Steps in reverse transcription of viral genomic RNA to proviral DNA, slightly modified fröm Junghans et al. Svoboda (page 17).

S. Karger Med ica l and Scientific Publishers Basel • M ü n c h e n • Paris L o n d o n • New York N e w D e l h i • Singapore Tokyo • Sydney

Vol. 26, 1986 Intervirology

Editor-in-Chief Fred Rapp

Department of Microbiology The Pennsylvania State University College of Medicine P.O. Box 850 Hershey, PA 17033, USA

Section Editors* DNA Viruses

Kenneth I. Berns (Section Editor) Department of Microbiology B204 Cornell University Medical College 1300 York Avenue New York, N Y 10021, U S A

Janet Butel, USA Richard J. Courtney, USA Samuel Dales, Canada Joseph C. Glorioso, USA Wain Mun Huang, USA Ronald Luftig, USA Haim Manor, Israel Robert Martin, USA Lois Miller, USA Anthony Minson, USA Edward S. Mocarski, USA Richard Moyer, USA Meihan Nonoyama, USA Ann Roman, USA James A. Rose, USA Gunter Siegl, Switzerland Saul Silverstein, USA Charles S.H. Young, USA Norton Zinder, USA

Füll addresses of Editorial Board members may be obtained from the oflfice of the Editor-in­Chie f or from the Publisher.

The Journal of the Virology Division, International Union of Microbiological Societies Founded 1973 by J.L. Melnick

Associate Editor Marian C. Horzinek

Institute of Virology Veterinary Faculty State University, De Uithof Yalelaan 1, Utrecht The Netherlands

Medical Virology Dr. Richard W. Whitley

(Section Editor) Department of Pediatrics and Microbiology The University of Alabama at Birmingham School of Medicine Children's Hospital Tower Suite 653 Birmingham, A L 35294, USA

Raphael Dolin, U S A Hugh J. Field, U K Martin S. Hirsch, USA Catherine M . Wilfert, USA

Plant Viruses Robert G . Milne (Section Editor)

Istituto di Fitovirologia Applicata Consiglio Nazionale delle Ricerche Via O. Vigliani, 104 10135 Torino, Italy

J. Allan Dodds, USA Roger Hull , U K Andrew O. Jackson, USA R. Koenig, F R G A.F . Murant, U K John W. Randles, Australia M.H.V. Van Regenmortel, France Ichiro Uyeda, Japan L. van Vloten-Doting,

The Netherlands

RNA Viruses David W. Kingsbury

(Section Editor) Department of Virology and Molecular Biology St. Jude Children's Research Hospital Memphis, T N 38101, USA

Michael Bratt, USA Michael J. Buchmeier, USA Richard W. Compans, USA Robert C. Gallo, USA Allan Granoff, USA Diane E. Griffin, USA Robert Haselkorn, USA Alice S. Huang, USA David C. Kelly, U K Michael Lai, USA John F. Longworth, New Zealand Brian W.J. Mahy, U K Herbert Meier-Ewert, F R G Frederick A. Murphy, USA Robert F. Ramig, USA M.E . Reichmann, USA William C. Russell, U K James H . Strauss, USA Victor M . Zhdanov, USSR

Taxonomy Fred Brown (Section Editor)

Welcome Biotechnology Pirbright, Woking Surrey GU24 O N G

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© Copyright 1986 by S. Karger A G , P.O. Box, CH-4009 Basel (Switzerland) Printed in Switzerland by Graphische Betriebe Coop Schweiz, Basel

Contents Vol. 26,1986

No. 1-2 Portraitsof Viruses Rous Sarcoma V i r u s

Svoboda, J 1 Tobacco Ratt ie V i ru s

Lister, R . M 61

Original Papers Epi top ic M a p p i n g o f Structural and Nonstructural Aleut ian Disease V i r u s Proteins

Kierek-Jaszczuk, D . ; Moenn ig , W . ; Stolze, B . ; Neth , R . ; T a n , S.; Greiser de W i l k e , I.; Kaaden , O . - R 74

Scrapie Incubation Periods and End-Poin t Titers in Mouse Strains DifTering at the H - 2 D Locus Carp , R . I . ; Cal lahan, S . M 85

Host Involvement in Vacc in i a V i ru s Repl icat ion Zaslavsky, V . ; Hofschneider, P . H 93

Short Communications H u m a n Papi l lomavi rus Type 38 Isolated from Patients wi th Epidermodysplas ia verruci­

formis Yutsudo , M . ; K a n d a , R . ; Tan igak i , T . ; K i t ano , Y . ; Hakura , A 104

Compara t ive Analys is o f the canAV-1 and c a n A V - 2 Genomes Jouvenne, P. ; H a m e l i n , C 109

Repl ica t ion o f the Alga l V i r u s P B C V - 1 in UV-Ir radia ted Chlorella V a n Etten, J . L . ; Burbank, D . E . ; Mein ts , R . H 115

E d i t o r s Note 120

No. 3 Original Papers Leukocyte Migra t ion Inhibi t ion Detects Cross-Reacting Antigens between Cells Trans-

formed by Epstein-Barr V i r u s ( E B V ) and E B V - L i k e S i m i a n Viruses Szigeti, R . ; R a b i n , H . ; T i m a r , L . ; K l e i n , G 121

Analysis o f Long-Term H u m a n Cytomegalovirus Latency in v i t ro Cockley, K . D . ; Rapp, F 129

Relat ionship o f Thei ler 's M u r i n e Encephalomyelit is Viruses to the Card iov i rus Genus o f P icorna viruses Ni tayaphan, S.; O m i l i a n o w s k i , D . ; To th , M . M . ; Parks, G . D . ; Rueckert , R . R . ; Palmen­berg, A . C . ; Roos , R . P 140

Specific Inhib i t ion o f Tobacco Mosa ic V i ru s Protein and Single-Stranded R N A Synthesis by Arabinofuranosyladenine Dawson, W . O . ; Lozoya-Saldana, H 149

Taxonomy o f Cucurbit-Infecting Tobamoviruses as Determined by Serological and Molec-ular Hybr id i za t ion Analyses Franck i , R . I . B . ; H u , J . ; Palukait is , P 156

IV Contents

Short Communications Membrane Carbohydrate Requirement for Rabies Vi rus B i n d i n g to Ch icken Embryo

Related Cells Con t i , C ; Superti, F . ; Tsiang, H 164

L A V / H T L V - I I I gag Gene Product p24 Shares Antigenic Determinants with Equine Infec-tious A n e m i a V i ru s but N o t wi th V i s n a V i r u s or Capr ine Arthr i t i s Encephali t is Vi rus Goudsmi t , J . ; Houwers, D J . ; Smit , L . ; Nauta , I . M 169

Epidemiology o f H u m a n Rotaviruses in Argent ina as Determined by R N A Genome Elec-trophoresis G ö m e z , J .A . ; Biscott i , E . L . ; Bercovich, J . A . ; Gr ins te in , S 174

No. 4 Original Papers Polyadenylated, Cytoplasmic Transcripts o f Varicella-Zoster V i r u s

Maguire, H . F . ; H y m a n , R . W 181 Studies on the Morphogenesis o f M u r i n e Cytomegalovirus

Wei land , F . ; K e i l , G . M . ; Reddehase, M . J . ; Kosz inowsk i , U . H 192 Adenovirus 3-7, an Intermediate Strain o f Subgenus B

Adr i an , T h . ; Wigand, R 202 Autographa californica Nuclear Polyhedrosis V i ru s Efficiently Enters but Does N o t Rep l i -

cate in Poiki lothermie Vertebrate Cel ls Brusca, J . ; Summers, M . ; Couch , J . ; Courtney, L 207

Short Communications Transforming Potential o f D N A o f the H u m a n P L C / P R F / 5 Hepatorna C e l l L ine

Iwamura, Y . ; Mi t amura , K . ; Yanagi , K . ; Hashimoto, T . ; Ka to , K 223 Genomic Differences between Strains o f Lactate Dehydrogenase-Elevating V i ru s

Contag, C . H . ; Retzel , E .F . ; Plagemann, P . G . W 228

Author Index 234 Subject Index 235

Intervirology The Journal of the Virology Division, International Union of Microbiological Societies Founded 1973 by J.L. Melnick

Editor-in-Chief Fred Rapp

Department of Microbiology The Pennsylvania State University College of Medicine P.O. Box 850 Hershey, PA 17033, USA

Section Editors DNA Viruses

Kenneth I. Berns (Section Editor) Department of Microbiology B204 Cornell University Medical College 1300 York Avenue New York, N Y 10021, USA

Janet Butel, USA Richard J. Courtney, USA Samuel Dales, Canada Joseph C. Glorioso, USA Wain Mun Huang, USA Ronald Luftig, USA Haim Manor, Israel Robert Martin, USA Lois Miller, USA Anthony Minson, USA Edward S. Mocarski, USA Richard Mover, USA Meihan Nonoyama, USA Ann Roman, USA James A. Rose, USA Gunter Siegl, Switzerland Saul Silverstein, USA Charles S.H. Young, USA Norton Zinder, USA

Associate Editor Marian C. Horzinek

Institute of Virology Veterinary Faculty State University, De Uithof Yalelaan 1, Utrecht The Netherlands

Medical Virology Dr. Richard W. Whitley

(Section Editor) Department of Pediatrics and Microbiology The University of Alabama at Birmingham School of Medicine Children's Hospital Tower Suite 653 Birmingham, A L 35294, USA

Raphael Dolin, USA Hugh J. Field, U K Martin S. Hirsch, USA Catherine M . Wilfert, USA

Plant Viruses Robert G . Milne (Section Editor)

Istituto di Fitovirologia Applicata Consiglio Nazionale delle Ricerche Via O. Vigliani, 104 10135 Torino, Italy

J. Allan Dodds, USA Roger Hull , U K Andrew O. Jackson, USA R. Koenig, F R G A.F . Murant, U K John W. Randles, Australia M.H.V. Van Regenmortel, France Ichiro Uyeda, Japan L. van Vloten-Doting,

The Netherlands

RNA Viruses David W. Kingsbury

(Section Editor) Department of Virology and Molecular Biology St. Jude Children's Research Hospital Memphis, T N 38101, USA

Michael Bratt, USA Michael J . Buchmeier, USA Richard W. Compans, USA Robert C. Gallo, USA Allan Granoff, USA Diane E. Griffin, USA Robert Haselkorn, USA Alice S. Huang, USA David C. Kelly, U K Michael Lai, USA John F. Longworth, New Zealand Brian W.J. Mahy, U K Herbert Meier-Ewert, F R G Frederick A. Murphy, USA Robert F. Ramig, USA M.E . Reichmann, USA William C. Russell, U K James H . Strauss, USA Victor M . Zhdanov, USSR

Taxonomy Fred Brown (Section Editor)

Welcome Biotechnology Pirbright, Woking Surrey GU24 O N G

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Intervirology 26: 192-201 (1986) © 1986 S. Karger A G , Basel

0300-5526/86/O264-O192S2.75/0

Studies on the Morphogenesis of Murine Cytomegalovirus

F. Weiland, GM. Keil, MJ. Reddehase, U.H. Koszinowski Federal Research Centre for Vi rus Diseases o f A n i m a l s , T ü b i n g e n , F R G

Key Words. Murine cytomegalovirus • Morphogenesis • Electron microscopy • Multicapsid virions

Summary. Two modes of assembly of murine cytomegalovirus (MCMV) were observed in cultured mouse embryo fibroblasts, generating two morphologically different types of viral particles: monocapsid virions and multicapsid virions. The assembly of nucleocapsids appeared to be the same for both types of morphogenesis. Three successive stages of intranuclear capsid formation could be distinguished: capsids with electron-lucent cores, coreless capsids, and capsids with dense cores. Some of the capsids were enveloped at the inner nuclear membrane to form monocapsid virions, which were first detectable in the perinuclear cisterna. Other capsids left the nucleus via nuclear pores and usually entered cytoplasmic capsid aggregates that received an envelope by budding into extended cytoplas-mic vacuoles, thereby forming multicapsid virions. Since the formation of multicapsid virions is not restricted to cell culture conditions and also occurs in vivo in immunosuppressed mice, multicapsid virions may play a role in the pathogenesis of cytomegalovirus infection.

Cytomegaloviruses cause severe clinical Symptoms only in the immunologically im-mature or immunosuppressed host [for re-view see Hamilton, 1982], indicating that in the immunocompetent host the immune Sys­tem is involved in the control of acute in­fection and the establishment of viral la-tency. Infection of mice with murine cyto-

Address inquiries to: D r . Frank Wei l and , Federal Research Centre for Virus Diseases o f A n i m a l s , P O Box 1149, D-7400 T ü b i n g e n ( F R G )

Received: June 20,1986 Revised: September 12,1986

megalovirus (MCMV) proved to be a con-venient virus-host model to study aspects of herpesvirus latency, recurrence, and patho­genesis [Brody and Craighead, 1974; Jordan et al., 1977; Mayo et al., 1977; Bräutigam et al., 1979; Shanley et al., 1982]. A morphologic feature special to M C M V is the preferential formation of multicapsid virions in addition to the regulär development of monocapsid virions during in vitro infection of embryonic fibroblasts [Hudson et al., 1976a], whereas the generation of twin capsid forms or oligo-capsid virions is a rare event in cells infected with herpes simplex virus [Nii et al., 1968; Watson, 1973] and also in cells infected with

Morphogenesis o f M u r i n e Cytomegalovirus 193

rodent cytomegaloviruses other than M C M V [Fong et al., 1979; Bruggeman et al., 1982]. Multicapsid virions of M C M V were not ob-served in salivary gland tissue during in­fection of immunocompetent mice. Only re-cently has it been observed that multicapsid virions are also formed in vivo in fibroblastic lung cells during infection of immunosup­pressed mice [Reddehase et al., 1985]. The origin of the virion envelope may be decisive for its molecular composition and thus for its immunogenic properties. Therefore, we studied the ultrastructural morphogenesis of both types of M C M V virions, monocapsid as well as multicapsid virions.

Materials and Methods

Virus and Infection Conditions M C M V (strain Smith , A T C C VR-194) was ob­

tained from the Amer i can Type Cul ture Co l l ec t ion , Rockwi l l e , M d . , U S A and was propagated in B A L B / c mouse embryo Fibroblast cells. Tertiary fi­broblasts were prepared from 15- to 17-day-old B A L B / c embryos as described [ K e i l et a l . , 1984]. Ce l l s were infected with M C M V by employing the tech-nique o f centrifugal (800 g for 30 min) enhancement o f infectivity [Osborn and Walker , 1968; Hudson et al . , 1976b], which resulted in an M O I o f 20 P F U per cel l . T o inhibit viral D N A synthesis, cells were infec­ted with M C M V in the presence of 250 | i g / m l phos-phonoacetic acid or 20 mM hydroxyurea ( H U ) and maintained in the presence o f the inhibi tor .

Electron Microscopy For electron microscopy, in situ Fixation o f cel l

monolayers (16-28 h postinfection) was performed wi th 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer ( p H 7.2) for 2 h. After r insing with cacodylate buffer, the cells were gently scraped off, collected by centrifugation, and postfixed in 1% osmium tetroxide. Specimens were dehydrated in acetone and em-bedded in Arald i te . Sections were cut wi th a Reichert O m U 3 ultramicrotome. Semithin sections (1 u,m) were stained with toluidine blue to select sites o f interest by

l ight microscopy. Ul t ra th in sections o f selected sites were then stained wi th uranyl acetate and lead citrate, and examined i n a Siemens 101 electron microscope at 80 k V .

Results

Assembly of Nucleocapsids The technique of centrifugal enhance­

ment of infectivity [Osborn and Walker, 1968; Hudson et al., 1976b] was applied to synchronize the inoculation. Two hours after infection, more than 90% of the mouse em­bryo fibroblasts in a semiconfluent mono-layer expressed the first gene product of M C M V , the immediate-early phosphopro-tein IE pp89 [Keil et al., 1985] in the nucleus, as detected by indirect immunofluorescence (not shown). This finding proved that viral replication started with a high degree of syn-chronicity. Viral D N A synthesis defining the beginning of the late phase of viral replica­tion occurs at about 16 h postinfection [Ebel-ing et al., 1983], and at that time ultrastructur­al alterations could be detected by electron microscopy.

Fibroblasts displayed enlarged nuclei with a pale nucleoplasm and delicately mar-ginated chromatin. Faint amorphous mate-rial was arranged in two or three focal areas in the nuclear matrix between the nucleoli. Coinciding with increasing staining affinity, the amorphous material acquired a more reti-cular appearance, and the first forms of viral capsids, typified by an electron-lucent core, emerged in the nucleus. Thereupon the focal areas fused to form a Single large inclusion body composed of diffusely or reticularly arranged amorphous material. This inclu­sion occupied the whole center of the nucleus and was surrounded by a clear zone of nucle­oplasm that separated the inclusion from the

194 W e i l a n d / K e i l / Reddehase/ K o s z i n o w s k i

Fig. 1. Capsids in the nucleus o f an M C M V - i n f e c t e d ce l l , a Caps ids with electron-lucent core. bl,2 In-completely formed capsids o f the type as shown in a. c l Coreless cap­sids; arrow: nucleocapsid. c2 Empty capsid possessing a distinct orifice. Bar marker represents 200 nm.

Fig. 2. Different stages in intra-nuclear incorporat ion o f spider-l ike filaments (deoxyribonucleop-rotein Strands) into coreless capsids finally forming the mature nucleoc­apsid. a Coreless capsid wi th an orifice. b Spider-l ike filamentous structure located in the nuclear ma-trix. c Contact between an osmio-ph i l i c 'spider ' and an empty capsid . d-i Different forms o f association o f 'spiders ' with empty capsids. k Mature nucleocapsid. Bar marker represents 200 nm.

nuclear membrane. This zone was identical to a halo observed by light microscopy. Fre-quently, Single capsids or Clusters of capsids were interspersed among the amorphous ma­terial of the inclusion body. Only in single cases were the majority of capsids located in the nucleoplasm of the halo region (data not shown).

At that stage three types of capsids were observed simultaneously in the nucleus of individual MCMV-infected cells: (i) Capsids with a diameter of 100 nm containing an electron-lucent core of hexagonal symmetry (flg. la). Incompletely formed crescent shaped capsids with crescent shaped cores

were encountered occasionally (fig. lbl,2). These putatively represented capsids in a stage of assembly. (ii) Capsids with a hex­agonal profile devoid of a core (fig. lcl). These capsids differed in size but usually were slightly larger than those described above. (iii) Capsids containing an electron-dense fuzzy or more Condensed core that was located in the center (fig. lcl , arrow; fig. 2k). Their diameters were 100 nm. These capsids were designated nucleocapsids and were considered to represent mature capsids con­taining viral D N A .

As already mentioned above, the first cap­sids emerging in the nucleus were those with

Morphogenesis o f M u r i n e Cytomegalovirus 195

F i g . 3. Nucleocapsids leaving the nucleus. a B ü d d i n g o f a nucle­ocapsid into the perinuclear Space, b Enve loped monocaps id v i r ion freely ly ing i n the perinuclear cist-erna. c, d Nucleocaps ids located i n the cytoplasm close to nuclear pores. Ba r marker represents 200 nm.

electron-lucent cores. The formation of nu­cleocapsids was selectively halted at this stage when cells were infected in presence of H U , whereas any capsid assembly was prevented when viral D N A synthesis was blocked with phosphonoacetic acid. Core­less capsids were formed only in the absence of H U when transition to the late (y) phase of viral gene expression was not inhibited. These findings strongly suggest that coreless capsids represent a more advanced stage in the assembly of nucleocapsids, but since in-termediate stages were not observed it could not be established whether capsids with elec­tron-lucent cores represented direct prede-cessors of coreless capsids or whether they were an independent structure of as yet un-known function.

Coreless capsids possessed a distinct ori­fice (fig. Ic2,2a). Electron-dense filamentous material with a spider-like appearance (most likely representing deoxyribonucleoprotein Strands) originated separately in the nuclear matrix (fig. 2b). These osmiophilic 'spiders' occasionally had fibers protruding towards the empty capsids (fig. 2c). Often they were

detected in different stages of association with coreless capsids (fig. 2d—i). Capsids with electron-lucent cores devoid of D N A also accumulated in the presence of H U in cells infected with herpes simplex virus and, after removal of the inhibitor, EDTA-sensitive nu-cleoprotein filaments have been observed in structural association with capsids, which appear to be stages in a process of entering [Friedmann et al., 1975]. It is plausible that the spider-like filaments of M C M V enter ra­ther than leave the coreless capsids, finally forming the electron-dense compact deoxy­ribonucleoprotein core that is characteristic of the mature nucleocapsid (fig. 2k). In con-clusion, apart from minor differences in ul-trastructural details, the assembly of M C M V nucleocapsids follows the scheme common to herpesviruses.

Formation of Monocapsid and Multicapsid Virions Nucleocapsids were detected in intimate

contact with the inner nuclear membrane (some of them during the process of budding) and were partly enveloped by a thickened

196 W e i l a n d / K e i l / R e d d e h a s e / K o s z i n o w s k i

area of the inner nuclear membrane (fig. 3a). The budding process resulted in the release of enveloped monocapsid virions into the perinuclear cisterna (fig. 3b).

In the cytoplasm, nonenveloped single nucleocapsids were not randomly distri-buted, but appeared in conspicuous proxi-mity to nuclear pores (fig. 3c, d), strongly suggesting that these nucleocapsids had just left the nucleus via that passageway. Passing through may be a rapid process, which would explain why nucleocapsids were not ob­served in the pore. The antithesis predicts de-envelopment of monocapsid virions [Severi et al., 1979] followed by penetration into the nucleus via the nuclear pores. Such a view, however, is neither plausible nor has any precedent in published reports on her-pesvirus morphogenesis.

Nucleocapsids aggregated in the cyto­plasm and were embedded in electron-dense deposits, consisting of an amorphous, fine granulär substance (fig. 4). At lower magnifi-cation such Clusters appeared as compact cytoplasmic inclusions. Frequently, the nu­cleocapsids were arranged predominantly at the margin of the inclusion. Only rarely were the amorphous matrix material and nucleo­

capsids seen separately in the cytoplasm. Be-sides mature nucleocapsids, coreless capsids also entered the Clusters, whereas capsids containing the electron-lucent altered form of the core described above (fig. la) were never encountered outside the nucleus.

In the Golgi region, Clusters consisting of variable numbers of embedded capsids (oc-casionally only one) were enveloped by bud­ding into cytoplasmic vacuoles (fig. 5a, b), thereby forming either multicapsid virions or monocapsid virions that contained a signifi-cant amount of matrix material as tegument, in contrast to those monocapsid virions that were enveloped at the inner nuclear mem­brane. Multicapsid virions as well as mono­capsid virions formed by either mechanism were enclosed in smooth cytoplasmic vacu­oles (fig. 5c, d) and released into the extracel-lular space (fig. 5e). Studies on virion mor­phogenesis in various tissues of virus-infec-ted immunosuppressed mice have shown that in the absence of immune control, M C M V replicates in vivo exactly as demon-strated for cultured fibroblasts. The presence of monocapsid virions in the perinuclear cist­erna and the release of multicapsid virions have been shown for infected fibroblastic

Morphogenes is o f M u r i n e Cytomegalovirus 197

Fig. 5. Envelopment o f cyto­plasmic nucleocapsids. Simultane-ous budd ing o f nucleocapsids into cytoplasmic vacuoles thereby form-ing mul t icaps id virions containing various numbers o f nucleocapsids a,b. M u l t i c a p s i d c and monocaps id d v i r ions enclosed in cytoplasmic vacuoles and released into the ex-tracellular space e. Bar marker rep­resents 200 nm.

cells of the alveolar septa during interstitial M C M V pneumonia [Reddehase et al., 1985]. From our findings, we envision the course of M C M V morphogenesis as schematically outlined in fig. 6.

Discussion

M C M V differs from other members of the herpesvirus family by producing two differ­ent types of viral particles simultaneously in the same individual cell: monocapsid virions and multicapsid virions. This uncommon

feature may account for properties important for the induction of an immune response and for the course of pathogenesis. To clarify the origin of the virion envelopes we studied the ultrastructural morphogenesis of M C M V .

It was found that both types of virion have the first steps of morphogenesis in common -the assembly of nucleocapsids in the nuclei of infected cells. In accordance with findings of Lussier et al. [1974] and Kurimura et al. [1977], three types of capsids were observed: capsids with electron-lucent cores, coreless capsids, and capsids with dense cores. Con-sistent with the assembly of herpes simplex

198 W e i l a n d / K e i l / R e d d e h a s e / K o s z i n o w s k i

Fig. 6. a Part o f an M C M V - i n f e c t e d cel l . (1) Intranuclear capsids in different stages o f assembly. (2) Accumula t ion o f cytoplasmic capsids. (3) B ü d d i n g of cytoplasmic capsids. (4) Intravacuolar mul t icaps id v i r ion . (5) Extracel lular monocapsid v i r ion . (6) Extracellular mul t icaps id v i r ion . Bar marker represents 1 Jim. b Schematic representation o f the supposed course o f M C M V morphogenesis.

virus capsids [Schwartz and Roizman, 1969], capsids with electron-lucent cores were never seen as free particles or enveloped virion-like particles outside the nucleus, whereas for the infection of human fibroblasts with strains of human cytomegalovirus (HCMV), Irmiere and Gibson [1983] have reported noninfec-tious enveloped particles (NIEPs) which are characterized by capsids containing a core similar in structure to that of intranuclear H C M V B-capsids, which correspond to the M C M V capsids with electron-lucent cores.

In a more recent report the same authors provide evidence that NIEPs are in fact enve­loped B-capsids and suggest that an assem­bly protein forming the core before D N A packaging is modified or removed in con-junction with D N A packaging [Irmiere and Gibson, 1985]. Our studies with H U and re-sults obtained for herpes simplex virus by Nii et al. [1968] and Friedmann et al. [1975] allow the analogous conclusion, namely that cap­sids with electron-lucent cores do not contain the viral genome and represent a premature

Morphogenesis o f M u r i n e Cytomegalovirus 199

stage in nucleocapsid assembly. Likewise it is now established for cytomegaloviruses [Ka-nich and Craighead, 1972; Haguenau and Michelson-Fiske, 1975; Kurimura et al., 1977] and for herpes simplex virus [Friedmann et al., 1975; Luetzeler and Heine, 1978] that viral deoxyribonucleoprotein Strands enter the coreless capsids to form mature nucleocap­sids.

Büdding at the inner nuclear membrane is the most frequent way of monocapsid virion formation for most herpesviruses [Shipkey et al., 1967; Darlington and Moss, 1968]. Cy­tomegaloviruses represent an exception to the rule since virions receive the envelope predominantly by budding into cytoplasmic vesicles [Todd and Storz, 1983]. Another characteristic of cytomegalovirus infections is the formation of cytoplasmic inclusions consisting of embedded nucleocapsids [Luse and Smith, 1958; Ruebner et al., 1966; Bere-zesky et al., 1971; Craighead et al., 1972; Hud­son et al., 1976a; Fong et al., 1979; Storz et al., 1984]. Release of enveloped nucleocapsid aggregates containing 1 to more than 20 nu­cleocapsids, i.e. multicapsid virions, into the extracellular Space is, however, a prominent feature only during M C M V infection [Hud­son et al., 1976a]. The occasional detection of bicapsid virions in other rodent cytomegalo­virus infections [Fong et al., 1979; Brugge-man et al., 1982] can be explained by inciden-tal budding of two nucleocapsids into the same vesicle.

The site of envelopment of M C M V nucle­ocapsid aggregates remained to be eluci-dated. Because it was considered unlikely that extended aggregates bud at the nuclear membrane or into cytoplasmic vesicles in the way single nucleocapsids do [Leestma et al., 1969], budding at the cell membrane ap­peared to be a plausible alternative. None-

theless, our studies have now established that the aggregates do not bud at the cell mem­brane, but bud exclusively into extended cytoplasmic vacuoles derived from the Golgi apparatus. On the other hand, monocapsid virions developed only incidentally by this mode from nucleocapsids that did not join aggregates. M C M V monocapsid virions were primarily generated by budding at the inner nuclear membrane. Al l three types of M C M V virions were transported through the cytoplasm enclosed by cytoplasmic unit membranes and were released into the ex­tracellular space by exocytosis. In conclu-sion, neither multicapsid virions nor the two types of monocapsid virions received the en­velope by budding at the cell membrane, and the origin of the multicapsid virion envelope differed from that of most monocapsid vir­ions.

These results could have an influence on the interpretation of recent findings. First, production of multicapsid virions in fibro-blast cells in culture and of monocapsid vir­ions in vivo in cells of the salivary glands has been related to attenuation and virulence, respectively [Osborn and Walker, 1970; Chong and Mims, 1981; Jordan and Takagi, 1983]. Since monocapsid virions and multi­capsid virions gain their envelope at different sites it is possible that they differ in the com-position of their envelopes and hence also in their properties. Second, none of the three types of virions is enveloped at the cell mem­brane. It is therefore conceivable that anti-gens which are derived from the virion enve­lope after integration into the cell membrane from without during penetration of the virion are not necessarily presented at the cell mem­brane in an antigenic form during intracellu-lar virion assembly. This might explain why a cytolytic T-lymphocyte clone specific for vi-

200 W e i l a n d / K e i l / R e d d e h a s e / K o s z i n o w s k i

rion structural antigen, clone Sl [Reddehase et al., 1986a], did not detect this antigen in the late phase of infection at a time when high amounts of virus were produced and released [Reddehase et al., 1986b].

Acknowledgments

W e thank M s s . T . Messerschmid, G . Rohrs , H . Spitzer and A . Straubinger for expert technica l assist-ance. This work was supported by grant K o 571/8 from the Deutsche Forschungsgemeinschaft.

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