Replication of animal viruses as studied by electron microscopy

Replication of Animal Viruses as Studied by Electron Microscopy* SAMUEL. DALES, PH .D .

New York, New York

brief survey aims to acquaint the reader with some morphologic aspects, particularly those observed by electron microscopy, of the replication of various animal viruses . It has been possible to demonstrate by recently developed procedures the sites within the host where viral materials accumulate (viroplasmic pools) and to visualize stages in the subsequent transformation of such materials into progeny virus particles . However, an important limitation of the current methods relates to intracellular localization of the sites of viral macromolecular synthesis ; thus, with a few exceptions, it has not yet been possible to trace precisely in situ the earliest stages of virus replication . This limitation has created a gap between the biochemical and cytologic findings, and has brought about much uncertainty regarding the chemical nature of certain structural components of viruses or viral precursors observed in the electron microscope . Outstanding among the exceptions are the poxviruscs, for which a good correspondence between morphogenesis and the synthetic events has been observed . Undoubtedly a similar integration will be attained in the near future for the other types of viruses . In the available space it will be possible to describe only the salient features of cell-virus interactions, which will be illustrated with appropriate examples . Among the topics to be considered briefly is penetration of inoculum virus into host cells, the sequence of stages in morphogenesis among different types of viruses, release of progeny particles and, finally, changes in the cellular fine structure of the host .

cell, animal viruses are transferred intact, with their genetic component still protected, into phagocytic vacuoles . Such transfer involves an active but apparently unspecific process of engulfment . Each type of virus studied to date, once it has been incorporated by phagocytosis, interacts with the host cell in a characteristic manner . For example, the DNA of vaccinia virus is released in a three-stage process involving uptake of intact virus, breakdown of an outer coat which enables the DNA-containing core (fig . 20 and 21) to enter the cytoplasmic matrix, and separation of the DNA from the core . The labeled DNA derived from the inoculum subsequently replicates in cytoplasmic factories where vaccinia develops . Rupture of viral protective coats within phagocytic inclusions is indicated also for poliovirus, reovirus type 3 and the myxoviruses . Particles of inoculated adenovirus 7 penetrate, without apparent rupture of their capsids, to the nucleus of HeLa cells, indicating that release of the DNA of this nuclear virus may occur in the vicinity of or within the nucleus . A more detailed consideration of virus penetration into host cells will be found elsewhere [1j .

HIS

REPLICATION AND MORPHOCENESIS

Small RNA Viruses. Agents exemplifying this group are the viruses of poliomyelitis and encephaloinyocarditis (EMC, Mengo) . In the 8 hour grcnvth cycle of Mengovirus multiplying in strain L mouse fibroblasts . the first morphologic evidence of an accumulation of viral products i, ; the deposition in the cytoplasmic matrix of dense aggregates of fibrillar material (viroplasmcd ; this appears late, i .e ., at 8 hours after inoct lation . Within this viroplasm it is possible to discern a paracrystalline organization of the dense component (Fig . 1) which

PENETRATION INTO CELLS

The preponderance of available evidence indicates that upon attachment to host cells, which involves specific receptors on the surface of the

* From The Rockefeller Institute, New York, New York . Council of the City of New York, Contract No . U-1141 . VOL .

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Ch chromosome G Golgi vesicles

M mitochondrion

N nucleus Va vacuole

Fio. 1 .

Portion of the cytoplasm of a strain L cell obtained 10 hours after infection with Mengovirus . large mass of viroplasm having a paracrystalline organization occupies the center of the field . Original magnification X60,000 [2] . A

precedes by about 2 to 4 hours the formation of crystals of virus particles . (Fig. 3 .) Presumably these viroplasmic foci, which were shown by cytochemical methods to contain RNA and viral protein, are the precursor material for mature Mengovirus, which appears later in crystals [2] . With poliovirus developing in HeLa or monkey kidney cells, the cycle of replication is complete in about 6 hours [3], In this system, scattered individual progeny virus and aggregates of a few particles become numerous in the cytoplasm as soon as 5 hours after inoculation . Viroplasmic foci like those appearing with Meagovirus become evident also after infection with poliovirus type 1, but earlier, 3 hours after inoculation, and continue to increase in size for at least 2 hours more, and by 7 hours large virus crystals are formed in the cytoplasm [4] .

As with Mengovirus, the viroplasmic foci apparently contain precursors of the poliovirus, which subsequently appears in the crystals . Thus, during multiplication of poliovirus and probably of other small RNA viruses, one population of particles, scattered in the cytoplasm, may be assembled soon after formation of the macromolecular products during the rapid synthetic period, while assembly of the other population takes place several hours later within foci of viroplasm . RNA Viruses with Lipoprotein Envelopes . In this category are vesicular stomatitis virus (VSV), rabies virus and the myxoviruses typified by influenza virus . In the case of influenza virus, the "soluble" viral antigen, representing the protein moiety of the helical nucleoprotein inner component [5], has been shown, by immunofluorescent technics [6] and AMERICAN JOURNAL OF MEDICINE



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Plc . 2 . 1 he centrosphere region of an L cell obtained 12 hours after infection with Mengov'irus . The arrows point toward aggregates and individual virus particles in the cvtoplasmio matrix and inside small membrane-bounded bodies . Original magnification X 50,000„ insert X 185 . 000 . Fiu. 3 . Portions of the nucleus and cytoplasm from a cell infected and obtained as in Figure 2 . , rwuadjacent ervstalsofvirusareshown . Original magnification X (,4 .(1011 .

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Fin . 4 and 5 . Whole mount o monke dney cell cultured in a viewed by negative contrast, showing the emergence of long filamentous particles of influenza A2 virus . Arrow indicates the point of emergence of two (filaments . Original magnification X 50,000 . Insert [5] shows the emergence at higher resolution . Note the spikes on the two particles and an area on cell surface, indicated by arrows, where spikes may be present . Original magnification X 140,000 . From Cuoartrv. P . W. Virology, 21 : 278, 1963 [40] .

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. 6 and 7 . Vesicular stunmtitis virus particles emerging fruin the outer surface of L cells 5 hours er inoculation of the culture . Double arrows indicate the region of apparent continuity between the cellular and viral membranes . The single arrows point towards spikes on the outer viral surface . Regularly packed inner nueleocapsid component is clearly evident in Figure 7 . Original niagnilication X 450,000 . Preparation, courtesy of S . C . Sih-erstein . F

by ferritin-antibody conjugates employed in conjunction with electron microscopy [7], to accumulate in dense aggregates within nuclei of infected cells . The specific site of the viral RNA synthesis has not been determined unequivocally at the present time but is also likely to be intranuclear . The hemagglutinin component of influenza virus, equivalent to the spikes present on the surface [ .5,8], has been shown to accumulate exclusively in the cytoplasm following the appearance of the soluble antigen . The final assembly of influenza virus occurs at the cell surface [71 . where the inner nucleoprotein component, having presumably migrated out of the nucleus, becomes enclosed within a cellderived {9] lipoprotein envelope . At some stage during the wrapping process, there are integrated into this coat small projections or spikes of ltemagglutinin (Fig . 4 and 5) and perhaps also the vital neuraminidase . The final stages in the morphogenesis of VSV also occur at membranous cell surfaces [10J, where the inner nucleo-capsid is arranged as a tightly packed helix inside a membranous envelope, apparently continuous with the cell voi . . 38,

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membrane . (Fig . 6 and 7 .) The similarity between formation of VSV and influenza virus is very striking . The development of rabies virus within nerve cells and astrocytes of infected mouse brains has been illustrated with a comprehensive series of electron micrographs [11] . This agent induces formation of extensive viroplasmic foci in the cytoplasm of host cells . Within such foci appear elongated cylindrical structures, each 100 to 1,300 A wide, presumed to be the virus . In this system there is evidence that in the formative stage the outer viral membranes are continuous with membranes of the smooth cisternac of the endoplasmic reticulmn (ER) which lie adjacent to the virus factories . Other viruses in this general category, which have been shown to form by a process of extrusion or budding at cellular membrane surfaces, are the oncogenic agents of Rous sarcoma and Friend leukemia [13,73], and the arboviruses [14] . The morphologic evidence on the formation of arboviruses is supported by biochemical studies in which it was shown by means of labeling experiments with radioactive precursors that

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The spindle and chromosomes of an L cell undergoing mitosis in a culture sampled 12 hours after infection with rcovirus . Aggregates of virus are closely associated with tubules of the spindle which have a dense coating on them (arrows) . Original magnification X 22 .000 [79] . FIG . S .

Sindbis virus derives the phospholipid moiety in its membranous coat from cellular materials [15] . In this respect acquisition of a membranous envelope by Sindbis virus appears to involve a process analogous to that reported for influenza virus 191 . In general, therefore, it appears that RNA viruses possessing lipoprotein outer coats become assembled at membranous surfaces of the cell and, at least in some cases, derive the material of their coats from cellular components . Reoviruses . These agents form a group of icosahedral particles measuring approximately 750 A in diameter and containing doublehelical RNA [16] . The host range of reoviruses

is extensive and includes L cells . With 1, cells as the host, initiation of replication is evident at 8 hours after inoculation, when small foci of dense viroplasmic matrix appear in the vicinity of the mitotic spindle elements . (Fig . 8 .) In these foci lie scattered dense viral nucleoids and partially assembled outer capsids . Progression of the infection becomes manifest by enlargement of the initial foci into large viral inclusions (Fig . 9), which eventually fuse into one another to form a continuous band of developing virus which circumscribes the nucleus [17] . The perinuclear zone, where reoviruses develop, has been shown by fluorescence microscopy to be related to the general position of the mitotic apparatus [18] AMERICAN JOURNAL OF MEDICINE



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Fin . 9 . A large perinucIear inclusion of rcnoirus particles, most possessing dense nucleoids . sari) ph-d as in Figure 8 . The arrows point toward coated spindle tobulns . Original magnification X ?5,000 . Fin . 10 . A spindle tubule shown at a higher resolution in a cell sampled as in Figure 8 . Several virus particles arc embedded within a dense material and among kinky filaments (arrows) . Uncuatcd segments of spindle tubules are indicated by double arrows . Original magnification . X 90,000 181 .

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and, more specifically, to the microtubular elements of the achromatic spindle component [ 19] . At sites where the virus progeny is developing there appear masses of dense twisted or kinky filaments, each 50 to 80 A in width, and tubules of the mitotic spindle coated by a finetextured dense material . (Fig . 8 and 9 .) The material coating the spindle tubules is probably antigenically related to the protective viral coats, as indicated by tagging experiments with ferritin-antibody conjugates [20] . Even after elimination of the microtubules by means of antimitotic agents such as colchicine, which does not inhibit the multiplication, kinky filaments remain in the viroplasmic foci, indicating that they may be required either for synthesis of some viral products or for the assembly of reoviruses into mature particles . Thesee kinky filaments may be derived from filaments of similar dimensions which occur in wavy bundles as a normal cytoplasmic component of uninfected mammalian cells . Nuclear DNA Viruses . This group comprises icosahedral particles varying widely in size and DNA content . Among the smallest is the Kilham virus, only 200 to 250 A in diameter [21,22] . Next on the ascending scale are members of the papova group of oncogenic agents [23], 450 to 500 A across, then adenoviruses approximately 700 A in diameter, and finally viruses of the herpes group which measure about 1,000 A in diameter . All these types of virus have been observed by electron microscopy to develop inside nuclei, where synthesis of viral components is thought to take place, as indicated by biochemical and cytochemical evidence . Thus, during infection with pseudorabies the nascent viral DNA appears in the nucleus [24], the compartment which has also been identified as the site where enzymes presumably related to the reproduction of herpes virus become concentrated [2,5] . Using combined Feulgen staining for DNA and immunofluorescent methods, it was shown that DNA and viral antigen of adenoviruses accumulate in crystals [26], identifiable by electron microscopy as aggregates of progeny particles [27J . (Fig . 11, 12 and 13 .) Poxriruses . The DNA-containing poxviruses are large, possess a distinctive morphology, and multiply in the cytoplasm, whereby it is possible readily to separate viral and host DNA synthesis [28] . These attributes have made the poxviruses a favorite material for investigation of viral replication and development .

The fact that viral DNA of this group is synthesized in the cytoplasm was appreciated many years ago by investigators who found, by light microscopy, that Feulgen-positive viral inclusion bodies develop after infection [29] . More recently it was demonstrated by cytochemical methods that initiation of viral DNA and protein synthesis occurs at about the same time in cytoplasmic factories [ .30] . Subsequent electron microscopic observations confirmed conclusively that morphogenesis of poxviruses occurs in the cytoplasm . The life cycle of vaccinia virus in synchronously infected L cells was studied by means of thin sections and electron microscopy . It was found that at an input multiplicity of about 10 plaque forming units (PFU) per cell, the inoculated virus initiates the formation, in less than 3 hours, of several independent foci or virus factories in the cytoplasmic matrix at sites where parental DNA genomes are released front viral cores [1,31] . Large ribosome aggregates (Fig . 14), which during the initial stages of replication become associated with each factory, may be polyribosomes implicated by biochemical studies as participants in virus-directed protein synthesis ]32] . Within 4 hours after inoculation, immature particles of vaccinia virus appear in the viroplasmic matrix of the factories . (Fig . 15 .) Such immature forms have circular profiles, are enclosed by a limiting three-layered membrane which is covered on its outer surface by dense material (Fig . 16 and 17), and possess dense internal nuclcoids . (Fig . 15 and 17 .) The electron microscopic evidence indicates that the viroplasmic matrix which becomes surrounded by the developing viral membranes (Fig . 15 and 16) is reorganized within the membrane into components of mature progeny (identifiable by the presence within the outer coat of two satellite bodies and a flat, plate-like central core enclosing the DNA) . (Fig . 19 to 23 .) Vaccinia particles undergoing maturation commence to migrate out of the factories and become scattered in other regions of the cytoplasm within 6 hours of inoculation . (Fig . 18 .) The appearance of the first mature progeny coincides with a rise in the infectious titer [311, implying that vaccinia particles must complete their maturation before they are capable of reinfecting cells . Replication, however, does not cease with the formation of the first wave of progeny but spreads outward from each factory until by 20 hours extensive portions of the cytoplasm have become converted into viroplasmic foci . AMERICAN JOURNAL OP MEDICINE



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lie . 11 . Portion of the nucleus and cytoplasm of a I IeLa cell 24 hours after inoculation of the culture kith adcnocirus type 7 . Large crystalline aggregates and isolated particles of progeny virus f arrows 1 are evident . Abnormal, fused mitoehondrial masses which occur in the cytoplasm arc illustrated at a higher nta ;;ni0catinn in the inter), Original magnifiearinn X 9 .000 ; insert X 14,000.

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FTC . 12 . A portion of the nucleus at higher resolution, preparation as in Figure 11 . Single arrows indicate long threads 15 to 30 A wide ; double arrows point towards dcnse clumps which may be a stage in the formation of viral nuclcoids . Original magnification X 104,000 . FIG . 13 . Portion of an intranuclear crystal of adenovirus 7 illustrates a close hexagonal packing of the icosahedral particles . In many, the dense central nueleoid and less dense capsid are discernible . Original magnification X 130,000 . AMERICAN JOURNAL OF MEDICINE

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Ft( ; . 14 . A virus factory in the cytoplasm of an I cell sampted 3 hours after inoculation with vacruua virus . Clusters of ribosorucs (arrows) within the viroplasoi and at the periphery are evident . Orutiual magnification X 70,000 [d7] . Fie . 15 . Lnmature for ins of vaccinia virus in the cytoplasm of an I, cell limn a culture sampled 8 hours after inoculation . The arrows point towards two viral nuclcoids which are not enclosed by viral membranes . Original magnification X 25,000 [ 17I .

Double

Infections

with

Unrelated Cytoplasmic

With strain L cells as the host for reovirus type 3, and input multiplicity of about 10 PFU per cell, 8 hours elapse before the progeny of reovirus appear in the spindle region . (Fig . 8 and 10 .) With vaccinia virus, however, as already noted, virus factories are discernible in less than 3 hours after inoculation, and mature progeny in less than 6 hours, suggesting that vaccinia virus may initiate synthesis of its products more rapidly than reovirus . Double infection can be initiated in a minority of the cells in a culture if L cells exposed 4 hours previously to reovirus are subsequently inoculated with vaccinia virus . When double infection occurs, particles of reovirus develop in the vicinity of spindle tubules and those of vaccinia virus in discrete factories . (Fig . 24 .) Thus under controlled conditions macromolecular synthesis of two unrelated viruses can be initiated in the cytoplasmic matrix of a single host cell . Viruses : Varcirrria Virus and Reovirus .

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MECHANISMS Of RELEASE

Although some animal viruses are known to be capable of spreading infection by being passed directly cell to cell, with most the transfer of progeny out of the host is obligatory for reinfection . Several mechanisms have evolved for setting free the progeny particles, either in a burst or by a slow and controlled process . In cells infected by small RNA viruses such as Mengo- and poliovirus, completion of replication cycles, which are of 6 to 8 hours' duration, is followed several hours later by a loss of regulation of permeability at the cell membrane [33] . Such changes in the physiologic properties of the membrane reflect some structural d amage. a s suggested by microscopic observations of single explanted monkey kidney cells previously infected with poliovirus and then placed into microdrop-cultures . In this study ]34] morphologic alterations were correlated with assays for free infectious progeny appearing in the medium, and it was established that the onset of



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Fie . 16 . Portion of an immature form of vaccinia virus of which the unit membrane is indicated by arrows . Note the layer of dense material external to the membrane . Original magnification. X 220,000 [1] . Fim . 17 . An immature particle of vaccinia virus possessing a nucleoid in which there occur 15 to 20 A wide filaments preferentially oriented parallel to the long axis of the nucleoid . Original magnification . X 270,000 [41] .

18 . Maturing or mature particles of vaccinia virus In the cytoplasm of an L cell sampled 8 hours after inoculation . Original magnification X 20,000 . FIG.

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Whole mount of a vaccinia virus particle viewed by negative contrast . Nwaerms ridges, which may he rodlets or tubules, are clearly risible on the surface . Original InagniIication X 150 .1100 11 . Fm . 19 .

Preparation as in Figure 19, the internal core and lateral bodies surrounded by the viral outer mc,ubrane as viewed from the narrow aspect . Original magnification X 140,000 [1 . Fio . 20.

Preparation as in Figure 19, the broad . rectangular side of vaccinia virus . Short projections emanating from the coat of the inner core are visible subjacent to the outer viral coat . Original magnification X 220,000 [1', . Fte . 21 .

Maturing or mature progeny particle of vaccinia virus lying in the cytoplasm of an L cell is partly surrounded by vacuoles of the cellular endoplasmic rcticuhnn . Original magnification F¢:. 22 .

X 120,000 [1' .

Fic . 23 . A mature particle of vaccinia lying between two adjacent L cells . A single envelope, presumably derived front the vacuoles of the endoplasmic reticulmn which enclosed the intracellular virus, is indicated by arrows. Original magnification X 120,000 [ 1] . cytopathic effects at the cell surface became evident at approximately the same time as release of poliovirus . Physical disruption of the outer cell membrane has been observed by electron microscopy in cells undergoing cytoparhic effects following infection by Mengovirus or EMC vines 12] . The progeny of viruses possessing lipoprotein envelopes, which are assembled at membrane surfaces, are released from the host by a direct process of "budding ." Thus, after completion of virus assembly, each particle becomes pinched VOL . 38 .

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off from the underlying host-cell membrane (Fig- 4 and 5 .) More elaborate processes are employed to carry vaccinia and herpes viruses out of the infected host . Commencing at about 6 hours from the time of inoculation, when the first wave of progeny appears, there is a continual release of particles from surfaces of HeLa or L cells during a further period of 10 or more hour duration until the infected cells degenerate . Herpes virus is transported through the cytoplasm inside cndoplasmic reticulum vesicles,

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FIG . 24 . An L cc]] sampled 12 hours after inoculation of the culture with rcovirus (R) and 8 hours after a second inoculation with vaccinia (V) virus, in which both viruses have multiplied in the cytoplasm . Arrows indicate cross sections of spindle tubules coated with a dense material . Original magnification X 40 .000 . which frequently surround the progeny in the cytoplasm as they emerge from the nucleus [35] . Maturing vaccinia progeny are sequestered in similar smooth-walled cisternae of the endoplasmic reticulum (Fig . 18 and 22) before being transferred to the cell surface . At the surface, the outer membrane of each cisterna enclosing the virus and the cell membrane presumably coalesce at the region of emergence so as to provide an opening for the escape of the virus, which passes to the outside enclosed by an inner membrane of the cisterna . (Fig . 23 .) ALTERATIONS IN CELLULAR STRUCTURE Many kinds of alterations in the fine structure of the infected host are known to be induced by viral infections but the most common among them involve formation of membrane complexes, which in some instances are elaborated as multilayered, parallel lamellas and in others as an extensive network of cisternae. Infection by poliovirus, Mengovirus, EMC virus and other small RNA viruses elicits the latter kind of response, which becomes manifest with formation of a multitude of small, membrane-enclosed

bodies, each 500 to 2,000 A in diameter . (Fig . 2 .) Such structures are developed rapidly in the centrospherc region during the period of completion of viral multiplication [2,3] . The development of these bodies is preceded by formation of a reticulum of cisternae which progressively envelop small segments of the cytoplasmic matrix and any progeny virus which happens to be lodged in the matrix . The small bodies which appear in cells infected by small RNA viruses may develop by a process analogous to that which brings about the wrapping and segregation of progeny particles of vaccinia and herpes viruses, described in the previous section . In addition to the cisternae which envelop progeny in the cytoplasm (Fig . 22), late in the infectious cycle in L cells other membranous components are developed, in the form of long cylinders, the walls of which are composed of concentrically arranged lamellas and flat vesicles [ .31] . The formation of similar cytoplasmic and intranuclear stacks of lamellas and cisternae has been observed following infection in other virus-cell systems . One should not overlook the fact, however, that AMERICAN JOURNAL OF MEDICINE



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Fm . 25 . An example taken from a monolayer culture preserved 24 hours after infection with vaccinia virus illustrates the phenomenon of fusion and formation of giant cells . At the lower right a cell of normal size is intimately associated via an interdigitating retic-ulmn of memhrares (arrows) . with a tin giant cell Original magnification X 14 .000 , .31 i .

penetration into cells of a large variety of toxic agents, among them colloidal silica 1 .36] and carcinogenic dyes [ .37], elicits the formation of similar multilaycred membrane complexes, which suggests that a basic response by the target cells is an attempt to segregate within membranes and later dispose of undesirable intracellular materials . However, even the extreme response to many viral infections appears to he inadequate because the host cell is killed . Another feature commonly observed after virus infection is a fusion of adjacent cells in monolayer cultures into multinuelcate giants, as has been observed with vaccinia virus [ .31], herpes virus [.3k], mumps virus [391 and other viruses . Along the surfaces of attachment a highly complex reticulum of interdigitating membranes develops, providing a large surface for intimate contact between the fusing cells . (Fig . 25 .) Complete integration of the attached cells may be achieved when the surface membrane-reticulum is ruptured at some points and is later transported into the cytoplasm of the giant cell . In considering the phenomenon of cell fusion, it seems appropriate to mention the VOL .

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analogous intracellular fusion of mitochondria, observed in HeLa cells undergoing infection with adenovirus 7 (Fig . 11), which heretofore has not been reported . A better understanding of these changes on a biochemical level must await future studies_ SUMMARY AND CONCLUSIONS

The most important conclusion arising from numerous cytologic observations on interactions between cells and animal viruses is that replication and morphogenesis are truly intracellular events, i .e ., they occur in the nuclear and cytoplasmic matrix compartments . Front this it follows that the nucleic acids of the inoculum must be released and penetrate through various cellular membrane-barriers into either of these compartments to initiate the genetic functions of the virus . Having thus gained access to the synthetic sites in the host, the viral templates are able to compete with the host cell and redirect the macrotnolecular synthetic machinery toward production of viral materials . With some types, for example the poxviruses, synthetic foci arc established at random throughout the cyto-



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plasmic matrix whereas other groups develop in specialized regions of the cell, as observed with reoviruses which are formed in the vicinity of the mitotic spindle apparatus . Several types of the icosahedral nuclear and cytoplasmic viruses arc assembled into large crystalline aggregates within a viroplasmic matrix . However, with the small RNA viruses, such as poliovirus, formation of large crystals does not occur until the exponential phase of replication is completed, whereas in the preceding period of exponential growth numerous scattered progeny particles are formed . This indicates that two populations of progeny may be produced during a one-step growth cycle . Release of progeny particles subsequent to their Formation can occur in several ways : the host cell membrane may disintegrate, scattering the virus in a burst ; or individual particles may become enveloped in a lipoprotein coat at cellular membrane surfaces, where the virus is assembled, and then separate from the cell ; or cisternae of the endoplasmic reticulum may sequester individual particles in the cytoplasmic matrix before they are moved to the cell surface prior to being eliminated . Cells frequently respond to viral infections by synthesizing large quantities of membranous material, which appears in the form of multilayered lamellas, cisternac of the endoplasmic reticulum, or in other forms . Since formation of extensive membranous complexes is elicited also by a variety of nonviral toxic agents, the changes observed after infection could be a manifestation of a basic mechanism developed to segregate and eliminate foreign material which has gained access into the cell interior . REFERENCES 1 . DALES, S . The uptake and development of vaccinia virus in strain L cells followed with labeled viral deoxyribonucleic acid . J. Cell Biol ., 18 : 51, 1963 . 2 . DALES, S . and FRANKLIN, R . M. A comparison of the changes in fine structure of L cells during single cycles of viral multiplication, following their infection with the viruses of Mengo and encephalomyocarditis . J. Cell . Biol ., 14 : 281, 1962 . 3 . KALLMAN, F ., WILLIAMS, R . C ., DULBEcco, R . and VOGT, M . Structure of changes produced in cultured cells sampled at specified intervals during a single growth cycle of poliovirus . J. Biophys. & Biochem . Cytol ., 4 : 301, 1958 . 4 . DALES, S ., EGOERS, H ., TAMM, 1, and PALADE, G . E . Unpublished observations . 5 . HORNE, R . W ., W.ATERsoN, A . F . . WILDY, P . and FARNHAM, A . E . The structure and composition of the myxoviruses . L Electron microscope studies

of the structure of myxovirus particles by negative staining techniques . Virology, 11 : 79 .1960 . 6 . WATSON, B . K . and Cooxs, A . H . Studies of influenza virus infection in the chick embryo using fluorescent antibody . J . Exper . Med ., 100 : 203, 1954. 7 . MORGAN, C ., RIFKIND, R . A . and ROSE, H . M . The use of ferritin-conjugated antibodies in electron microscopic studies of influenza and vaccinia viruses, Cold Spring Harbor Symposia . Quart Biol ., 27 : 57, 1962 . 8 . CnoppIN, P. W . and S1'OECKENIUS, W . Interactions of ether-disrupted influenza As virus with erythrocytes, inhibitors and antibodies . Virology, 22 : 482, 1964 . 9 . KATEs, M., ALLISON, A . C ., TvRELL, D . A . J . and JAMES, A. T . Lipids of influenza virus and their relation to those of the host cell . Biochim . et biophys . acto, 52 : 455, 1961 . 10 . HownrsoN, A . F . and WHIrMORE, G . F. The development and structure of vesicular stomatitis virus. Virology, 16 : 466, 1962 . 11 . MATSUMOTO, S . Electron microscopic studies of rabies virus in mouse brain . J. Cell . Blot., 19 : 565, 1963 . 12 . HAGLENAU, F ., FEBVRE, H . L. and ARNOLLT, J. An intracellular mode of formation of Rous sarcoma virus . The role of temperature on the multiplication of this virus . In : Proceedings of the Fifth International Congress on Electron Microscopy . Philadelphia, 1962 . Edited by Breese, S . S ., Jr . New York, Academic Press, Inc. 1962. 13 . DE HARVEN, E . and FRIEND, C . Electron microscopic study of a cell-fret induced leukemia of the mouse : a preliminary report . J. Biophys. & Boochem . Cytol., 4 :151,1958 . 14 . MORGAN, C ., Howa, C. and ROSE, H . M . Structure and development of viruses as observed in the electron microscope . V. Western equine encephalomyelitis virus . J. Exper . ,toed., 114 : 825, 1961 . 15, PFEFFERKORN . E . R . and HUNTER, H . S . The source of the ribonucleic acid and phospholipid of Sindbis virus . Virology, 20 : 433, 1963. 16 . GOMATOS, P . J . and TAMM, 1 . The secondary structure of reovirus RNA . Proc . Not . Acad . Sc ., 49 : 707, 1963 . 17 . TouRNIER, P . and PLISSIER, M . Le developpement intracellulairc du reovirus observe au microscopic. electronique . Presse mid., 68 ; 683, 1960 . 18 . SPENDLOVF, R . S ., LENF.rrE, E. II . and JOHN A . C . The role of the mitotic apparatus in the intracellular location of reovirus antigen . J. Immunol., 90 : 554, 1963 . 19 . DALES, S . Association between the spindle apparatus and reovirus . Proc. Nat. Acad. .Sc . . 50 : 268, 1963 . 20. DALES, S ., GOMATOS, P. .1 . and Hst, K . C . The uptake and development of reovirus in strain L cells followed with labeled viral ribonucleic acid and ferritin-antibody conjugates . Virology, 25 : 193, 1965 . 21 . DAL'TON, A . J ., KILt5AM, L . and ZEICEL, R . F . A comparison of polyoma, "K", and Kilham rat viruses with the electron microscope . Virology, 20 : 391, 1963 . 22 . BREESE, S . S ., JR., HoWATsoN, A . F . and CHANV, C . Isolation of virus-like particles associated with AMERICAN JOURNAL OF MEDICINE

Replication of Animal Viruses Kill tam rat virus infection of tissue cultures . Virology, 24 : 598, 1964 . 23 . MELNICK. .1 . L . Papova virus group . Science, 135 : 1128, 1962, 24. KAPl AN, A . and BEN-Ponar, T. '['he pattern of viral and cellular DNA synthesis in pseudorabies virusinfected cells in the logarithmic phase of growth . l''nole-v, 19 : 205, 1963 . 25 . KEIn, II . M . and Gol .n. E . Deoxyribonucleic acid nucleotidyltransferase and deuxyribonuclease front cultured cells infected with Herpes simplex virus, Bzoeham . et baophys . aria, 72 : 263, 1963, 26 . AovER, G . S . . DENNV, F . W . and GINSBERG, Ii . S . Intracellular localization of type 4 adenovirus . n . Cytological and Iluorescein-labelled antibody studies. .1 . Exper . Med., 109 : 85, 1959 . 27 . MORGAN, C ., GOnMAN . G . C ., BREITENFELD, P . and Rose . H . M . A correlative study by electron microscopy and light microscopy of the development of type 5 adenovirus . J. Expect Med ., 112 : 373, 1960 . 28 . MAGEE . \V . E . and SAGIK, B . P ., The synthesis of deoxyribonucleic acid by HeLa cells infected with vacciuia virus . Virology . 8 : 134, 1959 . 29 . BLAND. J . () . IV . and RontNOw, C . F . The inclusion bodies of vacciuia and their relationship to the elementary bodies studied in cultures of rabbit's cornea . J . Palh . Bar? ., 48 : 381, 1939 . 30 . CAIRNS, 1 . '1' he initiation of vaccinia infection . Virology, l1 : 603, 1960 . 31 . DALES, S. and SIMINOVrs'CH, L . The development of vacciuia virus in Earle's strain cells as examined by electron microscopy . 1 . Riophys. & Riodiem . Wnl . . 10 : 475, 1961 . 32 . SCHARrr . M . D . . SmvtKIN, A . J . and LEVINow, L .

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Association of newly formed viral protein with specific polyribosomes. Proc . -Val . .lead. Sr ., 50 : 6P6, 1963 . 33 . SANDERS, F . K . . HUPPERT, .1 . and HosxiNS, J . M . R epl ication of an animal virus . Svnrp . So,. Exp . Riot, 12 : 123, 1958 . 34 . Lwola, A . . DULBECCO, R ., Voce, M . and Lwoee, M . Kinetics of the release of poliomyelitis virus from single cells . Virology, 1 : t28,1955 . 35 . MORGAN, C ., Rose, H . M . . I [OLDEN . M . and JONES, E . P . Electron microscope observations on the development of herpes simplex virus . J . Exper . Med., 110, 643, 1959 . 36 . PoLLICARn. A ., COLLE'r, A . and PREOERMAN, S . Etude au microscope electronique de la lipophancrose eytoplasntique . 4th International Conference on Electron Microscopy, Berlin, 1958 . Edited by Bargmann, W ., Peters, D ., and NN"olpers, C . Berlin . 1960 . Spinger Verlag . 37 . EMMELOT, P . and BENEDETTI, E . L . Changes in the line structure of rat liver cells brought about by dimethylnitrosamine . J. R ophys . .? Biochcm . Cytel ., 7 : 393, 1960 . 38 . Rou.MAN . B . Polykaryocytnsis . Cold Spring Harbor Symposia Quanl . Biol., 27 : 327 . 1962 . 39, RAPP- N . Observations of measles virus infection of human cells . u s . Correlation of properties of clones of H .Ep-2 cells with the susceptibility to infection . Virology . 10 : 86, 1960 . 40 . Cuorrw, P . W . On the emergence of inllucnza virus filaments from host cells . Verologr . 21 : 278, 1963 . 41 . KAIIOKA, R ., SsMlNOVrrcH . L . and DALES, S . The cycle of multiplication of vacciuia virus in Earle's strain 1 . cells . i t . Initiation of DNA synthesis and merpho; cress . Virology. 24 : 205, 1964 .