JOURNAL OF VIROLOGY,0022-538X/98/$04.0010

Dec. 1998, p. 9738–9746 Vol. 72, No. 12

Copyright © 1998, American Society for Microbiology. All Rights Reserved.

Viral Glycoproteins Accumulate in Newly Formed AnnulateLamellae following Infection of Lymphoid Cells

by Human Herpesvirus 6GIORGIA CARDINALI,1 MASSIMO GENTILE,2 MARA CIRONE,1 CLAUDIA ZOMPETTA,1

LUIGI FRATI,1,3 ALBERTO FAGGIONI,1 AND MARIA ROSARIA TORRISI1*

Dipartimento di Medicina Sperimentale e Patologia1 and Istituto di Virologia,2 Universita di Roma “La Sapienza,”Rome, and Istituto Neurologico Mediterraneo “Neuromed,” Pozzilli,3 Italy

Received 28 May 1998/Accepted 14 September 1998

Ultrastructural analysis of HSB-2 T-lymphoid cells and human cord blood mononuclear cells infected withhuman herpesvirus 6 revealed the presence, in the cell cytoplasm, of annulate lamellae (AL), which were absentin uninfected cells. Time course analysis of the appearance of AL following viral infection showed that no ALwere visible within the first 72 h postinfection and that their formation correlated with the expression of thelate viral glycoprotein gp116. The requirement of active viral replication for AL neoformation was furtherconfirmed by experiments using inactivated virus or performed in presence of the viral DNA polymeraseinhibitor phosphonoacetic acid. Both conventional electron microscopic examination and immunogold fracturelabeling with anti-endoplasmic reticulum antibodies indicated a close relationship of AL with the endoplasmicreticulum and nuclear membranes. However, when the freeze-fractured cells were immunogold labeled with ananti-gp116 monoclonal antibody, AL membranes were densely labeled, whereas nuclear membranes andendoplasmic reticulum cisternae appeared virtually unlabeled, showing that viral envelope glycoproteinsselectively accumulate in AL. In addition, gold labeling with Helix pomatia lectin and wheat germ agglutininindicated that AL cisternae, similar to cis-Golgi membranes, contain intermediate, but not terminal, forms ofglycoconjugates. Taken together, these results suggest that in this cell-virus system, AL function as a viralglycoprotein storage compartment and as a putative site of O-glycosylation.

Annulate lamellae (AL) are stacks of narrow membranecisternae that are often disposed in parallel, are usually local-ized in the cell cytoplasm, and contain numerous pore com-plexes (20). They are frequently seen as extensions of roughendoplasmic reticulum (RER) cisternae, implying a strict re-lationship between the two membrane compartments. How-ever, the presence on AL of pore complexes similar in struc-ture and composition to those present on nuclear membranes(23) and the recent observation that AL are disassembled andassembled during mitosis concomitantly with the nuclear mem-branes (11) argue for a greater similarity of AL to the nuclearenvelope. Initially considered to be an ultrastructural charac-teristic of rapidly growing germ and tumor cells, they seemmore likely to be cell type specific and represent one of the lastcellular organelles with no specific assigned function. The pres-ence of AL in virus-infected cells has also been reported, buttheir possible relationship to the infection process has not beeninvestigated (20).

We have recently studied the intracellular maturation pro-cess of two herpesviruses, Epstein-Barr virus (EBV) (37) andherpes simplex virus type 1 (HSV-1), by immunoelectron mi-croscopy (13, 38). Whereas the presence of AL in cells infectedwith those two herpesviruses was never noticed, when westarted to analyze cells infected with a more recently discov-ered herpesvirus, human herpesvirus 6 (HHV-6) (31), we ob-served numerous cytoplasmic AL as a striking ultrastructuralfeature related to viral replication. HHV-6 is a T-lymphotropicvirus which causes exanthem subitum in infants (41) and has

also been of growing interest because of its potential role as acofactor in the etiopathogenesis and progression of AIDS (22).Despite numerous studies on the immunologic and molecularaspects of HHV-6, relatively little is known regarding the in-tracellular maturation pathway followed by the virions and byviral glycoproteins (5, 27). We reported recently that a peculiarcharacteristic of HHV-6-infected cells is the absence of viralglycoproteins over the cell plasma membrane (10), in contrastto what was observed for other members of the Herpesviridaefamily, such as EBV and HSV-1. Since this atypical finding mayreflect an unusual intracellular transport mode and fate of viralglycoproteins, the present study was undertaken to investigateby immunogold electron microscopy whether the phenomenonof AL neoformation in the course of HHV-6 infection couldcorrelate with viral envelope glycoprotein expression. We re-port that AL are neoformed only in cells with active viralreplication and that these cytoplasmic structures represent asite of viral glycoprotein accumulation and of possible initia-tion of O-glycosylation.

MATERIALS AND METHODS

Cells and infection. HSB-2 cells and human cord blood mononuclear cells(CBMC) were cultured in RPMI 1640 medium supplemented with 10% fetal calfserum plus antibiotics. CBMC, isolated from the umbilical vein after delivery,were prepared by Ficoll-Hypaque (Pharmacia, Uppsala, Sweden) centrifugation,and monocytes were depleted after adherence to plastic. CBMC were activatedwith phytohemagglutinin (PHA) at 5 mg/ml (Difco, Detroit, Mich.) for 48 h andcultured in the presence of 1 IU of human recombinant interleukin-2 (GenzymeDiagnostics, Cambridge, Mass.) per ml. The GS strain of HHV-6 was employedin this investigation and was propagated in HSB-2 cells. Briefly, the virus stock(titer, 105 50% tissue culture-infective doses) was obtained from the 7-day su-pernatant of infected cells, when more than 80% of the cells showed a cytopathiceffect. Cell-free culture fluid was harvested, filtered through a 0.45-mm-pore-sizefilter, and pelleted by centrifugation at 25,000 3 g for 90 min at 4°C. Forinfection, 5 3 106 pelleted cells were incubated with an appropriate dilution ofthe virus stock. After 4 h at 37°C, the cells were washed once and resuspended

* Corresponding author. Mailing address: Dip. Medicina Sperimen-tale e Patologia, Viale Regina Elena 324, 00161 Rome, Italy. Phone:396-4468450. Fax: 396-4468450 or -4452850. E-mail: torrisi@axrma.uniroma1.it.

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in complete medium. Uninfected HSB-2 cells and mock-infected CBMC, acti-vated with PHA and interleukin-2, were used as controls. For heat inactivation,the HHV-6 stock was incubated for 60 min in a water bath at 56°C. For UVinactivation, the virus was directly exposed to a UV light source, receiving a doseof 230 mW/cm2 for 5 min, as previously described (12).

Surface immunolabeling. HHV-6-infected HSB-2 cells, collected at 7 dayspostinfection, were incubated either before or after fixation (0.5% glutaralde-hyde in phosphate-buffered saline [PBS; pH 7.4] for 1 h at 4°C) with an anti-gp116 monoclonal antibody (MAb; 1:20 in PBS; Virotech, Rockville, Md.) for 1 hat 4°C. The anti-gp116 MAb binds to conformational epitopes (6a) and, as shownby immunoprecipitation, recognizes precursor, as well as mature, forms of theprotein (2, 3, and data not shown). Cells were then labeled with colloidal gold(prepared by the citrate method) conjugated with protein A for 3 h at 4°C.

Fracture labeling. HHV-6-infected HSB-2 cells, collected at 7 days postinfec-tion, were fixed with 0.5% glutaraldehyde in PBS (pH 7.4) for 1 h at 4°C,impregnated with 30% glycerol in PBS, and frozen in Freon 22 cooled by liquidnitrogen. Frozen cells were fractured in liquid nitrogen by repeated crushing witha glass pestle and gradually deglycerinated. Fractured cells were incubated withHelix pomatia lectin (HPL)-colloidal gold (10 nm) conjugates (Sigma ChemicalCo., St. Louis, Mo.) at 1:5 in PBS–0.15 M NaCl–0.5% albumin–0.05% Tween 20for 1 h at 37°C. Control experiments were preincubated in 100 mM N-acetyl-galactosamine (GalNAc) for 30 min at 37°C. Alternatively, freeze-fractured cellswere incubated in a solution of 1-mg/ml wheat germ agglutinin (WGA; SigmaChemical Co.) in 0.1 M Sorensen’s phosphate buffer–4% polyvinylpyrrolidone(pH 7.4) for 1 h at 37°C and labeled with colloidal gold (18 nm, prepared by thecitrate method) conjugated with ovomucoid for 3 h at 4°C. Control samples werepreincubated in 0.4 M N-acetyl-D-glucosamine for 15 min at 37°C, treated withWGA in the presence of the competitor sugar for 1 h at 37°C, and labeled withovomucoid-coated colloidal gold as described above. In some experiments,freeze-fractured cells were directly incubated with WGA-colloidal gold (10 nm)conjugates (Sigma Chemical Co.) at 1:2 in 0.1 M Sorensen’s phosphate buff-er–4% polyvinylpyrrolidone (pH 7.4) for 1 h at 37°C. For immunogold labeling,fractured samples were incubated with an anti-endoplasmic reticulum (ER)polyclonal antibody (15) at 1:50 in PBS for 1 h at 25°C. Alternatively, sampleswere incubated with an anti-gp116 MAb at 1:20 in PBS for 1 h at 25°C. Allsamples were labeled with colloidal gold (prepared by the citrate method) con-jugated with protein A for 3 h at 4°C.

Processing for EM. Unlabeled, surface immunolabeled, and fracture-labeledcells were processed for thin-section electron microscopy (EM) by postfixing with1% osmium tetroxide, staining with uranyl acetate (5 mg/ml), dehydration inacetone, and embedding in Epon 812. In some experiments, samples were ad-ditionally stained en bloc with 0.1% tannic acid in Veronal acetate buffer, pH 7.4,for 30 min at 25°C. Thin sections were examined unstained or after staining withuranyl acetate and lead hydroxide.

Postembedding. HHV-6-infected HSB-2 cells were fixed with 0.5% glutaral-dehyde in PBS (pH 7.4) for 1 h at 4°C, partially dehydrated in ethanol, andembedded in LR White resin. Thin sections were collected on nickel grids andlabeled with HPL-colloidal gold (10 nm) conjugates (Sigma Chemical Co.) at 1:5in Tris buffer–0.15 M NaCl–0.5% albumin–0.05% Tween 20 for 1 h at 37°C.Control thin sections were preincubated in 100 mM GalNAc for 30 min at 37°C.All sections were stained with uranyl acetate and lead citrate before examinationby EM. For immunogold labeling, sections were incubated with MAb 414, whichis specific for nuclear pore complex proteins (Berkeley Antibody Co., Richmond,Calif.) at 1:50 in PBS for 1 h at 25°C, followed by goat anti-mouse immunoglob-ulin G (IgG)-colloidal gold conjugates (British Biocell International; Cardiff,United Kingdom) at 1:10 in PBS for 30 min at 25°C.

Immunofluorescence assay. When a cytopathic effect was visible, infected cellswere tested for the presence of viral antigens by an indirect immunofluorescenceassay. Briefly, infected cells were washed in cold PBS, fixed in cold acetone onTeflon-coated slides, and incubated with anti-HHV-6 MAb p41/38 (Abi, Colum-bia, Md.) (8) or an anti-gp116 MAb. After two washes in PBS, the cells wereincubated with an appropriate dilution of fluorescein-conjugated goat anti-mouse IgG for 45 min at 4°C.

For a double-fluorescence assay, cells were incubated with the anti-gp116MAb (1:50 in PBS) and visualized with anti-mouse IgG-Texas red at 1:50 in PBS(Jackson Immunoresearch, West Grove, Pa.) and then incubated with the lectinHPL-fluorescein isothiocyanate (1:10 in PBS; Sigma Chemical Co.).

RESULTS

Neoformation of AL in HHV-6-infected cells. Morphologicalanalysis of HHV-6-infected HSB-2 T-lymphoid cells revealedthe presence of cytoplasmic AL, characterized by numerousstacked narrow cisternae containing pore complexes. We foundthat AL in HHV-6-infected cells represented a frequent ultra-structural feature related to the presence of viral particles atdifferent stages of maturation and intracellular transport. Infact, at later than 7 days postinfection, when more than 80% ofthe cells appeared to be infected, approximately 75% of the

infected cells displayed ultrastructurally recognizable AL, asassessed by random analysis of 400 ultrathin cell sections 15 to20 mm in diameter and crossing the nucleus. Parallel observa-tion of control uninfected cells, either in separate samples oramong the infected cells, did not provide evidence of the pres-ence of these structures.

In HHV-6-infected HSB-2 cells, AL showed typical ultra-structural features (Fig. 1a to d) previously described in othercell systems (19, 20) and appeared mostly in proximity or incontinuity with RER cisternae (Fig. 1a and b). Immunogoldlabeling of thin sections of resin-embedded HSB-2 cells with aMAb which recognizes nuclear pore complex proteins showeda positive reaction with AL (data not shown), further confirm-ing their identity as AL. In heavily infected cells, these struc-tures were prominent, occupying large areas of the cytoplasm.Their distribution inside the cells was either peripheral orperinuclear, and they were equally prevalent in both intracel-lular areas. Occasionally, tegumented nucleocapsids in the cy-toplasm and enveloped virions inside vesicles were foundamong the AL. Since the appearance of AL in infected HSB-2cells could represent a cell type-specific, instead of a virus-specific, phenomenon, we performed additional experimentswith another target of HHV-6 infection, human CBMC. Again,high expression of AL was detected in infected cells (Fig. 2aand b), whereas these structures were never observed in PHA-stimulated, uninfected control cells, demonstrating that theobserved AL neoformation is dependent on viral replication.

To better characterize AL in our cell system, we applied thefracture-labeling technique, a method which combines freeze-fracturing with immunogold EM and provides full access to thelabeling of large areas of intracellular membranes exposed bythe fracture process (35, 36, 39). To do this, cells were fixed,freeze-fractured, immunolabeled, resin embedded, and thinsectioned. We immunolabeled infected cells with anti-ERpolyclonal antibodies, which have been shown to label the ERand nuclear membranes specifically (21). We observed thatfreeze-fractured AL membranes exposed on the fracture planeof HSB-2 infected cells were strongly immunolabeled (Fig. 3ato c). A similar pattern of labeling was observed only on thefreeze-fractured ER (Fig. 3d) and nuclear membranes (notshown), whereas Golgi membranes appeared to be unlabeled(Fig. 3c), as expected (21). Quantitation of the immunogoldparticles associated with intracellular membrane profiles ex-posed on the fracture plane is shown in Table 1, providingsupport for the specificity of our immunolabeling procedure.Thus, at least in this cell system, AL seem to be not onlymorphologically but also antigenically related to the ER andnuclear membranes.

AL are induced late in the viral replication cycle. To analyzethe kinetics of AL formation during infection, we performedtime course experiments correlating the number of AL struc-tures with the expression of early or late viral antigens atdifferent times postinfection. Cells were tested at daily inter-vals by immunofluorescence analysis for the expression of anearly-late phosphoprotein of HHV-6 (p41) and of a late viralglycoprotein (gp116) and by parallel conventional thin-sectionEM for the presence of AL. The results are shown in Table 2and indicate that no AL were visible within the first 72 hpostinfection and that their formation correlated with the in-duction of gp116. In addition, the presence of AL in the cellcytoplasm was associated mostly with the contemporaneouspresence of nucleocapsids in the nuclei of the cells.

To confirm that viral DNA replication is needed for ALformation, the above time course experiment was also per-formed in the presence of the viral DNA polymerase inhibitorphosphonoacetic acid (PAA), which had previously been

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shown to inhibit HHV-6 replication (14). PAA treatment to-tally abrogated the expression of gp116 and significantly re-duced, but did not abolish, the expression of p41, consistentwith previous observations (12). Concomitant inhibition of AL

formation was observed (Table 3). A further confirmation thatviral replication is needed for AL formation was gained byexperiments using inactivated viral preparations. Neither UV-inactivated nor heat-inactivated virus was able to induce AL

FIG. 1. Morphological appearance of AL in HSB-2 cells infected with HHV-6. (a) Prominent AL stacks are present at the cell periphery in close proximity to aGolgi complex. Virions inside transport vesicles (arrowheads) and vacuoles (arrow) are visible in the same area. Immunogold surface labeling with an anti-gp116 MAbis dense over the extracellular virions but virtually absent on the cell plasma membrane. An immunogold-labeled extracellular virion at higher magnification is shownin the top left corner. (b) AL cisternae show numerous pore complexes and continuity with RER membranes. (c) Side view of parallel stacked AL showing numerouspore complexes, which appear to be structurally similar to the nuclear pores (arrowhead), with pore-associated fibrous material. (d) En-face view of tangentiallysectioned AL stack at a high magnification, revealing the octagonal symmetry of pore annular subunits (arrows) and a central electron-dense granule in some pores.Abbreviations: er, endoplasmic reticulum; G, Golgi complex; M, mitochondria; Nu, nucleus. Bars: a to c, 0.5 mm; d and inset in panel a, 0.1 mm.

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formation, ruling out an effect due to signal transductionevents following virus binding to the cell plasma membrane(Table 3).

Viral glycoproteins accumulate in AL. Immunogold labelingof the cell surfaces of unfractured HHV-6-infected cells, per-formed with an anti-gp116 MAb on either unfixed (data notshown) or prefixed (Fig. 1a) cells confirmed the virtual absenceof envelope proteins on the cell plasma membranes, which waspreviously demonstrated by using other antibodies and humanserum (10). The extracellular virions, however, appeared to bestrongly labeled with the anti-gp116 MAb when cells were fixedeither before (Fig. 1a) of after (data not shown) the immuno-labeling, testifying to the specificity of the labeling procedure.We therefore decided to perform a new immunoelectron mi-

croscopic analysis of infected cells to investigate the possiblepresence of viral glycoproteins over AL, ER, nuclear mem-branes, and Golgi cisternae. Again, the fracture-labeling tech-nique was selected to gain full access to the labeling of theexposed intracellular membranes, as was previously done forcells infected with other herpesviruses (37, 38). Gold labeling,performed by using the anti-gp116 MAb as described above,revealed no or little labeling over both freeze-fractured nuclearmembranes (Fig. 4a and Table 1) and ER cisternae (Table 1),whereas AL membranes appeared to be densely labeled (Fig.4a and b and Table 1), showing that these structures mayfunction as a storage compartment for viral glycoproteins. Incontrol experiments, performed by omitting the anti-gp116MAb from the immunolabeling procedure, a drastic reduction

FIG. 2. Morphological analysis of AL in CBMC infected with HHV-6. Tangential sections of the AL network are shown at low (c) and high (d) magnifications. Bars,1 mm. For definitions of abbreviations, see the legend to Fig. 1.

FIG. 3. Immunogold labeling of freeze-fractured HHV-6-infected HSB-2 cells using anti-ER polyclonal antibodies. AL, exposed on the fracture plane, are denselylabeled (a to c), whereas freeze-fractured Golgi membranes appear to be unlabeled (c). Cross-fractured ER cisternae are labeled (d). In panel c, nucleocapsids insidethe nucleus (arrowheads) testify to the viral infection. Bars: a to c, 0.5 mm; d, 0.2 mm. GM, Golgi membranes. For definitions of other abbreviations, see the legendto Fig. 1.

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of gold labeling was observed over AL. Specific labeling withthe anti-gp116 MAb was also observed over freeze-fracturedGolgi cisternae (Fig. 4c, arrow, and Table 1) and on the innersurface of cross-fractured Golgi cisternae (Fig. 4c and d, ar-rowheads), as well as on post-Golgi compartments, such asendosomal and lysosomal membranes (Table 1).

AL components are at an intermediate step of glycosylation.The combined application of lectin cytochemistry and the frac-ture-labeling technique was previously used for the character-ization of intracellular membrane compartments (34). Here, todetermine the glycosylation stage of AL membrane compo-nents, freeze-fractured HHV-6-infected cells were labeled withHPL and with WGA. HPL is specific for terminal unsubsti-tuted GalNAc, which is the first residue added during O-linkedglycosylation. This lectin is therefore able to bind intermediateforms of glycoconjugates in early locations along the exocyticpathway, such as at the cis-most cisternae of the Golgi complex(28). Alternatively, freeze-fractured cells were labeled with thelectin WGA, which is specific for sialic acid and is thereforeable to recognize terminally glycosylated components (4). Weused HPL directly conjugated with 10-nm colloidal gold par-ticles (Fig. 5) and WGA either directly (data not shown) orindirectly (Fig. 5) conjugated to 18-nm ovomucoid-coated col-loidal gold. Freeze-fractured AL membranes were densely la-beled with HPL (Fig. 5a), whereas fracture faces of both inner(Fig. 5b) and outer (not shown) nuclear membranes and ERcisternae appeared to be unlabeled. Comparable observationswere obtained when HPL-gold labeling was performed on thinsections of resin-embedded, unfractured, infected cells. In Fig.5c, AL are densely labeled, whereas nuclear membranes andthe ER appear to be unlabeled. Freeze-fractured cis-mostGolgi cisternae (not shown), as well as the inner surfaces ofcross-fractured cis-Golgi cisternae (Fig. 5d), in which gold par-ticles were allowed to penetrate, were labeled as expected (13,28). Labeling with WGA was virtually absent over freeze-frac-tured AL membranes (Fig. 5e and f), as well as on nuclearmembranes and the ER (not shown), whereas the unfracturedcell plasma membranes were densely labeled (Fig. 5e and f), asexpected (13, 34). Specificity of lectin labeling was determinedby examining 25 membrane profiles, exposed on the fractureplane, for each intracellular membrane compartment andcounting the gold particles associated with each profile. Valuesof less than 1% were considered background nonspecific la-beling. The absence of WGA labeling on freeze-fractured ALmembranes, as well as on freeze-fractured nuclear membranes,as previously reported (25, 33), is not at odds with reportsshowing WGA binding sites over AL pore complexes (1) andnuclear pores (17, 30). In fact, in these cases, WGA bindingsites appear to be associated mostly with the fibrous materialpresent at the pore margins and exposed on the cytosolic side,whereas with our freeze-fracturing method, we label WGA

binding sites present on membrane components and exposedon the lumenal cisternal side. Occasionally, however, in ourexperiments, very low WGA labeling in small clusters couldalso be detected over AL, probably associated with nuclearpore components. In control experiments for either HPL orWGA labeling (i.e., freeze-fractured cells preincubated withthe competitor sugar and then treated with the lectin in thepresence of the competitor sugar), the gold density was re-duced by more than 90%. Thus, these results suggest that ALmembranes contain intermediate, but not terminal, forms ofglycoconjugates and may represent a site of initiation of O-glycosylation.

Double-immunofluorescence experiments performed onHHV-6-infected HSB2 cells stained for HPL and gp116showed colocalization of the two signals in large dots (Fig. 6,arrows), which may correspond to AL. In control uninfectedcells, HPL staining was confined to typical Golgi structures(data not shown). Thus, the gp116 molecules present on ALare likely at an intermediate step of glycosylation.

DISCUSSION

Morphological alteration of the cellular structures or newappearance of ultrastructural features is frequently observedduring viral infection; however, in most cases, no relationshipwith the mechanism of cell infection or viral maturation hasbeen proposed. Among these, AL formation has been de-scribed in several viral systems (19, 20). The novelty of ourfinding on the induction of AL in HHV-6-infected cells isrepresented by the observation that AL neoformation occursas a late event of the viral replication cycle and correlates with

TABLE 1. Percentages of immunogold particles associated withintracellular membranes exposed by the fracturing process

Antibody (no.)a% of particlesb associated with:

AL ER-NM G E-Ly M PM

Anti-ER antibody (25)c 61 36.5 0.9 0.1 0.6 0.9Anti-gp116 MAb (25)d 62.8 1.9 6.3 28.3 0.2 0.5

a Number of membrane profiles exposed on the fracture plane.b Values of ,1% were considered to be nonspecific background labeling. NM,

nuclear membrane; G, Golgi cisternae; E-Ly, endosomes-lysosomes; M, mito-chondria; PM, plasma membrane.

c Total no. of gold particles, 1,872.d Total no. of gold particles, 5,124.

TABLE 2. Time course of expression of early (p41) and late(gp116) HHV-6 antigens and AL appearance in

HSB-2 cells following viral infection

Time (h)postinfectiona

Mean % ofpositive cells 6 SEMc Mean % of cell

sections withAL 6 SEMb

p41 gp116

0 0 0 024 0 0 048 3 6 0.6 ,1 072 7 6 1.4 2 6 0.4 1 6 0.296 15 6 2.5 3.5 6 0.7 6 6 1.2

108 30 6 4.2 8 6 1.2 11 6 2.7

a As assessed by indirect immunofluorescence.b Percentage of cell sections in which AL were present at EM observation.c For each time point postinfection, 1,000 cells were randomly analyzed by

immunofluorescence assay and 50 cell sections (15 to 20 mm in diameter andcrossing the nucleus) were randomly analyzed by EM. The results are means ofthree different experiments.

TABLE 3. Effects of viral inactivation and treatment with PAA onviral antigen expression and AL formationa

Treatment of HSB-2 cells

% of cellspositive for: % of cell sections

with ALp41 gp116

HHV-6 35 10 12UV-inactivated HHV-6 1 ,1 0Heat-inactivated HHV-6 ,1 ,1 0HHV-6 1 0.8 mM PAA 5 0 0

a Results of one of three experiments are shown. Cells were tested at 5 dayspostinfection. For details of the experimental procedure, see the footnotes toTable 2.

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the expression of gp116. Furthermore, we propose that thesestructures may play a role as a storage compartment for viralglycoproteins and as a putative site for addition of O-linkedoligosaccharides. Although AL formation has never been ob-served in cells infected with other members of the Herpesviri-dae family, different morphological alterations possibly corre-lated with viral glycoprotein accumulation following heavyinfection have been reported. In EBV-producing cells, multi-layered nuclear membranes seem to contain a high concentra-tion of the major viral envelope glycoprotein gp350/220 (37),consistent with the suggested role of these membrane areas assites of active viral maturation. In addition, in HSV-1-infectedcells, the transport of huge amounts of viral glycoproteinsthrough the Golgi apparatus during late stages of infectionmay be responsible for the induction of Golgi fragmentationand of dispersal of Golgi enzymes and glycosylation products(6). The presence of gp116 in AL not only reflects an increasein protein synthesis and transport but might also play a role in

the viral maturation process (39a). The occurrence of cytoplas-mic nucleocapsids and enveloped virions inside vesicles sur-rounded by AL structures argues in favor of a role in viralassembly.

The present results show that AL, in our cell-virus system,are antigenically related and frequently in continuity with ERcisternae. Several reports have described the accumulation ofmembrane components in smooth tubular extensions of theRER when their transport to the Golgi apparatus is arrested(18, 40). However, the lack of immunoreactivity of those struc-tures when the cells were labeled with the same anti-ER poly-clonal antibody used in the present work seems to exclude thepossibility that those structures correspond to AL. In addition,the accumulation of HHV-6 glycoproteins observed by us isnot a consequence of a block of transport to the Golgi, sinceboth Golgi complexes and post-Golgi compartments containHHV-6 glycoproteins.

It has been recently shown that major histocompatibility

FIG. 4. Immunogold labeling of viral envelope glycoprotein gp116 on freeze-fractured HHV-6-infected HSB-2 cells. Freeze-fractured AL appear to be denselylabeled (a and b), whereas nuclear membranes are unlabeled (a). In panel b, the arrow points to an enveloped virion inside a vesicle surrounded by AL. Freeze-fracturedGolgi cisternae (c, arrow) and the inner surfaces of cross-fractured Golgi cisternae (c and d, arrowheads) are densely labeled. Bars, 0.5 mm. NM, nuclear membrane.For definitions of other abbreviations, see the legend to Fig. 1.

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FIG. 5. Lectin-cytochemical labeling of HHV-6-infected HSB-2 cells. (a to d) HPL-gold (10 nm) is very dense over AL exposed either in freeze-fractured cells (a)or in resin-embedded, thin-sectioned cells (c). Nuclear membranes in freeze-fractured cells (a and b) and in resin-embedded cells (c) are unlabeled. ER membranesalso appear to be unlabeled (c). In panel d, a freeze-fractured cis-Golgi cisterna is labeled (d). (e and f) Labeling with WGA-gold (18 nm) is virtually absent overfreeze-fractured AL membranes, whereas unfractured plasma membranes are positively labeled. Bars, 0.5 mm. PM, plasma membrane; INM, inner nuclear membrane.For definitions of other abbreviations, see the legend to Fig. 1. The arrows in panel b indicate nucleocapsids.

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complex class I molecules may accumulate in a membranenetwork extending from the ER, where ubiquitin-dependentdegradation of the accumulated proteins occurs (26). Deter-mination of whether AL have a similar function in disposal,instead of storage alone, requires further investigation.

Despite the close antigenic and morphologic relationshipbetween AL and the ER, AL membranes, unlike the ER,contain intermediate forms of glycocomponents, as revealed bythe positive labeling with HPL, suggesting that they representsites of initiation of O-glycosylation. Although the addition ofGalNAc in O-linked glycoconjugates is generally thought tooccur in the cis-Golgi cisternae (28, 29), O-glycosylation mayinitiate in ER subregions in dependence on cell differentiation(24) or in an intermediate budding compartment in virus-infected cells (32). We cannot exclude, however, the possibilitythat intermediate forms of glycosylation are present on ALcisternae as a result of the retrieval of initially glycosylatedcomponents from the cis-Golgi, but this possibility seems veryunlikely because of the great amount of labeling and becauseof the normal appearance of the Golgi complex. Although wecannot conclude that the viral glycoproteins which accumulatein AL are at an intermediate step of glycosylation, the compa-rable amounts of HPL and gp116 immunogold labeling and thecolocalization of the corresponding fluorescence signals stronglyargue in favor of this possibility.

Several reports have described the biochemical characteris-tics of HHV-6 gp116, which is considered to be the glycopro-tein B homologue of HHV-6 (7, 9, 15, 16). Briefly, gp116 is atype 1 glycoprotein of 830 amino acids, which carries bothhigh-mannose and complex-type N-linked oligosaccharides (9,16) and possesses putative O-glycosylation sites near the trans-membrane region. The presence of complex-type oligosaccha-rides on gp116 is consistent with its intracellular localization,showing transit through the Golgi complex. In addition, theputative sites of O-glycosylation are compatible with the ob-served colocalization in AL of gp116 and HPL binding sites.

AL formation is not the only peculiar finding in HHV-6-infected cells. In fact, we have recently reported that an atyp-ical characteristic of HHV-6 infection is the virtual absence ofviral glycoproteins over the cellular plasma membrane (10). Ifthese events are correlated, one might envision intracellulartrafficking of viral glycoproteins quite distinct from that of themajority of herpesviruses. In addition, we showed here thatHHV-6 glycoproteins, at variance with what we observed byusing an identical approach with EBV- and HSV-1-infectedcells, are not detected on the inner and outer nuclear mem-branes, which are thought to play a fundamental role in viralreplication as sites of viral budding and as intracellular viralprotein locations. Again, this observation suggests that HHV-6has a maturation pathway different from that of other membersof the Herpesviridae family (39a). The results shown here aredirect evidence of the involvement of AL as a putative storagecompartment of viral glycoproteins and as a site of O-linkedoligosaccharide addition to the glycoprotein chain. In addition,since a biochemical assay for analysis of AL formation hasrecently been described (23), the high expression of HHV-6glycoproteins in infected cells could allow immunoisolation ofAL following cell fractionation for further characterization ofthe resident proteins of this cytoplasmic organelle.

ACKNOWLEDGMENTS

We thank D. Louvard for the generous gift of anti-ER polyclonalantibodies. We also thank Giuseppe Lucania and Lucia Cutini forexcellent technical assistance.

This work was partially supported by grants from MURST; fromAssociazione Italiana per la Ricerca sul Cancro (AIRC); from Minis-tero della Sanita, Progetto AIDS; from CNR (Target Project on “Bio-technology”); and from Istituto Pasteur Fondazione Cenci-Bolognetti,Universita di Roma “La Sapienza.”

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FIG. 6. Double-fluorescence staining with an anti-gp116 MAb (a) and HPL (b) of HHV-6-infected HSB-2 cells showing colocalization of the two signals in largecytoplasmic dots (arrows). Bar, 10 mm.

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