1 Laboratoire de Virologie, UPRES EA 3622, Faculté de Médecine Cochin, Université Paris V et Inserm U 567, Bâtiment Gustave Roussy, porte 636, 27 rue du Faubourg Saint Jacques, 75014 Paris, France
2 Laboratoire de Biologie Cellulaire de la Synapse Normale et Pathologique, Institut National de la Santé et de la Recherche Médicale U497, Ecole Normale Supérieure, 75005 Paris, France
Correspondence
Flore Rozenberg
flore.rozenberg{at}cochin.univ-paris5.fr
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ABSTRACT |
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Published ahead of print on 10 July 2003 as DOI 10.1099/vir.0.19279-0.
C. Potel and K. Kaelin contributed equally to this work.
Present address: Marie Curie Research Institute, The Chart, Oxted, Surrey RH8 0TL, UK.
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INTRODUCTION |
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METHODS |
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Hippocampal cell cultures.
Hippocampal neuron cultures were obtained as previously described (Goslin & Banker, 1989). Briefly, hippocampi were dissected from embryonic D18 rats, in HBSS-HEPES (1x HBSS, 20 mM HEPES). Cell suspensions were treated with trypsin 0·25 % for 15 min at 37 °C, followed by 0·9 g DNase I l-1 (Roche) for 5 min at 37 °C. Cell dissociation was obtained by trituration using a fire-polished Pasteur pipette. Cells were then plated onto polyornithine-treated glass coverslips in culture medium (Eagle's modified essential medium: 1 % L-glutamine, 0·6 % glucose, 0·2 % NaHCO3, 10-2 M HEPES, 5 UI penicillin and streptomycin ml-1) with 10 % horse serum. Approximately 20 000 cells were plated on a coverslip. After attachment of cells, coverslips were inverted and transferred onto a confluent monolayer of astroglial cells, and maintained in B27 Neurobasal medium supplemented by 1 % L-glutamine, 5 UI penicillin and streptomycin ml-1. Coverslips were maintained at 37 °C in a 5 % CO2 incubator. Neuronal cultures were infected 12 days after plating.
Antibodies.
Rabbit polyclonal anti-gB, anti-gD and anti-nucleocapsid antibodies (R69, R45 and NC-1, respectively) were generously provided by R. J. Eisenberg and G. H. Cohen (University of Pennsylvania, Philadelphia). Mouse monoclonal antibody directed against the microtubule-associated protein 2 (MAP2) was obtained from Sigma. Mouse monoclonal anti-GFP antibody was purchased from Roche. Rabbit polyclonal antibodies directed against Rab6 (C-19) or against the carboxy terminus of calnexin were obtained from Santa Cruz Biotechnologies and Stressgen Biotechnologies, respectively. Texas red-conjugated donkey anti-rabbit and anti-mouse immunoglobulin G (IgG) were obtained from Jackson ImmunoResearch Laboratories. The goat anti-rabbit IgG peroxidase conjugate and the goat anti-mouse IgG peroxidase conjugate were from Sigma.
Plasmids.
Plasmid pSR175 containing the UL27-gB coding region of the HSV-1 KOS strain under the control of the cytomegalovirus immediate-early promoter was kindly provided by R. J. Eisenberg and G. H. Cohen. Plasmid pgBct-GFP was constructed by an in-frame insertion of the GFP coding sequence at the NheI site of the UL27-gB gene in pSR175. To this end, the cDNA sequence encoding enhanced GFP was amplified by PCR from plasmid pEGFP-N2 (Clontech) using primers 5'-AAAAAAGCTAGCCGGAGGAGGAGTGAGCAAGGGCGAGGAG-3' and 5'-AAAAATGCTAGCCCACCACCCTTGTACAGCTCGTCCAT-3', which carry an NheI restriction site at their 5' ends. The primers were designed to delete the GFP gene initiation and termination codons and to introduce codons for three glycine residues at each extremity of GFP. The amplified and NheI-cleaved GFP DNA fragment was then transferred into the NheI site of pSR175.
Recombinant virus.
To construct the KgBct-GFP recombinant virus, subconfluent monolayers of Vero cells were co-transfected with 1 µg K082 genomic DNA and with linearized plasmid pgBct-GFP in a five-fold molar excess (250 ng) using LipofectAMINE according to the manufacturer's recommendations (Gibco-BRL). Plaques were screened 35 days after transfection for the presence of spontaneous fluorescence on Vero cells. Isolated KgBct-GFP recombinants were plaque-purified three times on D6 cells. Virus stocks were prepared as for KOS and K082. Viral DNA was analysed by PCR using gB-specific primers bracketing the gB gene NheI insertion sites and by Southern blotting as previously described (Potel et al., 2002).
Complementation.
Complementation assays were performed essentially as initially described (Cai et al., 1988a). Briefly, 4x105 Vero cells were seeded in 35 mm culture dishes and transfected the following day with 0·25 µg of pSR175 or pgBct-GFP using LipofectAMINE. The cells were infected 24 h later with K082 at an m.o.i. of 0·5 p.f.u. per cell. Extra-cellular virions were washed off 1 h post-infection (p.i.) by treatment with an acid/glycine saline solution, followed by two washes with PBS. Infected cell culture supernatants were harvested 24 h after infection, and titrated on D6 and Vero cells. The titre obtained from K082 virus complemented with native gB was taken as the reference value, and the complementing capacity of the gBct-GFP protein was indicated as a ratio with respect to this reference value.
Plasmid transfections, virus infections and brefeldin A treatment.
To analyse the expression of proteins, Vero cells were transfected with 1 µg of pgBct-GFP or pSR175 using LipofectAMINE and incubated at 37 °C for 24 h for immunofluorescence studies. To compare de novo synthesis of the proteins in the context of virus infection, Vero cells and hippocampal neurons were infected at an m.o.i. of 0·5 p.f.u. per cell and 3 p.f.u. per cell respectively with KgBct-GFP or KGFP-gB for fluorescence studies, and 5 p.f.u. per cell for immunoblot analyses. For brefeldin A (BFA) treatment, infected Vero cells were incubated for 14 h at 37 °C in culture medium containing 0·1 µg brefeldin A ml-1 (Sigma). Immunofluorescence studies were performed at 16 h p.i in Vero cells or 24 h p.i. in neurons. Immunoblotting of infected Vero cells was performed at 15 h p.i. To monitor the synthesis of the gB fusion proteins during an entire virus replication cycle, infected Vero and neuronal cells were examined every hour up to 25 and 30 h p.i., respectively.
Immunoblot analysis and endoglycosidase treatment.
Cells in 35 mm culture dishes were washed twice with PBS and lysed for 20 min on ice in 50 mM Tris/HCl pH 8, 62·5 mM EDTA, 1 % Nonidet P40, and 0·4 % sodium deoxycholate supplemented with 15 µg antipain dihydrochloride ml-1, 2·5 µg aprotinin ml-1, 2·5 µg pepstatin ml-1, 5 µg chymostatin ml-1, 2·5 µg leupeptin ml-1 and 100 µg Pefabloc ml-1 (protease inhibitors; Roche). Endoglycosidase H (Endo H) and peptide:N-glycosidase F (PNGase F) digestions were performed on about one-twentieth of the cell lysates for 2 h at 37 °C. Samples were run on denaturing SDS-10 % polyacrylamide gels and blotted onto nitrocellulose membranes. Membranes were saturated with 5 % non-fat milk in TBST (10 mM Tris/HCl pH 8, 150 mM NaCl, 0·05 % Tween-20) before incubation for 1 h with primary antibody at a dilution of 1 : 4000 (anti-gB) or 1 : 1000 (anti-GFP) in TBST/1 % BSA. The secondary antibodies, goat anti-mouse IgG or goat anti-rabbit IgG coupled to horseradish peroxidase, were diluted according to the supplier's instructions. Bound antibodies were detected by enhanced chemiluminescence (Amersham).
Immunofluorescence assays.
Transfected or infected Vero cells and neurons on glass coverslips were fixed with 4 % (w/v) paraformaldehyde in PBS for 20 min at room temperature and then permeabilized with 0·1 % Triton X-100 for 2 min for staining of internal antigens, or directly processed for surface staining. Cells were washed three times with PBS and then incubated with primary antibodies for 1 h at 37 °C. The gB, gD, calnexin and Rab6 antisera were diluted 1 : 300, the anti-nucleocapsid antibody was diluted 1 : 500, and the anti-MAP2 antibody was diluted 1 : 100 in PBS containing 10 % donkey serum. Coverslips were extensively washed with PBS, before labelling with the secondary anti-rabbit IgG antibody at a dilution of 1 : 300, or with the secondary anti-mouse IgG antibody at a dilution of 1 : 200 in PBS containing 10 % donkey serum for 1 h. After three rinses with PBS and one with water, coverslips were mounted onto glass slides and then analysed with a conventional Zeiss Axiophot fluorescence microscope. Spontaneous fluorescence was visualized using standard fluorescein isothiocyanate excitationemission filter sets, and Texas red signals were visualized using narrow-band Texas red filters. Images obtained with a Hamamatsu digital camera were processed using the Starwise fluostar program (version 5.9.2s).
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RESULTS |
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Subcellular localization of GFP-tagged gB in transfected and infected Vero cells
To further investigate the functional defect in gBct-GFP, the subcellular localization of the protein was visualized in transfected Vero cells, using both the spontaneously arising GFP fluorescence and the Texas-red coupled anti-gB staining. As controls, images were compared with those obtained with native gB and with the previously reported GFP-gB, which contains GFP fused to the ectodomain of gB. In permeabilized cells, gB and GFP-gB accumulate in the peri-nuclear region (Gilbert et al., 1994; Potel et al., 2002
), whereas gBct-GFP was visualized exclusively as a thin rim around the nuclear membrane and in a reticular structure of the cytoplasm (Fig. 1
A and B). In addition, in non-permeabilized cells, native gB and GFP-gB exhibit a punctate pattern at the cell surface (Potel et al., 2002
), whereas gBct-GFP was not detectable at the surface on any cell (Fig. 1C
).
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DISCUSSION |
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The long ectodomain of gB tolerates insertion of GFP, while preserving the functionality of the resulting protein and virus infectivity (Potel et al., 2002). Here, we showed that GFP-gB was biochemically processed similarly as native gB. In infected Vero cells, GFP-gB accumulated in the Golgi complex and at the cell surface. Its physical proximity to capsids as well as to another envelope glycoprotein, gD, strongly suggests that virus particle assembly occurs at these sites. In infected hippocampal neurons, however, GFP-gB puncta resembling vesicles were distinct from nucleocapsids and nucleocapsid proteins in the neuronal body as well as along the processes of neurons, and no obvious superimposition of nucleocapsid and gB markers could be detected by immunofluorescence staining. These observations are in agreement with the hypothesis first proposed by Penfold et al. (1994)
, that capsids are not enveloped in the cell body but rather move separately from other viral components, in order to acquire their final envelope near the axon terminal, thus permitting assembly of infectious particles at the site where the virus is to be delivered. Similar results were reported in a model of pseudorabies virus infection of sensory dorsal root neurons (Smith et al., 2001
). Exactly how and where assembly and egress of mature virions occur from these cells remains to be investigated.
The cytosolic domain of gB is one of the longest among herpesviruses glycoproteins, and has been extensively studied (Baghian et al., 1993; Cai et al., 1987
, 1988a
, b
; Gilbert et al., 1994
; Rasile et al., 1993
; Raviprakash et al., 1990
). With the aim to further investigate the function of this domain, we contructed a virus where GFP was inserted into the carboxy-terminal domain of the protein. Previous analyses of the post-translational processing of gB polypeptides in transfected cells showed that neither reduction of the cytoplasmic domain of gB by up to 43 residues (Butcher et al., 1990
) nor insertion of a few amino acids at position 45 of the cytoplasmic domain (Navarro et al., 1991
) prevented the intracellular transport of the glycoprotein. In this study however, insertion of GFP in-frame within the carboxy-terminal domain of gB resulted in a virus which was unable to propagate in non-complementing cells. This was due to gBct-GFP, as shown in a complementation assay where gBct-GFP provided in trans was unable to restore infectivity of the gB-null virus K082. Insertion of GFP might have changed a structural feature of gB, leading to misfolding of the fusion protein which would prevent its dissociation from ER chaperones, since mutated misfolded forms of gB are retained in the ER (Laquerre et al., 1998
; Navarro et al., 1991
; Zheng et al., 1996
). Retention of gBct-GFP in the ER might also be due to disruption of a targeting signal localized in the carboxy-terminal tail of gB, similar to those found in herpesviruses gB homologues (Heineman & Hall, 2002
; Heineman et al., 2000
; Meyer & Radsak, 2000
). Current studies are under way to further investigate this hypothesis. Whatever the mechanism underlying the block in gBct-GFP transport, the molecule was retained in the ER and did not undergo the final steps of glycosylation. However, the defect in gBct-GFP did not prevent the maturation and sorting of another envelope protein, gD, in cells infected with KgBct-GFP.
Embryonic hippocampal neuron cultures were used to analyse the transport of gB during HSV1 infection. As observed in epithelial cells, the punctate pattern of the functional GFP-gB in neurons suggested that the molecule exited from the ER, but whether this aspect is related to transport to the cell surface, endocytosis or elements located at the cell surface is not known. Local sites of brighter fluorescence observed in the long processes of neurons could be related to local enlargements due to attachment to the substratum, or to sites of contact with the processes of other neurons. The reason for using embryonic hippocampal neurons is that these cells can be stably polarized and well-differentiated under certain conditions (Dotti et al., 1988), and they have provided the support for studies focused on the sorting of viral membrane proteins. For example, using a temperature-sensitive mutant of vesicular stomatitis virus, glycoprotein G appeared to be polarized to the somato-dendritic domain, whereas haemagglutinin of a wild-type influenza virus was polarized to the axon. These studies suggested that similar mechanisms may control sorting in epithelial cells and in neurons, and showed that many apical and basolateral proteins are axonal and somato-dendritic respectively, although some proteins contradict this hypothesis (Dotti et al., 1993
; Dotti & Simons, 1990
; Jareb & Banker, 1998
; Ledesma et al., 1998
). In this study, by performing infection at low virus titres so that infected neurons are separated from each other, and by using a dendrite-specific marker, we could distinguish between the somato-dendritic and the axonal distribution of HSV-1 gB. In KGFP-gB infected cells, GFP-gB was sorted to the axon as well as the dendrites of hippocampal neurons. In contrast, in KgBct-GFP-infected cultures, gBct-GFP, which was retained in the ER, was restricted to the somato-dendritic compartment of neurons. Various axonal and dendritic sorting signals have been identified in membrane proteins and many of these are located in the cytosolic domains (Bradke & Dotti, 1998
). Herpesviruses homologues of gB have been shown to contain targeting sequences contained within their cytosolic domain. For example, an acidic cluster in the cytosolic domain of human cytomegalovirus gB is a key determinant for targeting gB to apical membranes in epithelial cells (Tugizov et al., 1996
). The role of such motifs in neuronal sorting has not been investigated. Axonal sorting of gB could be related to specific interactions with other viral proteins during infection. Recently, a model for pseudorabies virus sorting in sympathetic cervical neurons has been proposed, where membrane proteins such as gB would traffic in axons via an interaction with another viral protein, US9, yet still interact via their cytoplasmic tail with transport vesicles to traffic in other regions of the neuron (Tomishima et al., 2001
). KgBct-GFP, which contains a selective defect in gB maturation and subsequently axonal sorting, should prove useful in the analysis of the role of protein interactions in the sorting of viral components and subsequent virus maturation.
In summary, we showed that in infected hippocampal neurons, GFP-gB was transported separately from nucleocapsids and nucleocapsid proteins in the neurites, and was sorted to the axon. In contrast, the ER-retained gBct-GFP was restricted to the soma and dendrites of differentiated infected neurons. Future comparative studies of infection performed with the defective KgBct-GFP or the functional KGFP-gB should constitute a valuable model to study host and viral factors involved in the polarized transport of gB and in assembly of HSV-1 in neurons.
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ACKNOWLEDGEMENTS |
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Received 8 April 2003;
accepted 2 July 2003.