Intracellular Transport and Maturation Pathway of Human Herpesvirus 6

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1 Virology 257, (1999) Article ID viro , available online at on Intracellular Transport and Maturation Pathway of Human Herpesvirus 6 Maria Rosaria Torrisi,*,,1 Massimo Gentile,* Giorgia Cardinali,*, Mara Cirone,* Claudia Zompetta,* Lavinia V. Lotti,* Luigi Frati,*, and Alberto Faggioni* *Dipartimento di Medicina Sperimentale e Patologia, Università di Roma La Sapienza, Rome; Istituto Dermatologico San Gallicano, 00153, Rome; and Istituto Neurologico Mediterraneo Neuromed, Pozzilli, Italy Received November 10, 1998; returned to author for revision January 21, 1999; accepted March 10, 1999 A peculiar characteristic of cells infected with human herpesvirus 6 (HHV6) is the absence of viral glycoproteins on the plasma membrane, which may reflect an atypical intracellular transport of the virions and/or the viral glycoproteins, different from that of the other members of the herpesvirus family. To investigate the maturation pathway of HHV-6 in the human T lymphoid cell line HSB-2, we used lectin cytochemistry and immunogold labeling combined with several electron microscopical techniques, such as ultrathin frozen sections, postembedding, and fracture-label. Immunolabeling with anti-gp116 and anti-gp82-gp105 monoclonal antibodies revealed that the viral glycoproteins are undetectable on nuclear membranes and that at the inner nuclear membrane nucleocapsids acquire a primary envelope lacking viral glycoproteins. After de-envelopment, cytoplasmic nucleocapsids acquire a thick tegument and a secondary envelope with viral glycoproteins at the level of neo-formed annulate lamellae or at the cis-side of the Golgi complex. Cytochemical labeling using helix pomatia lectin revealed that the newly acquired secondary viral envelopes contain intermediate forms of glycocomponents, suggesting a sequential glycosylation of the virions during their transit through the Golgi area before their final release into the extracellular space. Immunogold labeling also showed that the viral glycoproteins, which are not involved in the budding process, reach and accumulate in the endosomal/lysosomal compartment. Pulse-chase analysis indicated degradation of the gp116, consistent with its endosomal localization and with the absence of viral glycoproteins on the cell surface of the infected cells Academic Press INTRODUCTION Herpesviruses are large DNA-containing enveloped viruses that replicate in the nucleus and acquire their envelope through budding at the inner nuclear membrane into the perinuclear space (Roizman, 1996). The pathways followed by either virions and viral glycoproteins during their maturation and intracellular transport to the plasma membranes along the exocytic route are still controversial: the viral envelope glycoproteins are processed to their mature forms by the addition of sugars during the intracellular transit, and glycosylation appears necessary for the exit of the virus from the cells. A possible model proposes that the virions are assembled in a single budding event at the inner nuclear membrane and transported inside vesicles and vacuoles to the Golgi complex and then to the cell surface for final release by exocytosis to the extracellular space. The above mechanism was first suggested for herpes simplex type 1 (HSV-1) (Roizman and Furlong, 1974; Johnson and Spear, 1982); our subsequent results on HSV-1 and Epstein Barr virus (EBV) infected cells (Torrisi et al., 1989, 1992; Campadelli et al., 1993; Di Lazzaro et al., 1 To whom reprint requests should be addressed at Dip. Medicina Sperimentale e Patologia, Viale Regina Elena 324, Roma, Italy. Fax: or torrisi@axrma.uniroma1.it. 1995) were also consistent with this pathway. An alternative model, which involves an initial envelopment at the inner nuclear membrane, followed by de-envelopment by fusion with the outer nuclear membrane or with endoplasmic reticulum cisternae, and re-envelopment of cytoplasmic naked nucleocapsids at the trans cisternae of the Golgi complex has been described for varicellazoster virus (VZV), pseudorabies virus (PrV) and human cytomegalovirus (HCMV) (Jones and Grose, 1988; Whealy et al., 1991; Card et al., 1993; Gershon et al., 1994; Zhu et al., 1995; Radsak et al., 1996; Granzow et al., 1997). This mechanism has been also proposed by some authors for HSV-1 and EBV (Gong and Kieff, 1990; Browne et al., 1996). Among human herpesviruses, little is known regarding the maturation pathway of human herpesvirus 6, a T- lymphotropic herpesvirus that is the etiological agent of exanthem subitum and is highly suspected to play a cofactorial role in the etiopathogenesis and progression of acquired immunodeficiency syndrome (Lusso and Gallo, 1995). Few reports have described the ultrastructure characteristics of HHV-6 (Biberfeld et al., 1987; Nii et al., 1990; Roffman et al., 1990; Cirone et al., 1992). Morphologically, HHV-6 is characterized by the presence of a prominent tegument, which has been proposed to be acquired in a specialized region of the nuclear area of the infected cell (Roffman et al., 1990). In addition, HHV /99 $30.00 Copyright 1999 by Academic Press All rights of reproduction in any form reserved. 460

2 HHV-6 INTRACELLULAR TRANSPORT infected cells display at least two peculiar characteristics that we have recently described: the absence of viral glycoproteins over the plasma membrane of virusinfected cells (Biberfeld et al., 1987; Cirone et al., 1994), which might reflect an atypical pathway of viral intracellular transport different from that of all other members of the herpesvirus family, and the de-novo induction, upon viral infection, of cytoplasmic annulate lamellae, which appear densely labeled with antibodies directed against HHV-6 glycoproteins (Cardinali et al., 1998). Thus to further approach the issue of HHV-6 maturation, we performed an ultrastructural analysis of HHV-6 infected HSB-2 cells, using an immunoelectron microscopic technique, called fracture-label (Torrisi and Pinto da Silva, 1984; Torrisi and Mancini, 1996), which we have previously used to study the maturation pathways of HSV-1 and EBV (Torrisi et al., 1989, 1992; Di Lazzaro et al., 1995). Other approaches, such as gold immunolabeling of ultrathin frozen sections or biochemical assays to study the fate of viral glycoproteins, were also consistent with a novel mechanism of viral egress. RESULTS HHV-6 envelopment and tegument acquisition Morphological analysis of HHV-6 maturation has been carried out on conventional thin sections of HHV-6-infected HSB2 cells. Ultrastructural examination revealed the presence of numerous intranuclear naked nucleocapsids (Figs. 1a, 1d, and 1e, large arrows). Envelopment of the nucleocapsids by budding at the inner nuclear membranes was also frequently observed (not shown), and enveloped virions lacking a visible tegument were present in the space between the inner and outer nuclear membranes (Fig. 1a, small arrow). A second type of naked nucleocapsids, characterized by a thick tegument, was evident in the cell cytoplasm and localized mainly in close proximity or inside stacks of neoformed (Cardinali et al., 1998) annulate lamellae (AL) (Fig. 1c, arrowheads) and in the Golgi area (Figs. 1e and 1f, arrowheads). Enveloped virions with a thick tegument were contained in vesicles either surrounded by AL cisternae (Figs. 1b and 1c, small arrows) or in proximity and occasionally in continuity with Golgi cisternae (Fig. 1d, small arrows). Closer inspection of the enveloped virions located in the perinuclear space compared with the enveloped virions located in cytoplasmic vesicles during their transit to the plasma membrane and compared also with extracellular enveloped virions clearly show striking differences in size and in the presence of a tegument and of spike projections on the envelope: enveloped virions in the intranuclear space (Fig. 2a) were in fact smaller in size (130 nm), lacking tegument, and their envelope appeared devoid of spikes, whereas enveloped virions in cytoplasmic locations, such as those contained in vesicles surrounded by AL (Fig. 2b), and extracellular virions (Fig. 2c) were larger in size ( nm) and showed a thick tegument and numerous spikes projecting from their envelopes. The presence or absence of visible tegument and spikes seems to suggest that the two types of enveloped virions are assembled by at least two different budding events, the first occurring at the inner nuclear membrane and the second in cytoplasmic locations such as AL or Golgi cisternae. To verify if the two envelopes differ in viral glycoprotein content, we performed an immunoelectron microscopical analysis on ultrathin frozen sections using a mixture of anti-gp116 and anti-gp82-gp105 monoclonal antibodies directed against the two major envelope glycoproteins of HHV-6. No gold immunolabeling was observed over the cell plasma membranes (Fig. 3d), as previously reported (Cirone et al., 1994). To rule out the possibility that absence of labeling over the plasma membrane could be a generalized phenomenon, affecting other host cell glycoproteins, analysis of the expression of CD5 membrane antigen on HHV-6-infected HSB-2 cells was carried out by immunofluorescence. Full-ring-membrane staining by Leu-1 mab was equally detectable in 70% of infected and uninfected cells (data not shown). Gold immunolabeling of gp116 and gp82-gp105 was dense over extracellular virions (Figs. 3c and 3d, large arrows) and on enveloped virions in cytoplasmic vacuoles (Figs. 3a and 3d, small arrows). The membranes of the vacuoles (Figs. 3a, 3b, and 3d) and of the Golgi cisternae (Fig. 3b) appeared also strongly labeled. The nuclear membranes (Figs. 3a and 3e) and the enveloped virions located in the space between the inner and outer nuclear membranes (Fig. 3e) were virtually unlabeled. Only occasionally ( 7% of the total intracellular enveloped virions, as assessed by random analysis of 50 ultrathin frozen sections) did virions inside vesicles in the perinuclear area also appear unlabeled (Fig. 3b). Totally similar results were obtained using an HHV-6-positive human serum (data not shown). Thus the viral envelopes acquired by budding at the inner nuclear membrane do not appear to contain major viral glycoproteins, such as gp116 and gp82-gp105, which in contrast are present on the envelopes of extracellular virions or of virions located in peripheral cytoplasmic structures. This finding confirms the morphological observation of the presence or absence of spikes on the virions. Intracellular transit and sequential glycosylation of HHV-6-enveloped virions Lectin cytochemistry has been widely applied in the past in combination with fracture-label technique, which provides full accessibility to the labeling of intracellular membranes exposed by the freeze-fracture process (Torrisi and Pinto da Silva, 1984). Recently lectin cytochemistry in the fracture-label method has been used to determine the glycosylation stages of HSV-1 envelope com-

3 462 TORRISI ET AL.

4 HHV-6 INTRACELLULAR TRANSPORT 463 FIG. 2. Ultrastructural examination of enveloped virions in different locations during their intracellular transport. (a) Virions located in the intranuclear space possess an envelope smooth and devoid of surface projections. The lack of tegument is evident and the size of the virions appears smaller compared with the extracellular viral particles. (b) Virions contained within vesicles surrounded by annulate lamellae show numerous spikes projecting from the envelope. A thick tegument is visible and the size of the virions is comparable to that of extracellular particles. (c) Extracellular virions close to the plasma membrane are characterized by an envelope enriched of clearly distinguishable spikes. PM, plasma membrane. Bars: 0.1 m. ponents during the intracellular transit of the virions in the infected cells and to demonstrate a progressive viral maturation occurring by sequential transport through or interaction with Golgi cisternae (Torrisi et al., 1992; Di Lazzaro et al., 1995). We decided therefore to use the same approach to investigate the extent of glycosylation of HHV-6 envelope components in different intracellular locations. HSB-2 cells, infected with HHV-6, were fixed, freeze-fractured, and labeled with lectin-gold conjugates. Fracture-labeled samples were then processed for thin section electron microscopy (for a review on the fracturelabel method, see Torrisi and Mancini, 1996). Lectin labeling was also performed on resin-embedded thin-sectioned cells. Helix pomatia lectin, which binds terminal unsubstituted GalNAc, recognizes intermediate forms of glycoconjugates after O-linked addition of the sugar in cis-golgi cisternae (Roth, 1984). Wheat germ agglutinin, which is specific for sialic acid, binds terminally glycosylated components (Bhavandan and Kalic, 1979). In HSB-2 cells, infected with HHV-6, the enveloped virions present in the perinuclear space appeared negative for both HPL (Fig. 4b, arrowhead) and WGA (not shown). Virions located inside vesicles surrounded by annulate lamellae were positively labeled by HPL (Fig. 4b, arrows) but unlabeled by WGA (Fig. 4a). Enveloped virions positive for HPL were also observed in vesicles or larger vacuoles in the Golgi area (Fig. 4c, arrows). Extracellular virions appeared unlabeled by HPL (Fig. 4d) but densely labeled by WGA (Fig. 4e). HPL-gold particles were also present on AL cisternae (Fig. 4b and Cardinali et al., 1998) as well as on Golgi membranes and vacuoles (Fig. 4c). Quantitation of HPL-gold labeling, determined by counting the number of gold particles per m 2 SEM in 10 images of AL areas and of virions surrounded by AL, compared with cytosolic areas for background aspecific labeling assessment, revealed that density of labeling over the virions ( gold particles/ m 2 ) was significantly higher (P 0.05) than that over AL areas ( ) and over cytosolic areas ( ). Thus enveloped virions carrying glycoconjugates at an intermediate step of glycosylation, as assessed by HPL positive and WGA negative labeling, are detectable already in close proximity or inside the AL network as well as the Golgi complex, implying progressive glycosylation of envelope components during the intracellular transit of the virions through the Golgi area. Intracellular fate and degradation of viral envelope glycoproteins in infected cells To analyze the intracellular route of traffic of the viral glycoproteins not involved in the envelopment process FIG. 1. Morphological analysis of the intracellular maturation of HHV-6 in HSB-2-infected cells. In conventional thin sections of HHV-6-infected HSB-2 cells, intranuclear naked nucleocapsids are frequently visible (a, d, and e, large arrows) and enveloped virions budded at the inner nuclear membranes can be found in the perinuclear space (a, small arrow). Cytoplasmic naked nucleocapsids are also evident in proximity of prominent stacks of annulate lamellae (c, arrowheads) and in the Golgi complex area (e and f, arrowheads). These cytoplasmic nucleocapsids (arrowheads) are characterized by a thick tegument, which is absent on the intranuclear nucleocapsids (large arrows). Enveloped virions inside cytoplasmic vesicles are seen either surrounded by annulate lamellae (b and c, small arrows) or associated with Golgi cisternae (d, small arrows). AL, annulate lamellae; Nu, nucleus; NM, nuclear membrane; G, Golgi complex; M, mitochondrium. Bars: 0.5 m.

5 464 TORRISI ET AL. FIG. 3. Immunogold labeling of gp116 and gp82-gp105 viral envelope glycoproteins on ultrathin cryosections of HHV-6-infected HSB-2 cells. Sections were incubated with a mixture of anti-gp116 and anti-gp82-gp105 mabs, followed by protein A-colloidal gold. Dense labeling is observed on virions located inside intracellular vacuoles (a and d, small arrows) and in the extracellular space (c and d, large arrows). The membranes of the vacuoles, which contain the gold decorated virions (a, b, and d), and the cisternae of the Golgi complex (b) are also strongly labeled. Virions in the perinuclear space (e) appear unlabeled. Occasionally, virions inside vesicles in the perinuclear area (b, arrowheads) also appear unlabeled. Nuclear membranes (a and e) and plasma membranes (d) are virtually unlabeled. V, vacuoles. Bars: a, b, and d: 0.5 m; c and e: 0.1 m. and to follow their final destiny inside the cell, we performed immunolabeling with anti-gp116 mab on freezefractured infected cells. Dense gold labeling of gp116 was observed on AL cisternae (Fig. 5a and Cardinali et al., 1998), Golgi membranes (Fig. 5d) and on the membranes of large peripheral vacuoles (Fig. 5c); again (Cirone et al., 1994; Cardinali et al., 1998), nuclear membranes and endoplasmic reticulum cisternae (not shown) revealed none or little labeling, and plasma membranes (Fig. 5c) appeared virtually unlabeled.

6 HHV-6 INTRACELLULAR TRANSPORT 465 FIG. 4. Lectin labeling of HHV-6-infected HSB-2 cells: (a) WGA-gold (18-nm gold particles) labeling of freeze-fractured cells showed that the virions (arrow) inside vesicles surrounded by annulate lamellae are unlabeled. (b and c) Labeling with HPL-gold conjugates (10-nm gold particles) on resin-embedded, thin-sectioned cells reveals that several enveloped virions (arrows) inside vesicles or vacuoles are positively labeled, either when located in the area of annulate lamellae (b) or in the Golgi area (c), whereas virions in the perinuclear space appear unlabeled (b, arrowhead). Annulate lamellae (b), cisternae of the Golgi complex (c), and intracellular vacuoles in the Golgi area (c) are HPL-positively labeled. (d and e) Extracellular virions and the cell plasma membranes are negative for HPL (d) and positive for WGA (e). Bars: a, 0.1 m; b e, 0.5 m.

7 466 TORRISI ET AL. FIG. 5. (a c) Immunolabeling of gp116 on freeze-fractured HHV-6-infected HSB-2 cells: dense gold labeling is present on annulate lamellae (a) and peripheral vacuoles (c), whereas intermediate compartment (b) and plasma membranes (c) appear unlabeled. (d g) Double immunolabeling of gp116 (large golds) and human cathepsin D (small golds): dense labeling for gp116 is present on Golgi complexes (d) and observed in late endosomal structures, identified by their positive immunoreaction for human cathepsin D (e, f, and h). Virions are frequently seen in close proximity to (f, arrows) or inside (h) these endosomal structures. When exposed to the labeling, virions inside transport vesicles (g) and in endosomes are strongly labeled by anti-gp116 antibody (h). In (e), a tegumented naked capsid is visible in the cell cytoplasm (arrowhead). (i and k) Anti-cathepsin D immunolabeling on resin-embedded, thin- sectioned HHV-6-infected HSB-2 cells: enveloped virions (arrows) can be frequently found inside densely labeled endosomal or prelysosomal structures. IC, intermediate compartment; E, endosomes; Ly, lysosomes. Bars: a c, e, f, and i k: 0.5 m; d, g, and h: 0.1 m. The intermediate compartment, morphologically recognizable as a tubulo-vesicular structure, was also unlabeled (Fig. 5b). Therefore viral glycoproteins were mostly present on AL, Golgi cisternae, and vacuoles. To identify these vacuoles containing high amount of viral glycoproteins, we performed double-labeling with antihuman cathepsin D, a marker of late endosomes and lysosomes; the gold labeling was associated with these vacuolar structures (Figs. 5e 5h) and allowed us to conclude that gp116 reach and accumulate in late endoso-

8 HHV-6 INTRACELLULAR TRANSPORT 467 FIG. 6. Fluorogram of SDS-polyacrylamide gel containing immunoprecipitates obtained from lysates of 35 [S]methionine pulse-labeled and chased HHV-6-infected HSB-2 cells using anti gp116 mab (A) or using anti-transferrin receptor mab (B). mal (pre-lysosomal) structures where viral glycoprotein degradation may occur. Interestingly, enveloped virions, positively labeled for gp116 (Figs. 5g and 5h), were frequently observed near to (Fig. 5f) or inside (5h) late endosomes or pre-lysosomes. This localization of HHV-6 mature particles in endosomes or lysosomes was also observed in thin sections of resin-embedded cells labeled with anti-cathepsin D antibodies (Figs. 5i and 5k), further indicating that a portion of the enveloped virions may be sorted to the endosomal acidic compartment for degradation instead of being secreted by exocytosis. To demonstrate viral glycoprotein degradation, a pulse-chase experiment was carried out on HSB-2 cells at 7 days p.i. As observed in Fig. 6, the band corresponding to gp116 was clearly visible at 2-h chase, faintly detectable at 5-h chase, and undetectable at 18-h chase. As control, transferrin receptor, which is a recycling protein and does not undergo degradation, was still detectable at 18-h chase. DISCUSSION The maturation pathway of human herpesvirus 6 has not been studied in detail and is still poorly understood: one of the main reasons for the paucity of informations might be represented by the specific growth characteristics of HHV-6 in vitro, which replicates slower and less efficiently than other members of the herpesvirus family, such as herpes simplex type 1, varicella zoster virus, and pseudorabies virus. The results presented in this paper show that HHV6 follows an intracellular maturation pathway which differs from that of other herpesviruses and which appears to be characterized by (i) a first envelopment at the inner nuclear membrane with acquisition of an envelope devoid of spike glycoproteins; (ii) de-envelopment followed by tegument addition and re-envelopment at the level of neo-formed annulate lamellae or at the cis-golgi cisternae; (iii) transport and sequential glycosylation of the enveloped virions inside transport vesicles interacting with the Golgi complex; and (iv) release from the cell by exocytosis or sorting to the endosomal acidic compartment (Fig. 7). Our previous studies on EBV and HSV-1 indicated that envelopment at the inner nuclear membrane was the only budding event occurring during viral maturation (Torrisi et al., 1989, 1992; Di Lazzaro et al., 1995). Differently from EBV and HSV-1, the prominent presence of a thick tegument on HHV-6 virions, which is acquired in the cytoplasm, as shown by our observations in this cell system, might explain the need for a de-envelopment event: in fact, de-envelopment at the outer nuclear membrane, resulting in release into the cytoplasm of naked nucleocapids, might be required for tegument acquisition. Evidence for tegumentation of HHV-6 variant B in intranuclear compartments called tegusomes has been previously reported (Roffman et al., 1990); in the present study, we never observed such structures; this difference might be due to the different cell types employed (thymocytes vs HSB-2 cells) and different variants and strains of HHV-6. In that study, however, a first morphological evidence of re-envelopment of the tegumented capsids in cytoplasmic unidentified vacuoles was reported. Besides the abovementioned differences, our current model is in agreement with their initial observation of cytoplasmic re-envelopment. A second possible explanation to justify the requirement of a de-envelopment/re-envelopment process might be the lack of viral glycoproteins at the site of primary budding on the inner nuclear membrane and on the enveloped virions in the space between inner and outer nuclear membranes; this envelope is presumably composed of nuclear membrane lipids and may contain immature glycoconjugates, as reported for HSV-1 (Torrisi et al., 1992; van Genderen et al., 1994). Therefore reenvelopment at annulate lamellae, a cytoplasmic organelle which we have recently shown to be neoformed following HHV-6 infection and where viral envelope glycoproteins appear to accumulate (Cardinali et al., 1998), or at cis-golgi cisternae, must be required for the acquisition of a new envelope complete of spike proteins. In the EBV- and HSV-1-infected cells, in contrast, there is no apparent need for this re-envelopment event because viral glycoproteins are present on both the inner nuclear membrane of the infected cells and on the enveloped virions budded from it (Torrisi et al., 1989, 1992). The above observations seem to indicate that the early steps of the maturation process of HHV-6 are similar to those reported for VZV and PrV, where cytoplasmic de-

9 468 TORRISI ET AL. FIG. 7. Proposed model for the pathway of intracellular transport and maturation of HHV-6 in infected cells. and re-envelopment have been described. However, a major difference with the above proposed models is represented by the intracellular site where re-envelopment seems to occur: whereas for VZV and PrV reenvelopment at post-golgi compartments, such as trans- Golgi network, has been described (Whealy et al., 1991; Gershon et al., 1994; Harson and Grose, 1995; Zhu et al., 1995; Granzow et al., 1997), our observations are in favour of a re-envelopment occurring at the level of annulate lamellae or at the cis-golgi cisternae. In fact, the detection of intermediate forms of glycocomponents on enveloped virions, as revealed by our lectin cytochemical analysis, suggests that the HHV-6 virion envelope undergoes a progressive maturation of glycocomponents by sequential interaction with the Golgi complex. This is in agreement with our previous results with HSV-1 (Di Lazzaro et al., 1995). Finally, whereas most of the mature enveloped virions seem to exit the cell by exocytosis to be released in the extracellular space, the double-immunolabeling experiments with anti-cathepsin D and with anti-gp116 mabs, as well as the pulse chase analysis, strongly indicate that the viral glycoproteins not involved in the budding process accumulate in late endosomal or pre-lysosomal structures where degradation might occur. These findings may account for the previously described peculiar characteristic of HHV-6-infected cells, i.e., the virtual absence of viral glycoproteins on the plasma membrane (Cirone et al., 1994). Previously reported time-course experiments on the expression of HHV-6 gp116 demonstrated that also at early time points after viral infection viral gps appeared only intracellularly and could not detected over the cell plasma membrane (Cardinali et al., 1998 and data not shown). Although we cannot exclude that their absence on the plasma membrane and their presence in the endosomal compartment might be consequent to their rapid internalization from the cell surface, as described for the varicella zoster virus ge (Olson and Grose, 1997), this possibility appears unlikely due to the absence of endocytic signals in gp116 cytoplasmic

10 HHV-6 INTRACELLULAR TRANSPORT 469 tail. Furthermore because complete enveloped virions are also frequently observed, along with viral glycoproteins, inside endosomal-lysosomal structures, similarly to what has been recently reported for HSV-1 (Brunetti et al., 1998), the significance of a possible divergent sorting of some virions to the endosomal compartment for degradation remains to be clarified. Cells and infection MATERIALS AND METHODS HSB-2 cells were cultured in RPMI 1640 medium supplemented with 10% FCS plus antibiotics. The GS strain (Ablashi et al., 1991) of HHV-6 (variant A) was employed in this investigation and was propagated in HSB-2 cells. Briefly, the virus stock (titer 10 5 TCID 50 ) was obtained from 7 days supernatant of infected cells, when 80% of the cells showed cytopathic effect. Cell-free culture fluid was harvested and filtered through a 0.45-mm filter and made into pellets by centrifugation at 25,000 g for 90 min at 4 C. For infection, pelleted cells were incubated with an appropriate dilution of the virus stock. After 4 h at 37 C, the cells were washed once and resuspended in complete medium. For all experiments, cells were collected after 7 days p.i., when 80% of the cells appeared infected. Uninfected HSB-2 cells were used as controls. Ultrathin cryosections Cells were fixed with 8% paraformaldehyde in PBS ph 7.4for2hat4 C, washed, and embedded in 2% agarose low melting point (LMP) that was solidified on ice. Agarose blocks were infused with 2.3 M sucrose in PBS for 3 h at 4 C, frozen in liquid nitrogen and cryosectioned following the method described by Tokuyasu (1973). Ultrathin cryosections were collected using sucrose and methyl cellulose and incubated with a mixture of antigp82 and anti-gp116 mabs (Virotech, Rockville, MD). The antibody against gp82-gp105 binds to linear epitopes, located in the N-terminal end (Pfeiffer et al., 1993, 1995; B. Chandran, personal communication). The mab against gp-116, which corresponds to HHV-6 gb (Chou and Marousek, 1992; Ellinger et al., 1993) binds to conformational epitopes (B. Chandran, personal communication) and, as shown by immunoprecipitation, recognizes precursor as well as mature forms of the protein (Balachandran et al., 1989; our data, not shown). All samples were labeled with colloidal gold (prepared by the citrate method) conjugated with protein A (Pharmacia Fine Chemicals, Uppsala, Sweden) for 3 h at 4 C. Alternatively ultrathin cryosections were incubated with a high titered human serum. All sections were then labeled with colloidal gold (10 nm) conjugated with protein A (1:10 in PBS). Finally, ultrathin cryosections were stained with a solution of 2% methyl cellulose and 0.4% uranyl acetate before EM examination. Postembedding HHV6-infected HSB-2 cells were fixed with 0.5% glutaraldehyde in PBS ph 7.4 for 1 h at 4 C, partially dehydrated in ethanol and embedded in LR White resin. Thin sections were collected on nickel grids, labeled with HPL colloidal gold (10 nm) conjugates (Sigma Chemical Co., St. Louis, MO) (1:5 in Tris buffer 0.15 M NaCl, 0.5% albumin, 0.05% Tween 20) for 1 h at 37 C. Control thin sections were pre-incubated in 100 mm N-acetyl-galactosamine (GalNAc) for 30 min at 37 C. Thin sections were also labeled with anti-human cathepsin D polyclonal antibodies (kindly provided by Dr. Ciro Isidoro, University of Torino, Italy) (1:50 in PBS) for 1 h at 25 C and protein A-colloidal gold (18 nm prepared by the citrate method) for 30 min at 25 C. All sections were stained with uranyl acetate and lead citrate before examination with EM. Fracture-label HHV6-infected HSB-2 cells were fixed with 0.5% glutaraldehyde in phosphate-buffered saline (PBS) ph 7.4 for1hat4 C, impregnated with 30% glycerol in PBS, and frozen in Freon 22 cooled by liquid nitrogen. Frozen cells were fractured in liquid nitrogen by repeated crushing with a glass pestle and gradually deglycerinated. For lectin labeling, fractured cells were incubated with HPL colloidal gold (10 nm) conjugates (Sigma Chemical Co.) (1:5 in PBS 0.15 M NaCl, 0.5% albumin, 0.05% Tween 20) for1hat37 C. Control experiments were pre-incubated in 100 mm GalNAc for 30 min at 37 C. Alternatively, freeze-fractured cells were incubated in a solution of 1 mg/ml of wheat germ agglutinin (WGA, Sigma Chemical Co.) in O.1 M Sorensen s phosphate buffer-4% polyvinylpyrrolidone, ph 7.4, for 1hat37 C and labeled with colloidal gold (18 nm prepared by the citrate method) conjugated with ovomucoid for 3hat4 C. Control samples were preincubated in 0.4 M N-acetyl-D-glucosamine for 15 min at 37 C, treated with WGA in the presence of the competitor sugar for 1 h at 37 C and labeled with ovomucoid-coated colloidal gold as above. For immunogold labeling, fractured samples were incubated with anti-gp116 mab (1:20 in PBS) for 1hat25 C and labeled with colloidal gold conjugated with protein A for 3hat 4 C. In double-labeling experiments, fractured samples were treated with anti-gp116 mab and protein A-gold (18 nm) as above and then incubated with anti-human cathepsin D polyclonal antibodies (1:50 in PBS) followed by protein A-colloidal gold conjugates (10 nm; British Biocell Int., Cardiff, UK; 1:10 in PBS, for 30 min at 25 C). Processing for electron microscopy Cells and fracture-labeled samples were processed for thin section electron microscopy as follows: postfixed with 1% osmium tetroxide, stained with uranyl acetate (5

11 470 TORRISI ET AL. mg/ml), dehydrated in acetone, and embedded in Epon 812. In some experiments, samples were additionally 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 poststained with uranyl acetate and lead hydroxide. Density of the immunogold labeling, determined as gold particles/ m 2 and the statistical analysis were evaluated using a Sigma Scan Measurement System (Jandel Scientific, Corte Madera, CA). Pulse chase analysis HHV-6-infected HSB-2 cells (7 days p.i.) were kept in methionine-free RPMI 1640 for 30 min at 37 C. Subsequently the cells were pulse-labeled for 30 min at 37 C with 100 mci/ml of 35 [S]methionine. After labeling, one portion of the cells was frozen (pulse). The remaining cells were washed twice in RPMI 1640 supplemented with 10% FCS, suspended in the same medium and the incubation at 37 C was continued for 2, 5 and 18 h (chase). Cells ( /point) were solubilized in RIPA buffer (0,01 M Tris HCl ph 7.4, 0,15 M NaCl, 1% sodium deoxycholate, 1% Triton X-100, 0.1% SDS, 1 mm PMSF), immunoprecipitated with monoclonal antibodies against gp 116 (Virotech International, Inc.) or against transferrin receptor (Becton Dickinson) and analyzed by SDS PAGE. ACKNOWLEDGMENTS We thank Dr. C. Isidoro for the generous gift of anti-human cathepsin D polyclonal antibodies. We also thank Mr. Giuseppe Lucania and Ms. Lucia Cutini for excellent technical assistance. This work was partially supported by grants from MURST, from Associazione Italiana per la Ricerca sul Cancro (AIRC), from Ministero della Sanità, Progetto AIDS, from CNR (Target Project on Biotechnology) and from Istituto Pasteur Fondazione Cenci-Bolognetti, Università di Roma La Sapienza. REFERENCES Ablashi, D. V., Balachandran, N., Josephs, S. F., Hung, C. L., Krueger, G. R., Kramarsky, B., Salahuddin, S. Z., and Gallo, R. C. (1991). 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