inoculat,ed poliovirus directly penetrates the plasma membrane within a few minutes, rather than being taken up by phagocytosis as suggested by previous studies (Mandel, 1962; Dales et al., 1965). Following penetration, neither intracellular nor extracellular virus was found during 1 and 2 hours postinoculation. After 3 hours, however, progeny virus was found deep in the cytoplasm, free among vesicles, and within a tubular system opening through the plasma membrane. After 4 hours, virus was also occasionally found aligned on fibrils in the cytoplasm or in cytoplasmic blebs. Since the indirect immunoferritin technique allows identification and localization of viral antigens, both as assembled capsids and in unassembled form, this technique has been applied in a study of the period in which poliovirus is formed and released. It was found that individual full and empty virus particles could be identified and localized. Although the sites of viral antigen production were not localized to the ribosomal level, accumulations of viral antigen were found. MATERIALS
AND
AN11 WILLIAJIS
react.ions
METHODS
Sera. Antipolio rabbit sera were prepared by 3 weekly intramuscular injections, in 4 sites, of cesmm chloride-purified poliovirus (lo8 PFU/inoculation) emulsified in incomplete Freund’s adjuvant (Difco), a booster dose 1 month later, and bleeding the following week. The efficiency of infection was monitored by indirect immunofluorescence using the antipolio serum diluted 1:40 and an antiglobulin singly labeled with fluorescein. Infected cells fixed with cold acetone, or by the two-stage method used for immunoferritin -
FIG. 1. (a) Indirect immunofluorescence, human embryonic kidney cells infected with poliovirus, 4 hours. Rabbit antipolio serum plus fluorescein-labeled sheep antirabbit globulin. X 1600.‘(b) Indirect immunoferritin. Note preservation of fine structure in HEp-2 cell, poliovirus infected, 4 hours, subjected to fixation, permeabilization and antibody reactions. Infected cytoplasm is freely penetrated by ferritin marker (barely visible at this low magnification). Multiple vesicles (ves), viroplasm, and many polyribosomes are characteristic of poliovirus infection at this time. X 33,000. (c) Uninfected HEp-2 cell, treated as infected cell, with antipolio serum 1:lO and ferritin-labeled antiglobulin, 1.7%. Minimal nonspecific reaction, chiefly upon fibrils. rer, rough endoplasmic reticulum. X 91,000. (d) Degenerate, uninfected HEp-2 cell, same control preparation. Marked nonspecific ferritin ret)ention. X 91,COO.
IMMUNOFERRITIN
STUDY
FIG. 1
OF POLIOVIRUS
‘14
LEVINTHAL.
DUXNEBACKE,
37”, medium without serum was added. Addition of virus was counted as time zero. Infectivity titers mere determined by plaque assay on monolayer cultures of HEp-2 and BS-C-l cells. Electron microscopy. Seventy-four specimens for comparative morphologic study of uninfected and infected cells were taken at intervals from 2 min through 20 hours after inoculation. They were fixed as monolayers, after decanting the medium and floating dead cells, in 1.6% glutaraldehyde (from 25 % stock, pH 3.4 or greater) in phosphatebuffered saline pH 7.2 with added magnesium chloride 5 X lop3 M and calcium chloride 1 X 1O-4 il/ir (PBS MgCa). The fixed cells were then scraped and postfixed as a centrifuged pellet in 1% osmic acid in PBSMg Ca, dehydrated in a graded ethanol 0.15 M NaCl series, infiltrated with propylene oxide, embedded in Epon 82, and cut with diamond knives on an LKB ultramicrotome. Sections were stained with 2% aqueous uranyl acetate and alkaline lead citrate, mounted on bare or collodion-coated, carbonized copper mesh grids, and photographed in an Elmiskop 1. Indirect immunoferritin technique. Seventy-three specimens, taken at the same intervals as above for reaction with antibody, were prepared by a two-stage fixation: first, 0.01% formaldehyde (made from paraformaldehyde) and 0.008 % glutaraldehyde in PBS MgCa, then permeabilization with 1.2 X 10e4 M digitonin in PBS MgCa, and finally 1% formaldehyde (with or without 0.8% glutaraldehyde) in PBS MgCa, each applied for 1 min in succession, followed by 3 washes of PBS MgCa over a period of 1 hour to 7 days. The fixed cells were pelleted in McNaught protein tubes, and then dispersed with a platinum wire into 5 volumes of rabbit antipolio serum diluted 1: 10 to 1:80 in PBS MgCa. After reaction for 1 hour at room temperature, antibody
AND
WILLIAMS
was removed and the cells were washed vigorously 3 times in 6.5 ml of PBS MgCa. Ferritin-labeled antirabbit globulin (5 volumes) was added for 1 hour, and after antibody removal and 3 vigorous washes in PBS I\IgCa, the cells were pelleted and postfixed in 1% osmic acid in PBS MgCa prior to embedding in Epon and thin sectioning. Controls consisted of normal cells treated in the same way, and virus infected cells in which a 1: 10 to 1:40 dilution of normal rabbit serum was substituted in the indirect immunoferritin reaction. RESULTS
ImmunoJluorescence Standard indirect immunoffuorescence of infected HEK cells with antipolio rabbit sera is illustrated in Fig. la. At 4 hours, there is diffuse cytoplasmic fluorescence with small peripheral accumulations, probably representing cytoplasmic blebs. In the 3-6hour period after inoculation the intensity of the fluorescence increased with time, and in a few cells was concentrated in a diffuse paranuclear mass, probably representing areas of multiple vesicles. I,ndirect Immunoferritin Fine structure. Excellent retention of fine structure was accomplished by two-stage fixation as shown at low magnification in Fig. lb. Controls. Healthy, uninfected cells showed variable, but generally minimal nonspecific retention of ferritin labeled antiglobulin, chiefly upon fib& following reaction with normal or antiviral rabbit sera (Fig. lc). Degenerate, uninfected cells, where cell death was evident from gross distortion of normal fine structure and presence of lysosomes, often retained considerable amounts of ferritin-labeled antiglobulin after reaction with any rabbit serum, a finding similar to the
FIGS. 2-7. Cells are polio-infected and subjected to immunoferritin reactions. FIG. 2. (A) HEp-2 cells, 3 hours. Note absence of ferritin in nucleus, wide distribution
of ferritin in infected cytoplasm except for mitochondrion (M) and nuclear membranes. Vesicles (ves), fibrils (jib), and viroplasm (op), lightly coated with ferritin. Ferritin scattered among free ribosomes (rib). X 61,006. (BD) Details of same cell, including in (C) plasma membrane and extracellular ferritin ringed virus (arrow). x 94,000.
IMMUNOFERRITIN
STUDY
FIG. 2
OF POLIOVIRUS
215
FIG. 3. (A) Multiple vesicle area, HEp-2,4 hours, with ferritin around full virus particles (arrows) and empty ones (e). Ferritin on fibrils (fib), but not associated with rough endoplasmic reticulum (rer), mitochondria (M), aud vesicle surfaces (except at inter&ices). X 70,000. (B and C). Details of Fig. 3A. X 104,OCO. (D) Detail of Fig. 4A. X 128,000.
216
FIG. 4. Other cytoplasmic structures, HEp-2, 4 hours. (A) Ferritin on fibrils (Jib) lying between the clustered vesicles and the nuclear and plasma membrane. Virus particles shown by arrows. X 70,000. (B and C) Collared vesicles (outlined). (B) X 80,000; (C) X 100,000. (D) Virus particles (arrows) and fibrils (fib) show ferritin, but viroplasm (VP) has very little. 217
21s
LEVINTHAL,
MJNNEBACKE,
AND
WILLIAMS
FIG. 5. Entvbulated progeny virus (all HEp-2 cells). (a) Intracellular, entubulated virus near nucleus (N), alld vesicles (MS); 3 hours. X 100,000. (b) Extracellular ferritin only at sit.e of tubule opening, 4 holux. X 68,000. (c) Virus emerging at 4 hours from tubules are ferritin coated. A free polyribosome (pr) is coated, but the rough endoplasmic reticulllm (rer) is not. (d) Free extracellular particles at 4 hours are heavily coated; free intracytoplasmic particle (double arrow) less so. Particles in tubules are ferritin free. (e) Extracellular, ferritin-ringed virus adsorbed to debris. Free intracellular ferritin-coated virus (double arrow). Tangential cut (outlined) of entubulated virus. X 100,000.
IMMUNOFERRITIN
STUDY
OF POLIOVIRUS
219
FIG. 6. Alignment of poliovirus on cytoplasmic fibrils. (a) HEp-2 cell, 4 hours. Ferritin coats accessible particles (arrows) but nearby ribosomes (rib) and mitochondrion show no ferritin. X 91,906. (b) Progeny virus, full and empty, some ferritin coated (arrows), aligned on cytoplasmic fibrils near plasma membrane, HEp-2 cell, 6 hours. X 72,066. (c) HEp-2 cell, 4 hours. Projecting bleb contains both enfibrilated and entubulated virus (arrow). X 48,990.
220
LEVTNTHAL,
DUNNEBBCKE,
nonspecific staining of dead cells seen in immunofluorescence (Fig. Id). Virus infected cells reacted with normal sera during the 3-G-hour period of virus production by still living cells (impermeable to 0.5 % erythrocin B when unfixed) showed more nonspecific retention of ferritin in the cytoplasm than did uninfected cells. However, infected cells showed distinctly more ferritin retention after reaction with specific antiviral sera. At no time after infection was there retention of ferritin antiglobulin in the Golgi appanucleus, upon mitochondria, ratus, intact plasma membrane, or membrane bound ribosomes. Distribution of ferritin in infected cells reacted with speci$c antisera. At 3 hours, when extracellular, ferritin-coated progeny virus was first noted, the cells showed a fairly dispersed distribution of intracullular ferritin with some concentration about vesicles, fibrils and viroplasm (Fig. 2). In the 4-6-hour period, paralleling the increase in intensity of immunofluorescent staining, there was an increase in the amount of ferritin coating of cytoplasmic structures. Ferritin was also localized to material among the clusters of vesicles whose formation is a prominent feature of this period, and to single dense or empty particles among the vesicles, presumed, therefore, to be complete or incomplete progeny virus (Fig. 3). Sheaves of fibrils were found near the clusters of vesicles and were frequently coated heavily with ferritin (Fig. 4A). Ferritin was also found associated with some of the polyribosomes (Fig. 5c) and with occasional small vesicles that were surrounded by a collar of lightly staining material (Fig. 4B and C). In contrast to its reaction at 3 hours, viroplasm was variably and minimally ferritin-associated (Fig. 4D). Progeny virus enclosed in the tubulovacuolar excretion system (which appeared first at 3 hours and enclosed only full particles) were not ferritin coated (Fig. 5a and b) except where the
ANI)
WILLIAMF
tubules opened through the plasma membrane (Fig. 5c and d). Neither the walls of the tubules (approximately 50 m,u in diameter when sectioned transversely), nor the plasma membrane near the particles undergoing release, retained ferritin. Single, free, intracytoplasmic particles were frequently found, but not enclosed in tubules, near the plasma membrane; they were ferritin coated, but not as heavily as extracellular particles (Fig. 5d).The extracellular, ferritin-ringed, dense particles were often found adsorbed to cellular debris, or to plasma membranes (Fig. 5c, d and e; Fig. 2C); extracellular empty particles were rare. Particles, both full and empty, were found aligned in parallel array on fibrils after 4 hours, and were coated with ferritin where defects in the lattice were apparent (Fig. 6). Figures 6 and 7 show a sequence suggesting that alignment on fibrils is followed by migration of the complex to the cell surface, formation of a cytoplasmic bleb and final separation from the cell. The existence of ferritin-coated, free particles near the extruded bleb suggests that virus is eventually released by disintegration of the bleb (Fig. 7C). DISCUSSION
Infection by poliovirus results in an accumulation of viral proteins within host cells, followed by their structural degeneration, as evidenced particularly by permeability of the plasma membrane to vital dyes and to ferritin after about 6 hours. Since both ferritin and globulin can bind nonspecifically to structures within such degenerate cells, the localization of specific intracellular antigen can be fruitfully attempted only during a relatively short interval: from about 2.5 hours, when the structural viral protein first appears (Scharff and Levintow, 1963), to about 6 hours, the onset of membrane permeability. During this interval, some 90% of the structural protein is made (Darnell et al., 1960). Our observations were restricted
FIG. 7. Formation, separation, and dissolution of cytoplasmic blebs. (A) BS-C-1 cell, 4 hours. Free cytoplasmic bleb. X 72,000. (B) HEp-2 cell, 4 hours. Free cytoplasmic bleb, with nearby ferritin-coated virus (arrows), suggesting virus is liberated by breakdown of bleb. X 70,000. (C) HEp-2 cell, 4 hours. Two cytoplasmic blebs at cell surface, one containing full and empty particles on fibrils, the other, no visible particles. Entubulated and free virus particles (arrows) near site of bleb formation. X 57,ooO.
IMMUNOFERRITIN
STUDY
FIG. 7
OF POLIOVIRUS
221
to this 2.5-6 hour interval and were considered significant only for those cells that, although infected, showed good retention of their normal fine structure. Virus A necessary condition for the immunoferritin technique to be applicable to a search for viral antigens in a system devoid of morphologically distinctive “virus factories” is a satisfactory demonstration that particles, morphologically identifiable as virus, are closely associated with ferritin molecules. Such particles are the extracellular ones, and these were found, 3 or more hours after poliovirus inoculation, to be well ringed with ferritin. In view of the certainty of antigenic identification of these particles we make the plausible presumption that intracellular virus and aggregates of unassembled viral antigen can also be identified in the same specimens. Our results shed light upon the nature of the extracellular virus particles and the free intracytoplasmic particles, as found after 3 hours following inoculation. Holland and Hoyer (1962) determined that polivirus, adsorbed at 37” and subsequently eluted, loses its ability to react with antibody and to readsorb to cell membranes. Since we found that the 3-hour extracellular virus particles bind to ferritin-conjugated antibody and adsorb to extracellular membranous debris and plasma membranes, and since such particles are not seen from 1 to 2 hours postinoculation, we conclude that they are progeny virus rather than residual virus from the inoculum. It is also reasonable to conclude that the 3-hour, intracellular particles, free and ringed with ferritin, are progeny virus inasmuch as none of them were found in the control reactions with ferritin, nor were they found during the 1-2hour interval. The entubulated, ferritin-free particles found in the cytoplasm are most likely progeny virus, since they did not appear before 3 hours and, when found apparently extruded from the tubules into the extracellular space, were ringed with ferritin. Unassembled Structural Antigens From identification of single particles extracellularly, or of completed capsids in
the cytoplasm, to localization of poliovirus protcms prior t,o assembly into complete shells is a move into more hazardous territory. We do believe, however, that these speculations are plausible: Clustered vesicles. Full and empty ferritinringed capsids were so regularly found among the clustered vesicles in the 4-6-hour infections that some function in virus assembly may be ascribed to the vesicle surface, especially in view of the observations of Penman et al. (1964) that a membranous complex exists associated with viral RNA, RX-4 polymerase, virus protein, and newly formed virus particles. Ferritin molecules were consistently found dotting the interstices of the vesicIes, in addition to surrounding the full and empty particles similarly located, making it Iikely that accumulations of structural viral proteins were located in these regions prior to assembly into complete shells. Considerable evidence favors the notion of a proliferation of vesicles as a response to “toxic” viral proteins (Bablanian et al., 1965a; Amako and Dales, 1967), but such evidence does not preclude the possibility that the vesicle regions are also virus assembly sites. ViropZasm. Since specimens taken at 3 hours show some ferritin coating of viroplasm, while the 4-6-hour ones show little or none, association of viral antigen with these structures may be temporary. It may be that they are a cellular reaction product, perhaps the result of agglutination of degenerating ribosomes, rather than structures actively involved in virus production. Other cytoplasmic structures. The increased association, with time after inoculation, of ferritin molecules with fibrils, smooth ER, small vesicles, and cytoplasmic matrix implies that free viral protein increases in amount and is widely distributed. Although a portion of dispersed ferritin may represent nonspecific reactions in gradually dying cells, it consistently occurs in cells that retain good fine structure and an intact plasma membrane (as demonstrated by vital dye exclusion) . D. Virus release. The elimination of progeny virus from still viable cells appears to occur by two mechanisms. In the first, newly formed, complete virus particles are
IMMUNOFERRITIN
STUDY
enclosed in, and later released from, tubules whose diameter is fairly constant except near the plasma membrane where they occasionally appear to coalesce into vacuolar forms. The tubules are probably derived from the ER, as suggested also by Blinzinger et al. (1969), who observed membranes enclosing small, intracellular virus crystals. The role of membranous channels, both tubules and narrow cisternae, in virus release has previously been noted in infections with SV40 (Levinthal et al., 1967 b), and vesicular stomatitis (Zee et al., 1968). In the second mechanism of release, both full and empty particles are aligned on fibrils and subsequently eliminated from the infected cell in cytoplasmic blebs. REFERENCES AMAKO, Ii., and DALES, S. (1967). Cytopathology of mengovirus infection. II. Proliferation of membranous cisternae. Virology 32,201-215. BABLANIAN, R., EGGERS, H. J., and TAMM, I. (1965a). Studies on the mechanism of poliovirusinduced cell damage. I. The relation between poliovirus-induced metabolic and morphological alterations in cultured cells. Virology 26, lOO113. BABLANIAN, R., EGG~RS, H. J., and TAMM, I. (1965b). Studies on the mechanism of poliovirusinduced cell damage. II. The relation between poliovirus growth and virus-induced morphological changes in cells. Virology 26,114-121. BLINZINGER, K., SIMON, J., MAGRATH, D., and BOULGER, L. (1969). Poliovirus crystals within the endoplasmic reticulum of endothelial and mononuclear cells in the monkey spinal cord. Science 163, 1336-1337. DALES, S., and FRANKLIN, R. M. (1962). A comparison of the changes in fine structure of L cells during single cycles of virus multiplication, following their infection with the viruses of Mengo and encephalomyocarditis. J. Cell Biol. 14, 281-302. DALES, S., EGGERS, H. J., TAMM, I., and PALADE, G. E. (1965). Electron microscopic study of the formation of poliovirus. Virology 26, 379-389.
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DARNELL, J. E., SCHAHFF, M. D., and LEVINTOW, L. (1960). Poliovirus protein: source of amino acids and time of synthesis. J. Biol. Chem. 235, 74-77. DUNNEB.~CKE, T. H., LEVINTHAL, J. D., and WILLIAMS, IL. C. (1969). Entry and release of poliovirus as observed by electron microscopy of cultured cells. J. Viral. in press. HOLLAND, J. J., and HOYB;R, B. H. (1962). Early stages of enterovirus infection. Cold Spring Harbor Symp. Quant. Biol. 27, 101-111. KALLMAN, F., WILLIAMS, R,. C., DULBJXCO, R., and \r~~~, M. (1958). Fine structure of changes produced in cultured cells sampled at specified intervals during a single growth cycle of poliovirus. J. Biophya. Biochem. Cytol. 4,301-308. LEVINTHAL, J. D., CEROTTINI, J. C., AHMADZADXH, C., and WICK~X, R. (1967a). The detection of intracellular adenovirus type 12 antigens by indirect immunoferritin technique. Intern. J. Cancer 2, 85-102. LEVINTHAL, J. I)., WICKER, It., and CEROTTINI, J. C. (1967b). Study of intracellular SV40 antigens by indirect immunoferritin technique. Virology 31, 555-558. MANDEL, B. (1962). Early stages of virus-cell interaction asstudied by using antibody. Cold Spring Harbor Symp. Quant. Biol. 27, 123-131. PENMAN, S., BECICER, Y., and DARNELL, J. E. (1964). A cytoplasmic structure involved in synthesis and assembly of poliovirus components. J. Mol. Biol. 8, 541-555. PENMAN, S., and SUMMERS, D. (1965). Effects on host cell metabolism following synchronous infection with poliovirus. Virology 27,614-620. RIFKIND, It. A., GODMAN, G. C., HOWE, C., MORGAN, C., and ROSE, H. M. (1961). Structure and development of viruses as observed in the electron microscope. VI. ECHO virus type 9. J. Exptl. Med. 114, 1-12. SCHARFF, M. D., and LEVINTO~, L. (1963). Quantitative study of the formation of poliovirus antigens in infected HeLa cells. Virology 19, 491-500. ZEE, Y. C., HACKETT, A. J., and TALENS, L. T. (1968).Electronmicroscopicstudies on the vesicular exanthema of swine virus. II. Morphogenesis of VESV type Ha4 in pig kidney cells. Virology 34, 596607.
39, 211-223 (1969)
Study
of Poliovirus Cells
by Indirect
J. D. LEVINTHAL, Department
Infection
of Human
Immunoferritin
T. H. DIJNNEBACKE,
of Molecular Biology
and
Monkey
Technique’ AND
and Virus Laboratory, Berkeley, California 94720
R. C. WILLIAMS University
of California,
Accepted May $4, 1969
Primary human amnion, human chorion, and human embryonic kidney, continuous lines of human amnion, human HEp-2, and HeLa, and continuous line monkey BS-C-1 cells were infected with poliovirus type 1, Brunhilde strain at a multiplicity of 2@200 PFU/cell. After aldehyde fixation and digitonin permeabilisation, cells were reacted with rabbit antipoliovirus serum followed by ferritin-labeled goat antirabbit globulin. Full and empty particles, ferritin coated by this technique and presumed to represent complete and incomplete progeny virus, were found 3 or more hours after infection: (a) free in the extracellular medium, (b) emerging at the plasma membrane by way of a tubulovacuolar excretory system, (c) free in the cytoplasm near the plasma membrane and among clusters of vesicles, and (d) aligned on fibrils, in the cytoplasm and in cytoplasmic blebs. Fibrils, vesicles, and the smooth endoplasmic reticulum were progressively more heavily coated with ferritin over the 3-6period of poliovirus urotein nroduction. an observation interpreted as demonstrating iocal accumulations*of free viral protein. INTRODUCTION
Studies by electron microscopy (Kallman et al., 1958; Dales and Franklin, 1962; Dales et al., 1965) indicate that the features com-
mon to cytolytic picornavirus infection include disaggregation of host polyribosomes followed by appearance of viral polyribosomes, the formation of aggregates of irregularly dense, coarse, granular material which have been called viroplasm (Dales et al., 1965), and the proliferation of multiple, full or empty, single-walled vesicles in the cytoplasm. Membrane-bound bodies are formed that contain material resembling cytoplasmic matrix and ribosomes; the nucleus shrinks and is displaced; and loss of plasma membrane integrity is shown by gradual leakage of cytoplasmic matrix. The 1 This investigation was supported in part by U.S. Public Health Service Research Grant CA 02245 and Training Grant CA 05628 from the National Cancer Institute; and National Science Foundation Research Grant GB 6918.
identification of single viral particles is complicated by the fact that they are about the same size as ribosomes. Nonetheless, some of the progeny virus are recognized when (a) aggregated into intracytoplasmic crystals, (b) lying as free particles deep in the cytoplasm among membranous vesicles; (c) enmeshed in a network of membrane-bounded channels, and (d) aligned along fib& in the cytopIasm (Rifkind et cd., 1961). No obvious sites of viral assembly appear, however, nor is it evident what structural changes occur secondary to virus-induced interference with cellular RNA, protein, and lipid metabolism (Penman and Summers, 1965), or secondary to “toxic” viral products (Bablanian et at., 1956a,b; Amako and Dales, 1967). In order to clarify the mechanisms of the initial and final stages of the viral cycle, Dunnebacke, Levinthal, and Williams have studied~byelectrcm microscopy the adsorption and penetration of poliovirus and the 211
‘11’) - -
LEVINTHAL,
DUNNEBACKE,
(below), gave similar findings when stained by immunofluorescence, although the staining after the latter method was less brilliant. Ferritin and fluorescein double-labeled antirabbit globulin was prepared as previously described (Levinthal et ab., 1967a) from goat antirabbit globulin purchased from Antibodies, Inc., Davis, California, and used at a concentration between 0.6 and 1.8 % proteins (as measured by Perkin-EImer Hitachi hand refractometer). Cells. Cells from human amniotic membranes, human chorion, and human embryonic kidney (HEK), kindly made available to us by Dr. A. W. Xakepeace and Mrs. Sylvia Mcleod (Kaiser Foundation Hospital, Oakland, California), were trypsinized and cultured in medium 199 containing 10% human serum and 10 % calf serum. After the cells became established, the human serum was omitted from the medium. HeLa, FL, and Lad-Al (a strain of human amnion developed in this laboratory) were cultured in Medium 199 with 10% calf serum, KB in Eagle’s ME!M with 2% fetal bovine serum, and HEp-2 and BS-C-1 (African green monkey) in L15 (Leibovitz) with 10 % fetal bovine serum, and antibiotics. The KB, HEp-2, Lad-Al, and BS-C-1 lines with which most of the immunoferritin work was done were contaminated by PPLO, but this did not interfere with the course of the infection by poliovirus or with the reaction with antibody. The basic media were prepared from powered mixtures (Grand Island Biological Company). Cells were plated in plastic petri dishes and incubated in a moistened 5 % CO2 atmosphere, at 37”. Virus. Poliovirus, type 1, strain Brunhilde, 20-200 PFU/cell, was added to cultures after 2 washes of Dulbecco buffer. After 45 min at
releahc of progeny virus (1969). In brief, they found that inoculat,ed poliovirus directly penetrates the plasma membrane within a few minutes, rather than being taken up by phagocytosis as suggested by previous studies (Mandel, 1962; Dales et al., 1965). Following penetration, neither intracellular nor extracellular virus was found during 1 and 2 hours postinoculation. After 3 hours, however, progeny virus was found deep in the cytoplasm, free among vesicles, and within a tubular system opening through the plasma membrane. After 4 hours, virus was also occasionally found aligned on fibrils in the cytoplasm or in cytoplasmic blebs. Since the indirect immunoferritin technique allows identification and localization of viral antigens, both as assembled capsids and in unassembled form, this technique has been applied in a study of the period in which poliovirus is formed and released. It was found that individual full and empty virus particles could be identified and localized. Although the sites of viral antigen production were not localized to the ribosomal level, accumulations of viral antigen were found. MATERIALS
AND
AN11 WILLIAJIS
react.ions
METHODS
Sera. Antipolio rabbit sera were prepared by 3 weekly intramuscular injections, in 4 sites, of cesmm chloride-purified poliovirus (lo8 PFU/inoculation) emulsified in incomplete Freund’s adjuvant (Difco), a booster dose 1 month later, and bleeding the following week. The efficiency of infection was monitored by indirect immunofluorescence using the antipolio serum diluted 1:40 and an antiglobulin singly labeled with fluorescein. Infected cells fixed with cold acetone, or by the two-stage method used for immunoferritin -
FIG. 1. (a) Indirect immunofluorescence, human embryonic kidney cells infected with poliovirus, 4 hours. Rabbit antipolio serum plus fluorescein-labeled sheep antirabbit globulin. X 1600.‘(b) Indirect immunoferritin. Note preservation of fine structure in HEp-2 cell, poliovirus infected, 4 hours, subjected to fixation, permeabilization and antibody reactions. Infected cytoplasm is freely penetrated by ferritin marker (barely visible at this low magnification). Multiple vesicles (ves), viroplasm, and many polyribosomes are characteristic of poliovirus infection at this time. X 33,000. (c) Uninfected HEp-2 cell, treated as infected cell, with antipolio serum 1:lO and ferritin-labeled antiglobulin, 1.7%. Minimal nonspecific reaction, chiefly upon fibrils. rer, rough endoplasmic reticulum. X 91,000. (d) Degenerate, uninfected HEp-2 cell, same control preparation. Marked nonspecific ferritin ret)ention. X 91,COO.
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FIG. 1
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37”, medium without serum was added. Addition of virus was counted as time zero. Infectivity titers mere determined by plaque assay on monolayer cultures of HEp-2 and BS-C-l cells. Electron microscopy. Seventy-four specimens for comparative morphologic study of uninfected and infected cells were taken at intervals from 2 min through 20 hours after inoculation. They were fixed as monolayers, after decanting the medium and floating dead cells, in 1.6% glutaraldehyde (from 25 % stock, pH 3.4 or greater) in phosphatebuffered saline pH 7.2 with added magnesium chloride 5 X lop3 M and calcium chloride 1 X 1O-4 il/ir (PBS MgCa). The fixed cells were then scraped and postfixed as a centrifuged pellet in 1% osmic acid in PBSMg Ca, dehydrated in a graded ethanol 0.15 M NaCl series, infiltrated with propylene oxide, embedded in Epon 82, and cut with diamond knives on an LKB ultramicrotome. Sections were stained with 2% aqueous uranyl acetate and alkaline lead citrate, mounted on bare or collodion-coated, carbonized copper mesh grids, and photographed in an Elmiskop 1. Indirect immunoferritin technique. Seventy-three specimens, taken at the same intervals as above for reaction with antibody, were prepared by a two-stage fixation: first, 0.01% formaldehyde (made from paraformaldehyde) and 0.008 % glutaraldehyde in PBS MgCa, then permeabilization with 1.2 X 10e4 M digitonin in PBS MgCa, and finally 1% formaldehyde (with or without 0.8% glutaraldehyde) in PBS MgCa, each applied for 1 min in succession, followed by 3 washes of PBS MgCa over a period of 1 hour to 7 days. The fixed cells were pelleted in McNaught protein tubes, and then dispersed with a platinum wire into 5 volumes of rabbit antipolio serum diluted 1: 10 to 1:80 in PBS MgCa. After reaction for 1 hour at room temperature, antibody
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was removed and the cells were washed vigorously 3 times in 6.5 ml of PBS MgCa. Ferritin-labeled antirabbit globulin (5 volumes) was added for 1 hour, and after antibody removal and 3 vigorous washes in PBS I\IgCa, the cells were pelleted and postfixed in 1% osmic acid in PBS MgCa prior to embedding in Epon and thin sectioning. Controls consisted of normal cells treated in the same way, and virus infected cells in which a 1: 10 to 1:40 dilution of normal rabbit serum was substituted in the indirect immunoferritin reaction. RESULTS
ImmunoJluorescence Standard indirect immunoffuorescence of infected HEK cells with antipolio rabbit sera is illustrated in Fig. la. At 4 hours, there is diffuse cytoplasmic fluorescence with small peripheral accumulations, probably representing cytoplasmic blebs. In the 3-6hour period after inoculation the intensity of the fluorescence increased with time, and in a few cells was concentrated in a diffuse paranuclear mass, probably representing areas of multiple vesicles. I,ndirect Immunoferritin Fine structure. Excellent retention of fine structure was accomplished by two-stage fixation as shown at low magnification in Fig. lb. Controls. Healthy, uninfected cells showed variable, but generally minimal nonspecific retention of ferritin labeled antiglobulin, chiefly upon fib& following reaction with normal or antiviral rabbit sera (Fig. lc). Degenerate, uninfected cells, where cell death was evident from gross distortion of normal fine structure and presence of lysosomes, often retained considerable amounts of ferritin-labeled antiglobulin after reaction with any rabbit serum, a finding similar to the
FIGS. 2-7. Cells are polio-infected and subjected to immunoferritin reactions. FIG. 2. (A) HEp-2 cells, 3 hours. Note absence of ferritin in nucleus, wide distribution
of ferritin in infected cytoplasm except for mitochondrion (M) and nuclear membranes. Vesicles (ves), fibrils (jib), and viroplasm (op), lightly coated with ferritin. Ferritin scattered among free ribosomes (rib). X 61,006. (BD) Details of same cell, including in (C) plasma membrane and extracellular ferritin ringed virus (arrow). x 94,000.
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FIG. 3. (A) Multiple vesicle area, HEp-2,4 hours, with ferritin around full virus particles (arrows) and empty ones (e). Ferritin on fibrils (fib), but not associated with rough endoplasmic reticulum (rer), mitochondria (M), aud vesicle surfaces (except at inter&ices). X 70,000. (B and C). Details of Fig. 3A. X 104,OCO. (D) Detail of Fig. 4A. X 128,000.
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FIG. 4. Other cytoplasmic structures, HEp-2, 4 hours. (A) Ferritin on fibrils (Jib) lying between the clustered vesicles and the nuclear and plasma membrane. Virus particles shown by arrows. X 70,000. (B and C) Collared vesicles (outlined). (B) X 80,000; (C) X 100,000. (D) Virus particles (arrows) and fibrils (fib) show ferritin, but viroplasm (VP) has very little. 217
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FIG. 5. Entvbulated progeny virus (all HEp-2 cells). (a) Intracellular, entubulated virus near nucleus (N), alld vesicles (MS); 3 hours. X 100,000. (b) Extracellular ferritin only at sit.e of tubule opening, 4 holux. X 68,000. (c) Virus emerging at 4 hours from tubules are ferritin coated. A free polyribosome (pr) is coated, but the rough endoplasmic reticulllm (rer) is not. (d) Free extracellular particles at 4 hours are heavily coated; free intracytoplasmic particle (double arrow) less so. Particles in tubules are ferritin free. (e) Extracellular, ferritin-ringed virus adsorbed to debris. Free intracellular ferritin-coated virus (double arrow). Tangential cut (outlined) of entubulated virus. X 100,000.
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FIG. 6. Alignment of poliovirus on cytoplasmic fibrils. (a) HEp-2 cell, 4 hours. Ferritin coats accessible particles (arrows) but nearby ribosomes (rib) and mitochondrion show no ferritin. X 91,906. (b) Progeny virus, full and empty, some ferritin coated (arrows), aligned on cytoplasmic fibrils near plasma membrane, HEp-2 cell, 6 hours. X 72,066. (c) HEp-2 cell, 4 hours. Projecting bleb contains both enfibrilated and entubulated virus (arrow). X 48,990.
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nonspecific staining of dead cells seen in immunofluorescence (Fig. Id). Virus infected cells reacted with normal sera during the 3-G-hour period of virus production by still living cells (impermeable to 0.5 % erythrocin B when unfixed) showed more nonspecific retention of ferritin in the cytoplasm than did uninfected cells. However, infected cells showed distinctly more ferritin retention after reaction with specific antiviral sera. At no time after infection was there retention of ferritin antiglobulin in the Golgi appanucleus, upon mitochondria, ratus, intact plasma membrane, or membrane bound ribosomes. Distribution of ferritin in infected cells reacted with speci$c antisera. At 3 hours, when extracellular, ferritin-coated progeny virus was first noted, the cells showed a fairly dispersed distribution of intracullular ferritin with some concentration about vesicles, fibrils and viroplasm (Fig. 2). In the 4-6-hour period, paralleling the increase in intensity of immunofluorescent staining, there was an increase in the amount of ferritin coating of cytoplasmic structures. Ferritin was also localized to material among the clusters of vesicles whose formation is a prominent feature of this period, and to single dense or empty particles among the vesicles, presumed, therefore, to be complete or incomplete progeny virus (Fig. 3). Sheaves of fibrils were found near the clusters of vesicles and were frequently coated heavily with ferritin (Fig. 4A). Ferritin was also found associated with some of the polyribosomes (Fig. 5c) and with occasional small vesicles that were surrounded by a collar of lightly staining material (Fig. 4B and C). In contrast to its reaction at 3 hours, viroplasm was variably and minimally ferritin-associated (Fig. 4D). Progeny virus enclosed in the tubulovacuolar excretion system (which appeared first at 3 hours and enclosed only full particles) were not ferritin coated (Fig. 5a and b) except where the
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tubules opened through the plasma membrane (Fig. 5c and d). Neither the walls of the tubules (approximately 50 m,u in diameter when sectioned transversely), nor the plasma membrane near the particles undergoing release, retained ferritin. Single, free, intracytoplasmic particles were frequently found, but not enclosed in tubules, near the plasma membrane; they were ferritin coated, but not as heavily as extracellular particles (Fig. 5d).The extracellular, ferritin-ringed, dense particles were often found adsorbed to cellular debris, or to plasma membranes (Fig. 5c, d and e; Fig. 2C); extracellular empty particles were rare. Particles, both full and empty, were found aligned in parallel array on fibrils after 4 hours, and were coated with ferritin where defects in the lattice were apparent (Fig. 6). Figures 6 and 7 show a sequence suggesting that alignment on fibrils is followed by migration of the complex to the cell surface, formation of a cytoplasmic bleb and final separation from the cell. The existence of ferritin-coated, free particles near the extruded bleb suggests that virus is eventually released by disintegration of the bleb (Fig. 7C). DISCUSSION
Infection by poliovirus results in an accumulation of viral proteins within host cells, followed by their structural degeneration, as evidenced particularly by permeability of the plasma membrane to vital dyes and to ferritin after about 6 hours. Since both ferritin and globulin can bind nonspecifically to structures within such degenerate cells, the localization of specific intracellular antigen can be fruitfully attempted only during a relatively short interval: from about 2.5 hours, when the structural viral protein first appears (Scharff and Levintow, 1963), to about 6 hours, the onset of membrane permeability. During this interval, some 90% of the structural protein is made (Darnell et al., 1960). Our observations were restricted
FIG. 7. Formation, separation, and dissolution of cytoplasmic blebs. (A) BS-C-1 cell, 4 hours. Free cytoplasmic bleb. X 72,000. (B) HEp-2 cell, 4 hours. Free cytoplasmic bleb, with nearby ferritin-coated virus (arrows), suggesting virus is liberated by breakdown of bleb. X 70,000. (C) HEp-2 cell, 4 hours. Two cytoplasmic blebs at cell surface, one containing full and empty particles on fibrils, the other, no visible particles. Entubulated and free virus particles (arrows) near site of bleb formation. X 57,ooO.
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FIG. 7
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to this 2.5-6 hour interval and were considered significant only for those cells that, although infected, showed good retention of their normal fine structure. Virus A necessary condition for the immunoferritin technique to be applicable to a search for viral antigens in a system devoid of morphologically distinctive “virus factories” is a satisfactory demonstration that particles, morphologically identifiable as virus, are closely associated with ferritin molecules. Such particles are the extracellular ones, and these were found, 3 or more hours after poliovirus inoculation, to be well ringed with ferritin. In view of the certainty of antigenic identification of these particles we make the plausible presumption that intracellular virus and aggregates of unassembled viral antigen can also be identified in the same specimens. Our results shed light upon the nature of the extracellular virus particles and the free intracytoplasmic particles, as found after 3 hours following inoculation. Holland and Hoyer (1962) determined that polivirus, adsorbed at 37” and subsequently eluted, loses its ability to react with antibody and to readsorb to cell membranes. Since we found that the 3-hour extracellular virus particles bind to ferritin-conjugated antibody and adsorb to extracellular membranous debris and plasma membranes, and since such particles are not seen from 1 to 2 hours postinoculation, we conclude that they are progeny virus rather than residual virus from the inoculum. It is also reasonable to conclude that the 3-hour, intracellular particles, free and ringed with ferritin, are progeny virus inasmuch as none of them were found in the control reactions with ferritin, nor were they found during the 1-2hour interval. The entubulated, ferritin-free particles found in the cytoplasm are most likely progeny virus, since they did not appear before 3 hours and, when found apparently extruded from the tubules into the extracellular space, were ringed with ferritin. Unassembled Structural Antigens From identification of single particles extracellularly, or of completed capsids in
the cytoplasm, to localization of poliovirus protcms prior t,o assembly into complete shells is a move into more hazardous territory. We do believe, however, that these speculations are plausible: Clustered vesicles. Full and empty ferritinringed capsids were so regularly found among the clustered vesicles in the 4-6-hour infections that some function in virus assembly may be ascribed to the vesicle surface, especially in view of the observations of Penman et al. (1964) that a membranous complex exists associated with viral RNA, RX-4 polymerase, virus protein, and newly formed virus particles. Ferritin molecules were consistently found dotting the interstices of the vesicIes, in addition to surrounding the full and empty particles similarly located, making it Iikely that accumulations of structural viral proteins were located in these regions prior to assembly into complete shells. Considerable evidence favors the notion of a proliferation of vesicles as a response to “toxic” viral proteins (Bablanian et al., 1965a; Amako and Dales, 1967), but such evidence does not preclude the possibility that the vesicle regions are also virus assembly sites. ViropZasm. Since specimens taken at 3 hours show some ferritin coating of viroplasm, while the 4-6-hour ones show little or none, association of viral antigen with these structures may be temporary. It may be that they are a cellular reaction product, perhaps the result of agglutination of degenerating ribosomes, rather than structures actively involved in virus production. Other cytoplasmic structures. The increased association, with time after inoculation, of ferritin molecules with fibrils, smooth ER, small vesicles, and cytoplasmic matrix implies that free viral protein increases in amount and is widely distributed. Although a portion of dispersed ferritin may represent nonspecific reactions in gradually dying cells, it consistently occurs in cells that retain good fine structure and an intact plasma membrane (as demonstrated by vital dye exclusion) . D. Virus release. The elimination of progeny virus from still viable cells appears to occur by two mechanisms. In the first, newly formed, complete virus particles are
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enclosed in, and later released from, tubules whose diameter is fairly constant except near the plasma membrane where they occasionally appear to coalesce into vacuolar forms. The tubules are probably derived from the ER, as suggested also by Blinzinger et al. (1969), who observed membranes enclosing small, intracellular virus crystals. The role of membranous channels, both tubules and narrow cisternae, in virus release has previously been noted in infections with SV40 (Levinthal et al., 1967 b), and vesicular stomatitis (Zee et al., 1968). In the second mechanism of release, both full and empty particles are aligned on fibrils and subsequently eliminated from the infected cell in cytoplasmic blebs. REFERENCES AMAKO, Ii., and DALES, S. (1967). Cytopathology of mengovirus infection. II. Proliferation of membranous cisternae. Virology 32,201-215. BABLANIAN, R., EGGERS, H. J., and TAMM, I. (1965a). Studies on the mechanism of poliovirusinduced cell damage. I. The relation between poliovirus-induced metabolic and morphological alterations in cultured cells. Virology 26, lOO113. BABLANIAN, R., EGG~RS, H. J., and TAMM, I. (1965b). Studies on the mechanism of poliovirusinduced cell damage. II. The relation between poliovirus growth and virus-induced morphological changes in cells. Virology 26,114-121. BLINZINGER, K., SIMON, J., MAGRATH, D., and BOULGER, L. (1969). Poliovirus crystals within the endoplasmic reticulum of endothelial and mononuclear cells in the monkey spinal cord. Science 163, 1336-1337. DALES, S., and FRANKLIN, R. M. (1962). A comparison of the changes in fine structure of L cells during single cycles of virus multiplication, following their infection with the viruses of Mengo and encephalomyocarditis. J. Cell Biol. 14, 281-302. DALES, S., EGGERS, H. J., TAMM, I., and PALADE, G. E. (1965). Electron microscopic study of the formation of poliovirus. Virology 26, 379-389.
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DARNELL, J. E., SCHAHFF, M. D., and LEVINTOW, L. (1960). Poliovirus protein: source of amino acids and time of synthesis. J. Biol. Chem. 235, 74-77. DUNNEB.~CKE, T. H., LEVINTHAL, J. D., and WILLIAMS, IL. C. (1969). Entry and release of poliovirus as observed by electron microscopy of cultured cells. J. Viral. in press. HOLLAND, J. J., and HOYB;R, B. H. (1962). Early stages of enterovirus infection. Cold Spring Harbor Symp. Quant. Biol. 27, 101-111. KALLMAN, F., WILLIAMS, R,. C., DULBJXCO, R., and \r~~~, M. (1958). Fine structure of changes produced in cultured cells sampled at specified intervals during a single growth cycle of poliovirus. J. Biophya. Biochem. Cytol. 4,301-308. LEVINTHAL, J. D., CEROTTINI, J. C., AHMADZADXH, C., and WICK~X, R. (1967a). The detection of intracellular adenovirus type 12 antigens by indirect immunoferritin technique. Intern. J. Cancer 2, 85-102. LEVINTHAL, J. I)., WICKER, It., and CEROTTINI, J. C. (1967b). Study of intracellular SV40 antigens by indirect immunoferritin technique. Virology 31, 555-558. MANDEL, B. (1962). Early stages of virus-cell interaction asstudied by using antibody. Cold Spring Harbor Symp. Quant. Biol. 27, 123-131. PENMAN, S., BECICER, Y., and DARNELL, J. E. (1964). A cytoplasmic structure involved in synthesis and assembly of poliovirus components. J. Mol. Biol. 8, 541-555. PENMAN, S., and SUMMERS, D. (1965). Effects on host cell metabolism following synchronous infection with poliovirus. Virology 27,614-620. RIFKIND, It. A., GODMAN, G. C., HOWE, C., MORGAN, C., and ROSE, H. M. (1961). Structure and development of viruses as observed in the electron microscope. VI. ECHO virus type 9. J. Exptl. Med. 114, 1-12. SCHARFF, M. D., and LEVINTO~, L. (1963). Quantitative study of the formation of poliovirus antigens in infected HeLa cells. Virology 19, 491-500. ZEE, Y. C., HACKETT, A. J., and TALENS, L. T. (1968).Electronmicroscopicstudies on the vesicular exanthema of swine virus. II. Morphogenesis of VESV type Ha4 in pig kidney cells. Virology 34, 596607.