Electron microscopic study of the formation of poliovirus

Electron microscopic study of the formation of poliovirus

VIROLOGY 26, 379-389 (1965) Electron Microscopic Study of the Formation of Poliovirus ~ S A M U E L D A L E S , H A N S J. E G G E R S , I G O R T A ...

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VIROLOGY 26, 379-389 (1965)

Electron Microscopic Study of the Formation of Poliovirus ~ S A M U E L D A L E S , H A N S J. E G G E R S , I G O R T A M M , Axn G E O R G E E. P A L A D E The Rockefeller Institute, New York, New York Accepted March 17, 1965 HeLa cells infected with poliovirus were sampled at intervals during a single cycle of viral replication and examined in thin sections by electron microscopy. From the samples, stages in virus development and sequential alterations in cellular fine structure were reconstructed. Changes resulting from the infection comprised (1) the appearance, at 3 hours after inoculation, of large ribosomal aggregates; (2) an accumulation of dense aggregates, designated viroplasm, in the cytoplasmic matrix; (3) the appearance in the matrix of numerous recognizable progeny particles first as individual units or in small groups and later in large virus crystals; and (4) development in the eentrosphere zone of masses of small, membrane-enclosed bodies, some of which contained one or several virus particles. These morphological findings, especially observations on the membrane-enclosed bodies, are discussed in relation to biochemical studies already published on the same virus-host cell system. It is concluded that poliovirus shares with other cytoplasmic viruses the fundamental characteristic of developing within the cytoplasmic matrix proper.


time could not demonstrate intracellular virus and hence could not identify its exact site of assembly. The present study takes advantage of newer preparatory methods to find out whether poliovirus is formed at

Electron microscopic studies have established that the development of all animal viruses so far examined proceeds within the cytoplasmic matrix or in the nucleoplasm. As to the formation of small RNA viruses, such as 5.iengovirus, electron microscopic evidence indicates that it occurs in the cytoplasmic matrix (Dales and Franklin, 1962). Yet recent studies, using gradient sedimentation of homogenates of poliovirus-

intracellular sites which are in principle different f r o m the site of formation of all the other animal viruses so far examined. MATERIALS AND METHODS

infected t t e L a cells, h a v e led P e n m a n et al. (1964) to conclude t h a t poliovirus is m a d e within m e m b r a n e - b o u n d e d structures, rather t h a n in the cytoplasmic matrix. A l t h o u g h cells undergoing single cycles of poliovirus infection were previously examined b y electron microscopy (Kallman et al., 1958), the techniques available at t h a t This work was supported by grant U-1141, from the Health Research Council of The City of New York, a grant from The National Foundation, grant AI-3445 from the National Institute of Allergy and Infectious Diseases, USPHS, and training grant 5-TI-GM-577 from the National Institute of General Medical Sciences, USPtIS.

Experimental details of the methods employed h a v e been published previously (Dales, 1963). Virus and cells. Poliovirus t y p e 1, Brunhilde strain was used. I-IeLa cells, obtained t h r o u g h the courtesy of Dr. B. Mandel, were grown in monolayer cultures on 100 m m plastic petri dishes in Eagle's M E M m e d i u m (1959) supplemented with 10 % calf serum. H e L a cells were inoculated with poliovirus at an input multiplicity of 22 P F U / c e l l . After adsorption for 1 hour, the virus ino c u l u m was removed, and the cell monolayer washed once with phosphate-buffered saline (PBS, Dulbecco and Vogt, 1954). T e n milliliters of Eagle's m e d i u m was then




added to each plate and incubation was continued in a humidified atmosphere with 5 % CO2 in air. The whole procedure was carried out at 37 °. Inhibitory compounds. Additional information about virus development was sought by means of inhibitors whieh have a specific action. Actinomyein D was used at a eoneentration of 2 #g/ml and guanidine hydrochloride at 95 t~g/ml (1 mM). The compounds were included in the medium from the time of inoculation. Electron microscopy. About 5 to 8 X 106 attached cells were freed from the surface of petri dishes by scraping with a rubber policeman. The suspensions were centrifuged at 1500 rpm for 5 minutes and pellets of cells obtained in this way were fixed for 3-5 minutes with a 1% solution of glutaraldehyde (Sabatini et al., 1962) in PBS, pH 7, and postfixed with a 1% buffered solution of osmium tetroxide (Palade, 1952). Fragments of these pellets were embedded in epoxy resin mixtures (Luft, 1961) and sectioned with diamond knives. Contrast in sections was enhanced by double staining with aqueous 1% uranyl acetate for 1 hour followed by solution B (Karnovsky, 1961) of lead salts. For microscopy an Elmiskop I at instrumental magnifications of 800040,000 was used. RESULTS

The Uninfected Cell HeLa cells possess large, spherical or oval, centrally placed nuclei, numerous mitochondria distributed throughout the cytoplasm, a relatively well developed endoplasmic reticulum (ER) and a Golgi complex of usual appearance. In sections, the profiles of the ER vacuoles are numerous, small, predominantly circular, and randomly distributed in the cytoplasm. Some possess attached ribosomes (rough-surfaced ER), while others lack these particles (smoothsurfaced ER). Most of the ribosomes of the cell are not bound to the ER membranes but occur free in the cytoplasmic matrix, either singly or in groups of 2 to more than 20 particles (polyribosomes), which take the form of rosettes or chains. More detailed accounts of the organization of HeLa cells

and other permanent cell lines cultured in vitro have been published (Robbins and Gonatas, 1964; Dales and Franklin, 1962).

Changes Resulting from Infection with Poliovirus Cells infected with poliovirus at a multiplicity of 22 PFU/eell showed fairly synchronous alterations in fine structure over the period examined (3-7 hours postinoculation). These changes comprised the appearance of large clusters of ribosomes; the development of masses of small, membraneenclosed bodies in the centrosphere zone; the accumulation of dense aggregates, designated viroplasm, in the cytoplasmic matrix; and the appearance of recognizable progeny particles either as individual units or in crystals. About 20 % of the cell profiles in samples taken at 3 hours postinoeulation contained 1 or 2 poliovirus particles either partly enclosed within invaginations of the cell membrane or sequestered inside phagocytic vacuoles near the cell surface. Presumably these particles represented a fraction of the inoculum, estimated to be at most 10 % of the virus added, which had failed to progress beyond the initial phase. The finding is compatible with the view of Mandel (1962) that phagoeytosis of poliovirus is an essential step for initiating infection. Ribosomes. Within 3 hours following inoculation, large aggregates of ribosomes, presumably polyribosomes, were observed in the cytoplasmic matrix. These aggregates comprised more ribosomes per aggregate than their counterparts in uninfected cells, and were frequently clustered around rough or smooth-surfaced elements of the ER (Fig. 1). The micrographs indicate that some of the large aggregates were in contact with the elements of the ER, but the exact nature of this association as well as the structures involved therein remain to be determined by further investigation. Such arrangements were found to occur in a sizable proportion of the cell profiles in samples taken at this time and at 5 hours. Membrane-bounded bodies. Small bodies which measured 700-2000 A in diameter were observed in small groups in the perinu-



Key to figure abbreviations: N, n u c l e u s ; M , m i t o c h o n d r i o n ; va, vacuole; B, membrane-enclosed small body. FIG. 1. P o r t i o n of t h e nucleus and cytoplasm of a cell from a sample t a k e n 3 hours after inoculation of t h e culture. Clusters of ribosomes (polyribosomes) are indicated b y arrows. Viroplasmic foci (VP) a n d vacuoles are also evident. Magnification: X 25,000.


D A L E S , E G G E R S , T A M M A N D 1)ALADE

FIG. 2. P o r t i o n of the nucleus a n d cytoplasm of a poliovirus-infected cell from a 7-hour sample. T h e d e v e l o p m e n t of t h e small, membrane-enclosed bodies is illustrated. F r a g m e n t s of t h e cytoplasmic m a t r i x are enveloped to a varied e x t e n t b y a network of m e m b r a n e - b o u n d e d channels. Particles of poliovirus, either singly or in groups, are lodged in t h e cytoplasmic matrix. Occasionally t h e virus lies t r a p p e d within the small bodies (arrows) M.agnification : X 50,000; inset X 75,000.



F~Gs. 3 and 4. Two examples showing poliovirus in the cytoplasm of cells 5 hours after inoculation. Some virus particles lie within, others are outside the small bodies. The arrows point toward empty capsids. In Fig. 4 note the regions where the outer envelope of the cisternae remains unoccluded. Magnification: X 125,000. clear zone in the 3-hour samples. These bodies were limited by a single membrane and contained a material similar in density and texture to the cytoplasmic matrix. By 5 hours after infection these bodies had proliferated extensively, and by 7 hours they occupied nearly the whole central region of the cell (Figs. 2 and 7). Polyribosomes and large aggregates of viroplasm were generally absent from the regions occupied b y these structures. Apparently the rapid development of these bodies in the eentrosphere region brought about a squashing and displacement of the nucleus toward one side of the cell. As judged by their size, site, and time of appearance after infection, the bodies observed by us are undoubtedly homologous with the U bodies described by Kallman et al. (1958) in poliovirus-infected monkey kidney cells, and with similar

bodies observed in L-strain cells infected with 5~[engo or encephalomyocarditis virus (Dales and Franklin, 1962). These bodies appear to develop as the result of the formation of an extensive network of sacs or vacuoles bounded by smoothsurfaced membranes. The micrographs (see Fig. 2) suggest that channels developed around portions of the cytoplasm, partially isolating peninsular projections of the matrix. Later these projections appear to be completely enveloped by membrane and thereby transformed into islets within the cisternal space. The space around the body was obliterated, as demonstrated by partial or complete reorganization of the envelope into a quintuple-layered membrane through fusion of two unit membranes. 5Iany, but not all, of these bodies contained one or more virus particles presumably caught within



FIG. 5. An aggregate of dense, finely t e x t u r e d m a t e r i a l (viroplasm) in t h e cytoplasmic m a t r i x of a cell 3 hours a f t e r inoculation. Scattered ribosomes are also evident in this field. Magnification: X 110,000. FIG. 6. An aggregate consisting of finely t e x t u r e d dense m a t e r i a l and larger granules, in t h e cytoplasm of a cell sampled 7 hours after inoculation. T h e granules, 170-250 A in diameter, m a y be poliovirus particles a t a n i n t e r m e d i a t e stage of assembly. Magnification: X 105,000.



:FIG. 7. P o r t i o n of the centrosphere region and peripheral cytoplasm of a cell sampled 7 hours after inoculation of the culture. T h e membrane-enclosed small bodies are very numerous and occupy the whole central region of the cell. Two crystals of virus particles are present in t h e m a t r i x near the surface of t h e ceil. Magnification: X 22,000.



]FIG. 8. A small area of cytoplasm in a cell from a 7-hour sample. A large crystal of poliovirus occupies the center of this field. A small region within the crystal, presumably derived from t h e cytoplasm, is devoid of virus. Magnification: X 90,000.

STUDY OF THE FORMATION OF POLIOVIRUS the islets of cytoplasmic matrix at the time the latter became enveloped. It should be emphasized that the matrix between the membrane-bounded bodies contained numerous virus particles scattered either individually, or as small crystals (Figs. 2 and 3). Aggregates of viroplasm. Aggregates of dense material (Figs. 1 and 5) were found in all samples beginning with those taken at 3 hours after inoculation. The dense material consisted of tightly packed filamentous and granular elements, which, on account of their similarity to material in known viroplasmic foci in cells infected with other RNA or DNA viruses, were inferred to represent polio-viroplasm. These aggregates were invariably lodged in the cytoplasmic matrix and occurred in about one-half of the seetioned cells sampled at 3 and 5 hours after inoculation. At 3 hours the aggregates were small, averaging 0.2 t~ across. By 5 hours larger aggregates measuring over 1 ~ across were commonly present. At 5 and 7 hours following infection, there were associated with these viroplasmic loci dense particles, 170-250 A in diameter, comparable in size and density to viral nucleoids, but without the discrete outline of mature virus (Fig. 6). These particles could be poliovirus in an intermediate state of condensation. The frequency of occurrence of viroplasmie loci decreased at 7 hours, concomitantly with an increase in the frequency of virus crystals. Virus progeny. A large number of particles of progeny virus, each 260-280 A in diameter, was evident in the cytoplasmic matrix 5 and 7 hours from the time of inoculation (Figs. 2 and 3). These particles were predominantly localized in the centrosphere region where they appeared either trapped within membrane-bounded bodies or dispersed in the intervening cytoplasm. Empty eapsids of the same size as complete partides were also observed in these samples (Figs. 3 and 4), in the same location. Infectivity data obtained in the poliovirusHeLa cell system indicate that assembly of progeny particles occurs sometime between 3 and 5 hours. Five hours after infection, crystals con-




Hours after inoculation Parameter

Large ribosome aggregates Membranebounded small bodies

Virus particles (dispersed) Foci of viroplasm Virus crystals
















÷ I

a ÷ =present.

taining tightly packed virus particles were seen in less than 1% of the cell profiles. Their frequency increased rapidly thereafter so that by 7 (Fig. 8) hours they were present in about one-half of the profiles. At the times investigated we did not find any close spatial relationship among the enlarged ribosome aggregates, the viroplasmie foei, and the accumulations of progeny virus. A summary of the changes described above as a function of time is given in Table 1.

Observations with Guanidine and A ctinomycin When infected cells were treated with guanidine, none of the changes manifest within untreated infected cells were found, a finding that is in line with the fact guanidine inhibits poliovirus multiplication (Tamm and Eggers, 1963). However, in the infected HeLa cells treated with aetinomycin D, poliovirus and the small membrane-enclosed bodies developed in the same way as in the untreated cells, as was to be expected since aetinomycin D does not inhibit the multiplication of poliovirus (Shatkin, 1962). DISCUSSION

Judging by previous evidence obtained with polio-, Mengo-, and other small RNA viruses, two of the structures we have found in HeLa cells after infection with poliovirus are probably associated with viral synthesis:



the large ribosomal aggregates or polysomes and the loci of viroplasm. The polyribosomes, presumed to be involved in protein formation, were found in a sizable proportion of the cell profiles to contain more ribosomes per cluster than in uninfected cells, in agreement with results obtained by zone eentrifugation of homogenates of poliovirusinfected cells by Pemnan et al. (1963) and Seharff et al. (1963). The loci of viroplasm found in the present investigation are morphologically similar to those encountered in L cells infected with Mengovirus and known to contain RNA and viral protein (Dales and Franklin, 1962). In the case of Mengovirus such foei appear at least 2 hours before crystals of virus develop, suggesting that the process of self-assembly (Caspar and Klug, 1962) within the viroplasmic pools requires 2 or more hours. The situation is probably similar with poliovirus, since viroplasmie foei are already present at 3 hours after inoculation, but mature virus in crystals first appears at 5 hours. Biochemical studies with radioactive precursors have indicated, however, that after the eclipse, synthesis of poliovirus RNA and protein and the assembly of viral components into complete virus can be carried through in less than 30 minutes (Darnell and Levintow, 1960; Darnell et al., 1961). The large numbers of scattered virus particles which are evident by electron microscopy at 5 and 7 hours postinoeulation (Figs. 2-4) may be the rapidly assembled progeny. From the beginning of their detection the progeny virus particles are always located in the cytoplasmic matrix, either in its usual continuous phase or within islets of matrix segregated within membrane-bounded bodies in the eentrosphere region. The interpretation of our findings is rendered difficult by two circumstances: first, the presumed sites of synthesis or assembly of viral products (endoplasmie retieulum, polyribosomes, and viroplasmie pools) are spatially separated from the sites where progeny poliovirus is first recgonized, i.e., mainly in the centrosphere region. Second, there is no clear temporal separation between the appearance of progeny particles and of small membranebounded bodies. Yet a tentative interpretation can be at-

tempted on the following basis. Our positive salient observation is the finding of progeny virus particles in large numbers in the continuous phase of the cytoplasmic matrix from the earliest time of their appearance on. Also important is the evidence concerning the mode of formation of membrane-enclosed bodies in the centrosphere region: they develop by a mechanism comparable to that of the formation of "autolytic vacuoles" in other cells (Ericsson and Trump, 1964), and they are undoubtedly islets of segregated cytoplasmic matrix, not the vacuoles normally present. Even though some of these bodies contain progeny virus, the association between the membrane-bounded bodies and poliovirus cannot be obligatory since many virus particles are found free in the matrix. The information obtained previously (Dales and Franklin, 1962; Mandel, 1962; Becker et al., 1963; Baltimore et al., 1963; Penman et al., 1964), taken in conjunction with our present findings, ean be accounted for by the following hypothesis, which is admittedly highly tentative: after the uptake of the virus in phagocytic vesicles, and the release and penetration of its RNA into the cytoplasmic matrix, the viral genome becomes associated with the cytoplasmic side of the membranes of the endoplasmic retieulum (ER), where production of viral RNA polymerase and viral RNA is initiated. The viral RNA binds preexisting ribosomes into large polyribosomes which synthesize virus-directed proteins. Many of the viral polyribosomes are attached to elements of the ER. Viral coat proteins and RNA either eombine rapidly into virus particles which are moved to the eentrosphere region, or accumulate into pools of viroplasm where morphogenesis of new virus particles occurs much more slowly. Newly formed progeny particles which reach the centrosphere region may be sequestered within membrane-bounded bodies. The latter appear to represent a secondary response of the cell to infection. In the ease of poliovirus, this response takes extreme forms, yet it does not prevent the eventual lysis of the infected cells. This tentative interpretation of our observations would explain the observations of Home and Nagington

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