Biogenesis of poxviruses: Role of A-type inclusions and host cell membranes in virus dissemination

VIROLOGY

46,

507-532 (1971)

Biogenesis

of

Poxviruses:

Role

Membranes YASUO

ICHIHASHI,2

SEIICHI

of A-Type

in Virus

lnciusions

and

Host

Cel&

Dissemination’

NATSUMOT0,3

MV~ SAMUEL

DALES

Several variants of vaccinia and cowpox were employed to study the probiems of virus disserni~~atio1~ i~~~olvin~ wrapping in host n~ernbral~es or inte~ratiol~ within Atype inclusions (ATI). All the cowpox variants employed induce ATI and hemagglutinin (HA). The control of synthesis of HA and ATI and its relationship to the process of occlusion was examined by means of a variety of metabolic inhibitors. The experimental data indicated that ATI and HA are virus-directed late functions whose synthesis is under regulation that is distinctive from t,hat related to biogenesis of mature virus. Capability to become occluded within ATI, a heritable character possessed only by some strains, is termed the V factor (Ichihashi and Matsumoto, 1968). Expression of t,he V trait can occur in the presence of rifampiciIi, as revealed by integration into AT1 of immature V+ strain CP58 particles. The V+ trait can also be transferred to the V- strain CPRCl by complementxtion in a double infection with the rifampicinresistant Nit4 vaccinia. These observations suggest that V may be acquired as a component of the virus membrane. To study the fate of progeny not, involved in occlusion in ATT, we employed V;LDcinin IHD-W, which induces polykaryoeyte formation (F’)) but is HA-, and strain IHDJ, which is HA+ and I?. An orderly progression of events, particularly evident with IHD-W virus, was associated with the movement of progeny particles which were formed earliest. According to the recox~st.ructed sequence, the wrapping of individual virus particles in cell-derived membranes occurred prior to their release at the cell surface. The IHD-J progeny were usualiy not enclosed by cisternae and migrated to the cell surface as ~lnwrapped or nak~dparticles. Both the cell mexnbraI~cs which formed themselves into membranes of cisternse and certain defined regions of plasma membrane in contact with naked intracellular progeny virus possessed viru$specified antigen(s). INTRODUCTION

In recent years considerable at~entioI1 was given to t,he ir&ial stages and biosynthetic 1 A preliminary report on some aspects of this work was presented by one of us (S.D.) at the Amer. Assoc. Pathol. and Bacterial. Symp. “Cell Membranes: Biological and Pathological Aspects,” St. Louis, Missouri, March, 1970. 2 International Postdoctoral Fellow. Present address: institute for Virus Research, Kyoto University, Kyoto, Japan. s Institute for Virus Research, Kyoto University, Kyoto, Japan. * Address for reprint requests.

events occurring during the infectious cycle of the poxviruses, but, relatively little information was published concerning virus tlisseminat~ionas it is related to the spread of infection. It was, however, generally :kppr’eciated that cellular membranes and A-type inclusions, also termed Downie or Mar&l bodies, are involved in the segregation :md movement, of progeny particles to the eel1 exterior (~fatsumoto, 19.56;I&to et at., 1963; Dales and Siminovitch, 1961). With regwd to segregation, mature part~icles of certain poxviruses are occluded in the ~r(~teitla~e(~~~~ matrix of A-type inclusiorrs (RI:rtsumoto,

508

ICHIHASHI,

MATSUM~r~,

1%6), and some poxviruses cause a modificat#ion of the surfaces of host cells by inducing synthesis of a hemagglutinin (HA) (Driessen and Greenham, 1959; Kaku and Kamahora, 1964). Although the antigens of the A-type inclusions a,nd HA do not become components of the virus particle i&elf, both are most probably produced under the control of the virus genome, as demonstrated by the formation of recombinant types, identifiable by genetic analysis (Ichihashi and Matsumoto, 1969). In synchronously infected cells the HA and A-type inclusion antigen are dete~tabIe for the first time 2-3 hr after development of mature, infectious particles has commenced, indicating that the two materials may be synthesized as a distinctive class of virus products. In the first part of this study we describe experimental evidence concerning the regulation of the formation of these antigens. The second aspect deals with the genetic characteristics of the virus surface as they are related to occlusion of virus particles within A-type inclusions, and with modifications of the host membranes involved in transporting the progeny out of cells. The third section of this inquiry, described in the companion article (Ichihashi and Dales, 1971), deals with the intluence of poxviruses on cell-to-cell membrane interactions.

AND DALES

mented with 10 % fetal calf serum (FCS), as previously described (Dales and Siminovitch, 1961; Ichihash and Matsumoto, 1968; Dales and Mosbach, 1968). For synchronous infeetion, 5 PFU/cell of virus were added and infection was allowed to develop in NM containing 5 % FCS. Virus titers were determined on monolayers of CEF according to published methods (Ichihashi and Matsumoto, 1968). The progeny of mixed infection could be identified by the characteristic plaque type and as hemaggluti~n positive (HA+) or negative (HA-) by t~hehemadsorption method whereby White Leghorn chicken erythrocytes (RBCs) became attached to cells possessingHA on their surface (Oda, 1965). In Table 1 is given the list of the poxviruses employed and their genetic characteristics which have been described in previous reports (Hanafusa, 1960; Nagayama et al., 1970; I~hihashi and Matsumoto 1969). Antisera and immure leering. Specific antisera against vaccinia of type IHD-W (inducing polykaryocytosis and IHD-J (inducing HA production) were prepared in rabbits infected with purified suspension of either virus, as described previously (Gold and Dales, 1968; Ichihashi and Matsumot,o, 1966). For immune labeling of the surface components using t,he light ~eros~ope, the indirect method patterned after Coons (Goldst,ein et al., 1961) was employed. Cover MATERIALS AND METHODS glass cultures of cells were washed three Cells and viruses. Monolayer cultures of times with phosphate-buffered saline (PBS HeLa, Lz , or chick embryo fibroblast (CEF) of Dulbecco and Vogt, 1954), and then fixed cells were propagated in nutrient medium by immersion in acetone at -40” for 5 min (NM) consisting of Eagle’s MEM supple- then were hydrated in PBS. A thin overlay of either preimnlune or specific antiserum diluted 1:4 in PBS was spread to cover the TABLE 1 cells and incubated for 30 min at 37”. The GENISTIC CHARACTERISTICS OF THE unadsorbed antibody was washed away and POXVIRUSES EMPLOYED the cultures were incubated for 30 min at 37” Integration with an overlay of fluorescein-conjugated Rifaminto picin antirabbit goat globulin (Microbiological Virus type resistfig! ance Associates) diluted 1:4 with PBS. A similar sions w+> procedure was employed for electron microscopic immune labeling. Specific antisera Vaccinia IHD-W + Vaccinia IHD-J + + diluted 1: 20 and antirabbit sheep globulin Vaccinia NR4 + + conjugated with ferritin (kindly prepared by Cowpox CPRCl + + Dr. K. C. Hsu, Columbia University) diluted cowpox CP58 + -t + 1: 100 were absorbed to unfixed cells prior

to processing for embedding and thin sectioning. ~~~~~b~t~r~. In each case, inhibiti was initiated by adding to the infected cultures an overlay of pretva~rmed Nbz plus the inhibitor. To suppress DNA synthesis, hydroxyurea (HU) was added at 5 X 10v3 JZ, and cyt,osine arabinoside (CA), at 5 pg/ml. (Both drugs were purchased from Sigma Co.) The inhibition was ascertained by comparing the amount of acid-precipit,able radioactivity bound t.o cells using scintillation eounting or autoradiography. In the latter procedure, 10 &i/ml of thymidine-3H {Schwartz) were added at the time of ~nfectiol~ and maimained in the NM throughout the infectious cycle. Nonmacromolecular label was extracted with 5 7%:trichloroacetic acid at 0” and the cells were coated ~~&h photographic emuIsion (Sakura NRs or Ilford Kci> and exposed for about 2 weeks. Under these experimentsll conditions inhibition of DNA synthesis was found to be almost complete. To inhibit, RNA synthesis, 3 pug/ml of actinomycin D (gift from Merck & Co.) were added and to suppress protein synthesis, 10 &g/ml of puromycin (CalBiochem) were applied. R~farnp~ci~ (gift from Ciba Pharmaceutical Co.) was usually added at 100 fig/ml. E&ran microscopy. l?or routine examination by means of &in sections, pellets of infected cells were prepared as described previously (Dales, 1963b). To examine infected cells in the vicinity of plaques, monolayer cultures of HeLa cells were grown on 20 X 60 mm pieces of coverglass which had been coaied with a he?vy carbon film (approximately 200-506 A tZhick). Each monolayer was infected with about, 30 PFU of virus and plaques \vere allowed t,o develop for 45 hr under a mixture of 0.9% agar and NM. The plaques were fixed for 30 min in situ with 2.5 % glutaraldehyde in M/%0 PO, buffer, pH 7.0, the agar overlay was removed, and cells were washed with buffer, then postfixed with buffered 1% Os04 for 30 min. r~el~ydration conformed to the standard procedures. For embedding t,he monolayers were covered with m epoxy resin mixture which, aft,er polymerization, hardened into a layer 1-3 mm thick. Selected areas containing plaques were i~~entified under a binoc~~lar

microscope and dissected out’. 13:wh segnwnt, of the monolayer was oriented in parallel or perpendicl~l~~r t,o the flat. surface of an epox? resin block, cemented, and sectioned. ‘I’hc sect5ons passed either vertically or horium t.ally through the cells of the monolayt~r. RESULTS

Certain structures, some previously ctbserved in L cells infected with ectronwli;b virus (Ichihashi and Matsumoto, 19661, were also present, following infection of HeLa ~11s by co~\pox. Both the CP58 and CPRCl st.rains elicited the format,ion of large A-t.ypc cytoplasmic inclusions (ATI). Such inclusions had a dense, somelvhat granular homegeneous and compact, m&fix clearl? &tinguishable from the fibrous dense material t~f the viroplasmic matrices or factories (l’igs. l-5). It is known from previous studies that integration or occlusion of mature rctromelia and cowpox particles in ATI is tiet,crmined by the V+ marker (Ichihashi ~1 ZMatsumoto, 1969). The CP.58 strain of co\\-pox induced tile ~ormat.io~l of i~~~~usio~~~or Downie bodies (Kato et nl., 1963) ~~)n~,ai~~irlg masses of mature virus particles (lcigs. 1-4). Such inclusions, like the I\‘Iarchal bodies found in ectromelis i~lf~ct~~~l~ ~~lats~lr~~~)t[}, 1956), are formed under the regulation of virus possessing the V+ trait. By cont>r:wt, the CPCRl strain of cowpox, which is of tlw V- &ss, induced ATI. complefel:- devoid of oc~l~lded virus particles (Figs. 1 and .;i. However, the CP5S and CI’RCI. are cltwl~ related irnn~~~r~olo~icall~ (l~n~~llblisll(3(~ (tbser~at,ions~ . One prominent morphological fe~turr not, reported hitherto was the preaenot? ol’ i\umerous clusters of ribosomal particles :lt t 11(x surface of AT1 (Figs. l-5). By :ma,logy lvith similar, ~~biq~~t,o~~sribosomnl a~~reg:~tt~s~ t Iw clusters were identified as polyribosonrcs. The polyribosomes were frequently formed into zipperlike assemblies (Fig. :;I 1 indicat ing that they were arranged into heliws of t,he type previously observed in baet (&I, and eukaryotic cells (Dales Pt nl., 1Utirt ; Schwartz :md %inder, 1967; Wcias :~ntl Grover. 196s; F,chlin, 106.~~~antI ~)~~~~)~l~~~ti~~Tl~

Key to abbreviations: IV, nucleus; C, centriole; G, Golgi membranes; F, focus of viroplasm; I, immavirus; M, mature virus. FIG. 1. Portion of the nucleus and cytoplasm of a HeLa cell infected with CP58 cowpox for 24 hr. Several dense A-type cytoplasmic inclusions, each filled with mature particles, are present in the cytoplasm. Large numbers of polyribosomes (arrows) surround each inclusion. X 15,000. FIG. 2. Higher resolution image of a portion of the area illustrated in Fig. 1. X 30,000. ture

510

structure of polyribosomes and inclusions from infected cells (Ichihashi and Dales, to be published). When HeLa cells infected 13 hours previously with CP58 con-pox arc transferred to S 31 containing act,inomycin 1) and incubated for another 4 hr, polyribosomes were not evident in the vicin& of ATI. They were, however, present in Iargt: number after incubation for 17 hr in N\I with 100 pg/nil rifampicin. Another structure observed regularly after infection for lo-24 hr of HeLa and I, cells by CP.58 or CPRCl cowpox n-erc bundles of elongated, rigid tubular structures (Figs. -I and 6). The tubules had a uniform diameter of about, 500 A and dense walls 60.-SO A”z thick. Although t,heir length was variably, some were over 2.2 p long. Examination at higher resolution revealed a well-defined periodicity indicative of a helical arrangement of the basic m&s constituting the walls of the cylinder (E’ig. 6). Bundles of similar tubes were frequently observed in the cvtoplasmic matrix and occasionally were found within nuclei. The nature of the matSerial const’ituting the tubes is unknown at present. However, the tubes were assembled \vhen synthesis of DNA was blocked by HI,?, intlieating that material of tire tubes was sytithesized as an expression of someearly virus function. Similar but larger structures havtb been observed in Yaba viru:: infection ((I(% Harvrn ant1 Yolrtt, 1 %iti) .

Our previous studies using inhibitors of 1)X’.% synthesis demonstrated that, in the biogemSIS of mat,ure IHD-W virus, expression 01 early and lat,c functions is required (Pago of isohtted polyribosomes (Rich et al., 1963; and IMPS, 1969, 1971). Thus, virus-specified enveloljes belong in the class of cwly comShelton and Rut?‘, 1966). During normal infection with either CP.58 ponents and the four enzymatic activitiw residing in t,he virus core are among late or CI’R.Cl, the polpribosomes were situated factors associated with maturation. Sitw in the vicinity of developing ATI, covering CI’S and CI’RCI co\VJ>OX induced form;~ their entire surface (Figs. l-3, 5). LIorphotion of AT1 and HA while IHD-W vaccini:i logically these polyribosomes were indistinguishable from ribosomal clusters present, did not, we \vishctl to det,ermine ~vhether the throughout other regions of the c-&oplasmic two nonvirion materials belong in the c:itc,matrix. The intimate associat,ion bet’meen gory of early or late f:t&ors. The relat,ionship hc+\veetl a one-st,ep the polyribosomes and AT1 matrix could be dcmonstr:itcd b>- releasing the complex growth cycl(h of Cl’.% arid the formation of Frc;. 3. Another example illustrating ribosc~mes in the vicinity of an inclllsion.

lollg polyX 32,000.

512

DISSEMINATIOK

AT1 was ascertained as illustrated in P’ig. 7. increase in the infectious particles commenced at 4 hr but AT1 were detected for the first time 9 hr post infection. Thereaft.er, the AT1 developed rapidly and by 18 hr nearly all cells possessedthese inclusions. It should be emphasized that the linear increase in development of AT1 occurred 7--S hr later than the exponential production of Cl’;?8 cowpox (I<‘&. 7). The time course of appearance of HA closel\- paralleled that of AT1 development (Fig. S). The HA associated with CP%infected HeLa cells could be detect.ed initially S hr post, infection, rose exponentially in titer, and reached a maximum at 1s hr. To determine whether HA and ATI were early or late functions of the CP58 cowpox, the effects of DYA inhibitors were monit,ored in HeLa cells following kansfer into N3i ~o~ltail~ing eit,her HU or CA. The culture samples lvere exposed to either inhibitor at, 2-hr intervals commencing 2 hr after inoeulation and were t,lrereafter incubated for a total of 24 hr from the time of inoculation. The percentage of cells with clearly identifiable ATI was determined by phase-contrast microscopy. The results summarized in Fig. 9 sho\r;ed that treatment with either inhibit,or O-3 h-f after infection completely suppressed formation of the inclusions, but exposure to HU or CA at any period after 8 hr failed t,o suppressATI formation (Fig. 9). Therefore, in this system ATI development is contingent upon prior DNA synthesis during t,he initial 3-S hr of infection. ~uppre~ion of HA production by HU and CA followed a very similar pattern to that described for ATI. As shown in Fig. 8, appli#cation of HU O-3 hr after infection completely abolished HA formation. Presence of this inhibit*or 3-S hr post-infect’ion reduced proportionately the amount of HA formed ^...~_

-._-

(Q.

$8). These

observations detnonstrated that both HA and AT1 were produced only when DXA replication could occur and, hence, by definition, both materials belong in t’he r:ltegory of lat,e functions. Relationship to RNA and protek sgnth&s. Addition of ~~ct~irlornyci~lor puromycin t,o inhibit synthesis of RNA or protein also :tffect,ed t,he format~ion of HA and ATI. Application of either actinomycin D or puromycin atj any t,ime during the i&al S-hr period suppressed t)he formation of both materials (Fig. 10). Addit,ion of t#heseinhibit,ors ;tt later times, S-l 8 hr after infect,ion, decreased the HA t,it,er and t,he percentage of cells containing ATI, in relation to the time the inhibition was instituted. These combined results using a variety of metabolic inhibitors indicated t’hat synthesis of AT1 and HA started late, was contingent, upon DNA synthesis and was most probably dependent upon t8ranscription from progeny DNA. Once transcription and t ranslat.ion into these antigens had comi~~enced, the continued and uninte~upted synthesis of RNA and protein was required for the exponent,ial increase of the mass of these I\VO materials. BjTects of‘ rifampicin. Addition of rifampi& to infected HeLa cell cultures inhibits the formation of mature progeny poxviruses (Moss et ai., 1969; N~~gayama el al., 19701, but does not interfere with t,he developn~eI~t of HA or AT1 (Figs. S and 10). When HeL:t cells were infected with Cl?% virus for ci hi in t,he presence of rifampicin, then fransferred to NM lvithouf rifampicin, but, now containing 5 x lo-:’ M HI:, t hr HA :mtl

- -

C-6. Specific features of HelLa cells infected for a period of 17 hr with cowpox. An A-type cytoplasmic inclusion devoid of virus particles. Numerous polyribosomes stninclusion. A bundle of rigid tubular structures is indicakd by au arrow. X 19,000. The periphery of an A-type cytoplasmic inclusion illustrated at higher resolut,ion reveals fhr of polyribosomes with the surface of the dense matrix. X 37,000. A bundle of tubular structures illustrated at higher resolution. The well defined periodicity walls is indicative of a helical arrangement of the romponent,s constituting the wall of the 145,000. FIGS.

FIG. 4. round the FIG. 5. association FIG. 6. along the tubes. X

-__

depending on the time elapsed before HU was added. When cells were exposed to HU later than S hr post-inoculation there was no furt,her reduction in the final HA titer

514

ICHINASHI,

MATSUMOTO,

AND DALES

3 6 HOURS

9 12 IS 18 21 24 $0 POST

INFECTION

FIG. 8. Effect of HTJ (O-O), actinomycin D (U-U), puromycin (A-A), and rifampicin (X-X) on HA production in HeLa cells infected HOURS POST iNFECTION with cowpox CP58. Infected monolayers of HeLa cells were transferred to inhibitor-containing medium at the times indicated. All sampleswere suspended in 1 ml of PBS and sonicated for 1 min. FIG. 7. One-step growth cycle of CP58 cowpox Titers were determined using a-fold dilution and development of A-type cytoplasmic inelu- series on samples collected 24 hr after infection. sions (ATI). HeLa cells were infected with 5 Other cultures were harvested throughout the PFU/cell. Samplestaken at each time point conone-step growth cycle and assayed for HA prosistedof 2 petri dieh cultures (60mm) whieh were duction (O.-O). 0 4 8 i2 I6 20 24

washedin NM, scraped,pooled in 2 ml of PBS and sonically disrupted.Plaque assayswere made

of spicules (Dales and Mosbach, 1968). From the reconstructed sequence of virus morphopoeisis as examined by meansof thin sections, these membranes appear to be ATI inclusions developed at a normal rate unique to this agent and discontinuous from during the remaining 18 hr of the infectious any of the cellular membranes present during virus development. cycle. This showed that after reduplication As shown previously (Dales, 1963a), memof virus DNA in the presence of rifampicin, subsequent treatment with HU was com- branes of vaccinia and the other poxviruses pletely ineffectual in suppressingthe synthe- are assembled within viroplasmic matrices or factories which develop in the cytoplasm. sis of HA and ATI. First evidence of membrane formation is the Efects of Rifampicin on the Biogenesis of appearance of short,, arched segments conEnvelopes, Virus Maturation, and Inte- sisting of a unit membrane and spicule coat. gratis of Purt~~les ~.thi~ A-Type InDuring assembly of t,he membrane, the surClU~.~S face area increasesin all directions presumE$ects on membranes. The envelopes of ably by accretion of material around the poxviruses meet several of the criteria for initial site of condensation. The predetercellular membranes (Dales and Mosbach, mined curvature arising during self-assembly 196s). Chemically they are constituted from must determine the uniformity of the size protein and lipid, of which lecithin is a and hence the ultimate surface area of the prominent phospholipid. These envelopes envelopes of immat,ure vaccinia. It appears possessa trilaminar or “unit” membrane likely that the arching is imposed at the structure of approximately the same dimen- very beginning of membrane assembly by sions as that of host cell membranes. The the attached spicule layer because virus en‘?.mit” membrane is coated externally by a velopes with sparsely distributed spicules were irregular in shape (Fig. 21). Those closely packed and uniformly distributed dense layer (Fig. II), equivalent to the layer regions of the virus envelope which lacked using CEF and the frequency of cells containing ATI was ascertained by phase contrast microsCOPY.

DISSEMINATION

NOUNS

POST

OF POXVIl~U~F:S

INFECTION

9. Effects of HU (O-O) and CA (0-O) on formation of ATI. Coverglass cultures of HeLa cells were infected with CP58 virus then transferred IO medium with HU or CA at the times indicated. Samples were enumerated for the proportion of cells possessing A-type cytoplasmic inclusions (ATI) 24 hr after infection. Development of .kTI in the absence of inhibitors (O..O) was also monitored by sampling throughout the growth cycle at the times indicated. FIG.

spicuks were flexible, highly convolut,ed structures which frequently formed into sealed, small vesicles (Figs. J.1 and 12). Other features of the aberrant development of membranes of CP58 following rifampicin treatment are illustrated in Figs. 11-14. As was found previously with vaccinia (Dales and Mosbsch, 1968; Pennington et aZ., 1970; ~agaynma et a?., 1970), the sheets of flexible uncoated membrane can remain open ended, or become sealed into spherical vesicles or elongated flexible tubules possessing t.lre unit. membrane config~~ration. In some instances :I coI~t,i~~uity between segments of spicule-coated curved envelopes and uncoated membrane sheets was observed (Fig. 11). Att:lchment of long, flexible membranous elementz to t,he surface of AT1 was encountered frequently (Figs. 14 and 15). Perhaps this membrane mat,erial possessed an afinity for :L Ilydrophobic substance of t’he ATI. Another manifestation of abnormal membrane development, xx-as the great variability in the size of immature particles. The envelopes of such particles had sparsely distributed coating subst,ance (spieules?) (Figs. 16 and 17). With regard to the size, it was calculat,ed hhat tShesurface area of the largest, observed particle \vas 0.55 p2, while that of the smalIest only 0.05 ~2, while the average :\.rid highlg uniform surface area of normal

2 1.j

HOURS

POST

INFECTION

FIG. 10. F:ffrct of actinomycill 1) (~3 ;:I), puromycin (A- a), and rifampicitl (X >< J on formation of ATI. Iltfected coverglass rultltres were treated with act,inomyein D, puromycin. or rifa~lpicil~ and examined for the presence of incltlsions as described irk Fig. 0.

immature particles was 0.29 ~2. An abnormal coating of the unit membrane by t!he spicules may have affected the curvature of dewloping envelopes \r-hi& resulted in the siz,~ differences observed. EJltfPctsMLnzaturation. The CPRC~l variant of coupox is sensitive to rifnmpicin, induces AT1 and HA, but, is V- for integration of particles. The NR, mutant., described previously (~agay~~~~la61al., 1970), is resistant to rifampicin, fails to induce either AT1 or HA, but is V+ for integration (Table I). It was of interest to determine whether (a) immature particles are endowed with the V+ trait and hence could be occluded in .4TI and, (b) late funct,ior~s necessary for rnatur~~ti(~r~ could be derived from the SR, mutant, virus by complemerlt:~t,ion. We t,herefow infected HeLa cells \vitll 211: 1 mixture of CPKCl and NR, viruses and incubated the culturrs for 24 hr in the presence of the antibiotic. Singly infected ceils were used as the controls. As aI~ti~i~~:~ted,the NRi mutant mulCplied to high titers while the wscqkiblr CPIZCl virus ws suppressed (Table 2). In the mixedly infect,ed cultures, high yields \vere obtained. The titer of HA+ infectious particles constituted about, 10 ‘ii t)f t.he t-otal progeny and w.v:~,s more than loo-fold higher than titers obiained from cells singly infect.ed with CE’RCl (Table 2). To differentiate the t\z-o types of progeny virus, plaques were allowed t,o develop for 4S hr on CEF mortolayers covered by a liquid X:X I overlay. Then Iif3Cs were added to the monola~ws 1st

516

iderttif!, foci that were ~~ema~glutiIlin positive or negative. The HA+ and HA- plaques were isolated, grown into virus pools, md exnmined for the genet~ic characters of the progeny. It was found that lO/lO of HA+ plaques contained virus which w‘as rifampicin sensitive, induced HA a.nd AT1 but was Y- with respect to integration, i.e., possessed the same markers as CPRCl (Table 1). By contrast, lO/lO HA- plaques contained virus that was rifampicin resistant, failed tu induce HA or ATI and was W, i.e., like the N& mutant (Table 1). Since with the rtoxviruses maturation is required for infectivity D:lles and Siminovitch, 1961), it! appeared that functions required for maturntion could be provided by NR.8 and became incorporated int.o the drug-sensitive CPRCl particles. Therefore, NRJ could rescue CPRCl virus presumably by making nvailabie the necessary late maturation factors that I\-ere suppressed in the rifampicinsensitive &rain. iQfeceL:ts orz ~nte~rut~onwithin A -type inclusions. During normal infection, in the absence of rifampicil~, only mature CP58 particles were found in ATI, and none belonging to t,lte CPTXI. (V-) strain were occluded. I~ifarnpi~i~~ blocked t,he rn~tur~~io~ of both variants. Some of the immaOure CP5S particles which were formed in the presence of rifarn~ici~~ became integrated within the m&ix of A4TI (Iiig. Is), suggest’ingthat they possessedthe V” factor. W11er~arlls were doubly infect,ed in the

FIG. II. Aberrant morphopoeisis of cowpox CP58 in HeLa cells infected and treated with rifttrrll)ici I) for 14 hr. With the exception of one immature particle in the process of formation (short, arrow), t.he e?ttensive sheaths of membrane surrounding the viroplasmic matrix are without it uniform coatSing OT spicules (lctng arrows). Flexible tubular elements, possibly ~l~)~lol~~r~~~s with the unit rrtemhram~ of I hp virus envelope, are also evident. X 86,ooO. FIGS. 12 xnd 13. Comparison bet.ween the unit membrane of the virus envelope of immaturt: cowpoke CP58 (arrow) and the flexible tubular elements in mat,erial from t,he I~repar~t,ion described in Fig. 11. Both cross sections (Fig. 12) and longitudinal. cuts (Fig. 13) reveal the presence of a unit’ merrtbranc~ ol about the same dimensions as in the virus. X 140,000 and X 160,000. FIG. 14. Associat.ion between flexible tubules and ATI in HeLa cells infected for 17 hr wit.h cowpox CP58. Cozt~mencir~g with the t,ime of inoculation, t.he cells were t.rcated for 13 hr with riftzmpirin. Upo” washing out the rifampicin, actinomycin D was added to the cult,ure medium and incubation was r~)rttinued for an additional 4-hr period. Note the absence of polyribosotnes from the surfarc of t.hs inclusion. X 46,000. FIG. t5. Enlargement of a portion of the a,rea in Fig. 14. Nol e f he int imatc aasociat ion beiwc~t~ t lrcb tlLbulrs :tncl the surfacr of the inclusion. X 100,000.

518

ICHIHASHI,

~ATS~MUTU~

AND DALES

DISSEMINATION

OF POSVTIII:SES

Al!)

TABLE 2 L~ISSCUI~:

OF

RIF.\MPICIN-SI~NSITI~~

Controls Virus

(‘PR(11 x 1-L (‘PR(:l

type

Poxvrsus (PFU/ml)

HA+ 5.8

x 107 0 6.4 x 107

BY

THE

X1(,

I< IGSIST.INT

IZifampicin HA-

HZ\+

MIST

added

\SPP

(Pl;t‘/ml) I-.\-

0 2.1 x 108 1.0 x 108

R.6 x 10’ 0 0 X.6 X 10’ + NIL 1.3 x 100 5.!) x 10’ -.__ -~~ u ti0 mm petri dish monolayer cultures of 6 X lo6 cells/plate were infected singly with :< I’FU c,ell of either virus or doubly with 2 PFU/cell of each of CPRCl and NRa viruses. At 24 hr after inocrdatiou cells were washed, scraped, and suspended in 1 ml of PBS, then sonicated for 1 min to release intracellular virus. Plaque assays were made on CEF. The HA+ or HAplaques were identified by adsorp tion of chicken RRCs.

tion revealed that. individual mature particles were surrounded by several vesicles, sometimes contiguous Jvith Golgi cisternae and occasionally possessing a dense coating layer (coated vesicles) as illustrated in Figs. ‘“-%A, B. In samples preserved at lat’er times, 6-l” hr after inoculation, some virus particles were completely enveloped by a single flat cisterna (Fig. 24D). This evidence suggested that several of t,he smaller vesicles attached to the virus surface coalesced into one continuous sac investing the virus. It remains to be elucidated ho\v the virus regulates its own envelopment and whether fusion bet\veen the vesicles is controlled in the same manner as polykaryocytosis associated with infection by the IHD-W strain of vaccinia (Dales and Siminovit’ch, 1961; Ichihashi and Dales, 1971). The virus and associated cisternae migrated toward the cell surface in preparation for emergence (Fig. 24E). &casionally, membranes of cisternae facing the cyt,oplns-

mic matrix possessed a dense coating mlbst,ance like that observed on “coated vesicles” (E’ig. 24G and H), indicating t Ilat. smooth and coated vesicles can fuse with one another to become membranes of cisternae. Sometimes fusion between individual virus-associated vesicles was not completed by the time the virus reached tlw cell wrface, as evident, in E’igs. Z4B and C’. Usu;lll>-~ n-hen the outer membrane of t,he enveloping cisterna came into contact with the plasma membrane, there was a fusion betlvern tlw membranes in contact), thereby rxposing to the cell exterior the inner membrane of tht> cisterna and enclosed virus particle (E‘ig. 24F and H-J). During the last ,st:qtx, :LC cording to this reconstructed cycle, the vine n-as freed into the extracellular fluid I\-1wr1 still invested by tile inner membrantl of tlw cisterna, or in its unwrapped or nnlwcl statcb if the investing membrane became rul)turccl (I’ig. Z4K). The IHD-W virus progeny ww not, alwa,vs wrapped in host-derived men1 branes but, migrated as uncnvt~lopt~tl or

FIG. 16. Appearance of aberrant immature particles of cowpox CPRCl in HeLa cells illft3c.t (~1 t’tibr 2 I hr in the presence of rifampicin. Note the great variability in the size of the spheres. X 52,900. FIG. 17. Two aberrant particles from the preparation described in Fig. 16. Note the similarit>it! I h(* structlxre of the envelopes and disparity in the size of part,icles. The dense elements coatilrg the rlllil membrane do not appear to be packed as closely and uniformly as in normal, immatllrr fornrs of virtls. X 122.500. FIG. 18. A-type cytoplasmic inclusions from the preparat,ion described in Fig. 16. Thflr
Fro. 21. Portion of an L cell from a culture sampled 7 hr after ~R~c~~~t.ioR with vaccii~ia virus. In the cent~rosphere region are present cent2rioles and a large riumber of C3olgi vesicles continuous with those of t.he L:olgi complex (arrow-s). X 40,000.

DISSEMINATIOK

OF POXI’IRUSES

.-j:! 1

FIG. 22. Selected region from Fig. 21 shown at a higher magnification to illustrate the invthstmcrlt 01 mature vaccinia by host, cell cisternae. Note the presence of a coated vesicle near one virlls iuLrtic,lfb (arrow). X 92,000. FIG. 23. Another example, as in Fig. 21, selected to illust,rate the association between Colgi (*ist(~rtI:u~ and progeny particles. X 57,000.

FIG. 24. (A-L) Reconstructed sequence of the envelopment, migration to the surface, and release through the plasma membrane of progeny vsccinia. The examples were selected from cells preserved 6-8 hours after infection. X 106,660 (A and B) Envelopment by vesicles that have not fused into a continuous cisterna. (B) is near the cell surface; (C) two small vesicles interposed between the plasma membrane and a single membrane surrounding the discharged virus particle. (D) Virus particle lying in the centrosphere region surrounded by a flattened cisterna. (E) A vaccinia particle with its enveloping cisterna is presumed to have migrated to the vicinity of the cell surface. (F) Separation of the inner and outer membranes of the investing cisterna in the vicinity of the plasma membrane (arrows). 522

i(i) Portion of the cisterna membrane facing the cytoplasmic matrix possesses a dense coat (arrows). (H) Vacciitia in t,he process of release at the cell surface. A single membrane, originating from the inner membrane of the enveloping cisterna, now surrounds the pa’ticle facing toward 6he cell of exkrior. ‘l’hc otizter ~len~~~r~~l~e of t,he cisterna in the inderior of t.he cell possesses a dense coat, (arrows). (I and J i Stages in the process of release at; the cell surface, as in (H). (Kj Single membrane s~]rr~~Indii~g an extract4lr1I:tr vaccinia particle has become ruptured. Note rhe free end I)f the unit membrane clearly cviciclnt nr’nr t be arrow. 523

524

XCHIHASHI,

~~TSU~OTO,

naked particles to a zone subjacent to the plasma membrane. In other instances, naked particles were transported into microvilli (Fig. 24L). It is known that immature particles of vaecinia are not i~ect.ious (Dales and Si~novitch, 1961; Pogo and Dales, 1971). It should be mentioned that occasionally immature particles of the poxviruses studied emigrated from the factories, most notably in cells treated with HU. Such immature particles also became wrapped in smooth cisternae and migrated to the cell surface in the same manner as the mature progeny (Fig. 25). The process of wrapping and release of IHD-W vaccinia described in Figs. 2224A-L was in evidence during the earlier phases of the one-step growth cycle. Examination of samples preserved 14-24 hr after infection established that intracellular particles in large numbers were accumulated throughout the cytoplasm and remained in their naked or partially wrapped state, indicating that controlled emigration from the cell declined during the latter stages of the cycle. Examination of HeLa cells infected with the IHD-J strain of vaccinia showed that some of the early mature progeny were likewise wrapped and ~se~nated (Fig. 27). However, a great majority of IHD-J particles were moved toward the cell surface in their unwrapped or naked state (Figs. 26 and 28). Regardless of whether HeLa, or L cells were the host, the same quantitative differences in the number of wrapped and unwrapped particles belonging to the IHD-W or IHD-J strains of vaccinia were observed. This suggests that interaction between the virus and membranes of Golgi vesicles is characteristic of the virus strain and independent of the host cell employed. Dissemination of the virus progeny among neighboring cells was also examined after initiating infectious foci by a single PFU. For this purpose seIected individual plaques of IHD-J vaccinia deveIoping in HeLa cell monolayers were sectioned in a plane eit,her vertical to or in paralIe1 with the monolayer. Cells in the zone adjacent to the plaque contained many mature, predominantly naked, particles, some of which had migrated to the

AND

DALES

Fro. 25. Envelopn~ent and egress of immature vaccinia particles developing in an L cell incubated for 14 hr in the presence of 5 X 10-a M HU. Compare the appearaxe of the particles and associated membrane (arrows) with images in Figs. 24 A, B, and I. X 47,000. region of cytoplasm near the cell surface (Figs. 26, 28, and 29). The reIatively few extracellular particles, predo~nantly of the naked variety, were sequestered between membranes of adjacent cells. In contrast with the extensive and rapid cell fusion produced by the IHD-W strain, whether cells were infected at high or low virus multiplici-

1)ISSERIINATIOX

ties (Dales and Siminovitch, 1961), no evidence of intercellular membrane fusion or dissolution \vas found in the case of cells infect,& with the IHD-J strain (Figs. 26 and 29). Host

Cell

3 Iernbranes

Although the cellular responseto infection by t,he two vaccinia strains is different, infect,ivity of both IND-W and IHD-J virus could be neutralized with equal efficiency by atltiserun~ prepared against either agent, indicating that the same or very similar antigens were present on t,hese two strains. However, IHD-J induced the production of HA. To d&ermine whether antigens chnractrrist.ic for one strain and not the other apl)esred on host cell surfaces, intact infected HeLs. cells were mixed w&h homologous and l~~ter~)logousantisera and subjected to indirect in~m~Ixloferritin Iabeling experiments. Examination of thin sections by ele&ron microscopy showed that exposure of IHD-W infected cells to homologous antisera caused antibody binding to surfaces of naked extracellular virus and to t,he wrapping, i.e., inner membrane of t,he cisterna remaining nft,er ~~~igr~~,t,ion t,o the surface. In this case, tohere w:ks no cvidrnce of antibody binding onto the surface of plasma membranes. Controls run in parallel fashion with preimmune sera revealed absence of nonspecific binding to the virus or associated membrane. HeLa cells infect’ed witah IHD-W vaccinia and reaet,ed with IHD-,J antiserum were also sr~eci~cally labeled at, the surface where virus particles nut1 el~veloping membranes occurred (l:ig. 30). In reciprocal t&s, antibody t’o IHD-W virus was absorbed at regions of the cell surface immediately above mature naked IUD-J progeny particles and occasionall;\ onto particles t,runsported out, of the cell but nowhere else (Figs. X5-34). The rare immat,ure THD-J psrt~ielesthat were present at the cell surface also absorbed the IHD-W antihod)- (Fig. 35). When IHD-J infected HeLa cells were mixed wit,h ant’isera made against IHD-J virus, anhibody \vas absorbed onto t#lzeentire cell surface :md not exclusively 011the

OF POSVTRIXES

fir”;,

virus particles and associated membranes, as uxs tile casewit’11LHD-W infection (F’ig. 31j. The new antigen(s) Jvith extensiw tli~tribution tl~rougl~o~l~t.he cell surface tn:ty l~sve been the HA. When antisera to IIf D-J wrc absorbed with lysates of IHD-W infrcte(l HeLa cells, t’heir capacity to neutralize tlw infectivit,y ~-3s almost complet~rl~~w.2novttl without reducing appreciably :tntibotly :ICtivity blocking hemadsorption of chicken RBCs to the surface of infectetl cells jsec data in Table 1 of the following :trti& (Icllil~:~,slli and Dales, 1971)j.

In the studies reported in this nrticle tltta fate of progeny virus 1~~sexnminrtl in relation to t.he synthesis of HA and ATT, occlusion within ATI, and controlled wrapping and emigration through the cell surface. E’roduction of HA and -4TI appears to be under virus regulation which is dist,inet from that involving biogenesisof virus particles. Thus format,ion of ATT and HA starts S--9 hr after infection, while the appearance of mature particles commencesby 4-5 hr. Experiments using inhibit.ors sho\v that both the transcription and tr:mslation into the requisite RIZ’iZ and protein of HA and XT1 become active very lat,e, and are not dependent upon the formation of those RKA species t’hat are involved with biogenesis of mature virions. HA and ATI belong to t,he category of Me functions since they require prior T1S.4 synthesis for their expression. Yet both substances are s~l~tllesized in the presence of rifampicin, which inhibits late t,r~ltlscriptio~~ and virus maturation (?;agayam:t c>t nl,, 1970; I’ogo, 1971). This provided 11swith another distinction between the rcgulntion of the synt)hesis of virus compontir~ts mid ihese tn-o :mcillary substances. C’onccrning our current observations with (:I?% and CPRCl, the Me appearance of HA and the massive ATI in the presence of rif~~rn~)i~ir~ can explain -i&y there is a large difierenw between the degree of inhibition of protein synthesis in cowpox- nntl vlLccini:~-infect,etl cells (Tan and ~IcAuslan, 1970). Thus, Tart and McAuslan observed :I drastic reduction by 8-S hr of the overall protein syrithcsis in

526

ICHIHASHI,

~TSUM~~~,

WR vaccinia infection, but a much lesserreduction after cowpox infection, whether rifampicin was present or absent. Most probably much of the protein synthesized late in the cycle is the AT1 protein. Attachment of massesof pol~ibosomes to the AT1 indicates that protein is being synthesized on the surface of ATP, It may be possibleto ascertain through the experiments now in progress whether the polyribosomes are synthesizing the AT1 protein and, therefore, contain mRNA and nascent polypeptide characteristic of this antigen (Ichihashi and ~~atsumot,o, 1966). Increase in the mass of ATI occurs most rapidly during the period of the infectious cycle that is associated with virus occlusion in the proteinaceous matrix. Ability to become integrated is a heritable property of the virus, termed the V factor (Ichihashi and Matsumoto, 1969). NH, and CP58 particles are V+, while CPRCl are V- (Ichihashi and ~~atsumoto, 1968). Our current experiments show that V+ can be expressed in the presence of rifampicin. In the course of the infection with CP58 or double infect,ion with NRI and CPRCl, Borneimmature virus made in the presence of rifampicin is

AND DALES

occluded within AT1 showing that immature particles possessthe V+ factor, perhaps in the virus envelope. Concentration of the progeny by integration within massive AT1 ensures dissemination of packets of virus aft,er death and breakdown of cells. Concerning t,he release of individual particles out of host cells, two major routes are followed involving the participation of cellular membranes. Along the direct pathway, naked particles can move within the cytoplasmic matrix toward the plasma membrane and frequently pass int,o micro~lli. The progeny may be disse~nated by a terminal breakdown of the microvilli, although this has not been established clearly. In synchronously infected cells and cells in the vicinity of plaques most of the IHD-J progeny virus is naked, in contrast with IHD-W particles which undergo extensive wrapping. Immunoferritin labeling reveals that antigen(s) of the virion can be identified on the plasma membrane covering the virus particle but not elsewhere, implying that such virion antigen(s) can be inserted into the segment of cell membrane that comes int’o contact with t,he virus particle. This type of alteration appears to bear

FIG. 26. Section passing through the region of a plaque cut perpendicular to the monolayer of HeLa cells infected 4 days previously with 100 PFU/plate of J vaccinia. Each cell in the area contains predominantly unwrapped or naked mature virus. There is no evidence of cell fusion. The surface of the plastic dish was on the left. X 75M). Fm. 27. Single enveloped J vaccinia at the surface of a HeLa cell sampled 17 hr after infection. Description as in Fig. 24H. X 62,000. FIG. 28. Higher resolution image of a selected region from the same preparation as in Fig. 26 illustrates the presence of naked, mature particles in the cytopl~~c matrix subjacent to the cell membrane. x 62,000. FIG. 29. Section cut parallel to the monolayer of HeLa cells in a preparation as described in Fig. 2G. Each cell in the area contains mature, unwrapped particles. X 11,000. FIG. 30. Segment of the surface of a HeLa cell infected 17 hr previously with J vaccinia, following reaction with immune serum against IHD-J. Extensive distribution of ferritin (arrows) indicates uniform attachment of the antibody onto the entire cell surface, including regions covering the mature virus progeny. X 62,000. FIG. 31. Segment of the surface of a HeLa cell from a culture infected as described in Fig. 30. The cells were reacted with IHD-W vaccinia antiserum. The ferritin marker is present exclusively on the surface of virus particles or on the cisternal membrane acquired during emergence (see Fig. 24). X 62,CK@. FIGS. 33-35. Preparation as in Fig. 31. Fig. 32. Note the presence of a ferritin tag on the membrane directly above the naked intracytoplasmic virus. Fig. 33. The ferritin is dist,ributed on the cisterna membrane (see Fig. 24). Fig. 34. Ferritin label surrounds the virus surface of an extracellular particle. Remains of a cisterna membrane are indicated by an arrow. Fig. 35. Example illustrating labeling with ferritin-antibody of a rarely encount)ered extracellular immature IHD-J vaccinia. All figures: X 75,000.

528

ICHIHASHI,

MATSUMOTO,

AND DALES

530

ICHIHASHI,

MATSUMOTO,

some ~~a~t~ to the mo~ficatio~ of membrane arising from infection by Herpes (Nii et al., 1968; Schwartz and Roizman, 1969; Spear and Roizman, lQ70;), myxo- (Hi&, 1942; Hoyle et al., 1961; Morgan et aL, 1961), paramyxo- (Due-Nguyen, 1968; Compans et al., 1966>, and other agents (Oshiro et aE., 1969). The second and more complex process for exporting the virus involves membrane-tomembrane fusion and shrouding of individual particles. In the case of IHD-W vaccinia, the first particles to mature are moved to the centrosphere zone, where they become enveloped by cisternae formed from vesicles of the Golgi complex. From a reconstructed sequence of events one can presume that individual vesicles come into contact with the virus surface, then fuse with one another so as to generate a double membrane sac. Perhaps the initial interaction between the Golgi vesicles a,nd the virus surface involves a hitherto obscure phenomenon of specific recogrmion. Association after ~gr~tion of naked IHD-J particles with the plasma membrane may depend upon the same kind of recognition. These observations, together with the identi~~ation of a virion-speei~e antigen on the investing membrane of exported virus at the surface, imply that the virus surface stimulates and controls an interaction with Golgi or plasma membranes which may cause the insertion of a new antigen into host cell membranes. It is noteworthy that immature particles of IHD-W developing in the presence of hydroxyurea, or occasionahy in the normal course of infection, can also be segregated and eliminated by an identical process. A similar type of wrapping has been observed with herpes simplex virus (Lee&ma et at., 1969). Asymmetry of the inner and outer faces of cell membranes is one of their properties most probably related to the envelopment process. The surface of progeny virus interacts with vesicles or the plasma membrane on the side facing the c~opl~~c matrix but does not lyse these membranes. By contrast, infecting particles of the inoculum after encountering the outer face of the plasma membrane induce the membrane to invaginate so as to form a phagocytic vacuole. The membrane of the vacuole is subsequently

AND DALES

Iysed and the virus envelope undergoes a simultaneous rupture (Dales, 1963a). The membranes investing progeny virus possess a capacity for fusion. In this case when the virus and surrounding eisterna reach the cell surface, fusion occurs between the outer membrane of the cisterna and the plasma membrane. The host membrane-~rus interaction associated with shrouding and tra~port of IHD-W vaccinia is distinctive from cellular functions related to the packaging and export by secretion of normal cell products, which become concentrated inside smoothmembrane vesicles, as in the case of pancreatic zymogen (Jamieson and Palade, 1971), secretion granules of leukocytes (Bainton and Farquhar, 1968a, b), and formative lysosomes (Strauss, 1967; Cohn and Fedorko, 1969). A distinctive modification of the cell surface resulting from infection by IHD-J vaecinia and cowpox strains is the appearance of HA. The soluble form of HA is first evident in the cytoplasm, and later may be converted into a membrane-bound component distributed over the entire cell surface. The sibilance of poxvirus induced HA in fusion of membranes and virus ~sse~nation will be considered in the companion article (Ichihashi and Dales, 1971). In summary, four phases associated with transporting individual progeny particles out of cells can be recognized. In the first step, apparent recognition between surface of the virus and Golgi vesicles leads to fusion between the vesicles and formation of a continuous double-membrane cisterna. In the second stage, virus and cisterna migrate toward the surface. During the third stage, the outer wall of the cisterna fuses with the plasma membrane of the host creating a channel for the release of the virus enclosed by the inner cisternal membrane. Finally, the residual investing membrane may be ruptured, releasing the naked virus int,o the extraceIlular fluid, or the virus and covering membrane may separate as a unit from the cell. This process is particularly evident with IHD-W vaccinia, By contrast, unwrapped or naked particles of THD-J vaccinia usually migrate directly without becoming enveloped in cisternae. At regions of intima~,e as-

DISSEMINATION

sociat,ion bet lyeen unwrapped virus and the plasma membrane, t,he surface is modified by acquisit,ion of a virus antigen. ACKNOWLEDGMENTS We wish to thank Dr. K. C. Hsu, Columbia University, whose work was supported by grants from the U.S.P.H.S. HE-03929 and AM-13200, for providing the ferritin-conjugated antibody, and Miss Eva Nagy for proficient assistance. This work was supported by U.S.P.H.S. grant AI07477 to S.D. and by a U.S.P.H.S. International Postdoctoral Research Fellowship F05 TW 1544 to Y.I. R.EFERENCES BAINTON, D. F., and FARQUHAR, M. G. (1968a). Differences in enzyme content of azurophil and specific granules of polymorphonuclear leukocytes. I. Histochemical staining of bone marrow smears. J. Cell Biol. 39, 286-298. BAINTON, D. F., and FARQUHAR, M. G. (196813). Differences in enzyme content of azurophil and specific granules of polymorphonuclear leukocytes. II. Cytochemistry and electron microscopy of bone marrow cells. J. Cell Biol. 39, 299317. COHN, 2. A., and FF,DORBO, M. E. (1969). The formation and fate of lysosomes. “Lysosomes in Biology and Pathology” (J. T. Dingle and H, B. Fell, eds.), Frontiers of Biology, Vol. 144, pp. 43-133. North-Holland ResearchMonographs. COMPANS, R. W., HOLMES, K. V., DALES, S., and CHOPPIN, P. W. (1966). An electron microscopic study of moderate and virulent virus-cell interactions of the parainfluenza virus SV5. Virology 30,411-426. DALES, S. (1963a). The uptake and development of vaccinia virus in strain L cells followed with labeled viral deoxyribonucleic acid. J. Cell Biol. 18, 51-72. DALES, S. (196313). Association between the spindle apparatus and reovirus. Proc. Nat. Acad. Sci. U. S. 50, 268-275. DALES, S., and MOSBACH, E. (1968). Vaccinia as :I model for membrane biogenesis, Virology 35, 564-583. DALES, S., GOMATOS, P. J., and Hsu, K. C. (1965). The uptake and development of reovirus in strain L cells followed with labeled viral ribonucleic acid and ferritin-antibody conjugates. Virology 25, 193-211. DALES, S., and SIMINOVITCH, L. (1961). The development of vaccinia virus in Earle’s L cells as examined by electron microscopy. J. Biophys. Biochem. Cytol. 10, 475-503. DE HARTEN, E., and YOHN, D. (1966). The fine

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