Analysis of the role of the membrane-spanning and cytoplasmic tail domains of herpes simplex virus type 1 glycoprotein D in membrane fusion

Helena Browne, Birgitte Bruun, Alison Whiteley{dagger} and Tony Minson

Division of Virology, Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QP, UK

Correspondence
Helena Browne
hb100{at}mole.bio.cam.ac.uk


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Glycoprotein D (gD) of herpes simplex virus type 1 is a type 1 membrane protein in the virus envelope that binds to receptor molecules on the cell surface and which induces cell–cell fusion when co-expressed with gB, gH and gL. A chimeric gD molecule in which the membrane anchor and cytoplasmic tail domains were replaced with analogous regions from the human CD8 molecule was as competent as wild-type gD at mediating membrane fusion and virus entry. However, when gD was tethered to the membrane by means of a glycosylphosphatidylinositol (gpi)-anchor sequence, which binds only to the outer leaflet of the lipid bilayer, it was unable to function in cell–cell fusion assays. This chimera was incorporated into virions as efficiently as wild-type gD and yet virus particles containing gpi-linked gD entered cells more slowly than virions containing wild-type gD in their envelopes, suggesting that gD must be anchored in both leaflets of a lipid bilayer for it to function in both cell fusion and virus entry.

{dagger}Present address: School of Animal and Microbial Sciences, University of Reading, Whiteknights, PO Box 228, Reading RG6 6AJ, UK.


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The current view of herpes simplex virus (HSV) entry into host cells is that following attachment of the virus particle to cell surface glycosaminoglycans, the virus envelope fuses with the plasma membrane, releasing the tegumented nucleocapsid into the cytoplasm. This process is mediated by virion glycoproteins and viruses that lack gB, gD or the gHL heterodimer are unable to enter cells (Cai et al., 1988; Forrester et al., 1992; Ligas & Johnson, 1988). Furthermore, co-expression of this set of glycoproteins in transfected cells induces polykaryocyte formation (Muggeridge, 2000; Turner et al., 1998), suggesting that gB, gD and gHL constitute the minimal requirements for HSV-induced fusion. The finding that this cell fusion assay system does not require glycoproteins containing a syncytial mutation, nor is it dependent on additional glycoproteins known to be important for virus-mediated cell fusion but dispensable for infectivity, such as gE, gI, gM and the UL45 gene product (Balan et al., 1994; Davis-Poynter et al., 1994; Haanes et al., 1994), implies that transfection-induced cell fusion may share features in common with the fusion of virions with the plasma membrane rather than virus-induced syncytium formation, although this remains to be confirmed in further detail. Cellular receptors for gB and gHL have yet to be identified, but gD is known to bind to a number of structurally unrelated molecules to mediate HSV entry (reviewed by Campadelli-Fiume et al., 2000; Spear et al., 2000). These include HveA (a member of the TNF receptor family), nectin 1 (which shares homology with the immunoglobulin superfamily) and 3-O-sulphated heparin sulphate (Cocchi et al., 1998; Montgomery et al., 1996; Shukla et al., 1999). The three-dimensional structures of part of the ectodomain of gD, both bound to HveA and in its unbound form, have been reported recently (Carfi et al., 2001). However, the events that link the binding of gD to these receptors with the fusion of the virion envelope with the plasma membrane are not understood, although it has been shown that cell fusion requires the presence of a gD receptor on recipient cells (Browne et al., 2001; Pertel et al., 2001) and it has been proposed that receptor binding by gD may trigger the fusogenic activity of either gB and/or gHL. Previous studies have identified regions in the ectodomain of gD that are important for virus infectivity (Chiang et al., 1994; Muggeridge et al., 1988) and Feenstra et al. (1990) showed that the sequence encoding the cytoplasmic tail of gD can be deleted with no effect on function. It has also been reported that a recombinant virus expressing a gD molecule that contains the membrane anchor from the Golgi-resident enzyme {alpha}-2-6-sialyl transferase and the cytoplasmic tail of CD8 grew with wild-type kinetics and produced virions containing wild-type levels of gD (Whiteley et al., 1999), implying that the authentic transmembrane (TM) domain and cytoplasmic tail of gD are not required for either incorporation into virus particles or infectivity. However, since gD is involved in both receptor binding and fusion, it was of interest to determine whether the membrane anchor and cytoplasmic tail of gD play a role in the fusion process. Therefore, we replaced these regions with analogous domains from another type 1 glycoprotein, CD8, or with a glycosylphosphatidylinositol (gpi)-anchor sequence. gpi-anchor sequences are known to associate only with the outer leaflet of the membrane lipid bilayer and a previous study has demonstrated that a gpi-linked HSV gD molecule can be released from the plasma membrane by treatment with phosphatidylinositol-specific phospholipase C (Lisanti et al., 1989). Constructs expressing these chimeric molecules were tested in transient fusion assays, the ability of these proteins to complement a gD-null virus was measured and the rates at which virions containing these modified forms of gD were able to enter cells were determined.

Sequences encoding the TM and cytoplasmic tail regions of the human CD8 molecule, corresponding to the C-terminal 53 residues, were derived from the plasmid pS84 (a gift from S. Munro, LMB, Cambridge, UK) and were fused in-frame to sequences encoding the gD ectodomain (aa 1–340) from HSV-1 strain Patton in the plasmid pCDNA3 (Invitrogen). The resulting construct was called pCDNAgDCD8CD8. The sequence encoding the gpi-anchor domain of decay-accelerating factor (DAF), which is composed of the C-terminal 37 aa residues, was derived from the plasmid gD1-DAF (Zurzolo et al., 1993) and was also fused in-frame with the sequence encoding the ectodomain of gD in a pCDNA3-based vector to generate pCDNA3gDDAF. Replacement of the authentic gD TM domain with that of either CD8 or DAF had no effect on the expression of these chimeric molecules on the plasma membrane of transfected cells (Fig. 1a).



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Fig. 1. (a) Chimeric gD molecules are expressed on the plasma membrane of transfected cells. 293T cells were transfected with plasmids expressing wild-type gD, gDCD8CD8 or gDDAF for 24 h. Cells were fixed in 2 % paraformaldehyde and gD hybrid molecules were detected by immunofluorescent staining using monoclonal antibody LP2 followed by incubation with FITC-conjugated rabbit anti-mouse. (b) Entry rates of pseudotyped virions containing gDCD8CD8 and gDDAF. Approximately 300 infectious progeny virions from complementation experiments were allowed to adsorb to monolayers of gD-positive cells for 1 h at 4 °C. At various times after transfer to 37 °C, unpenetrated virions were inactivated with a citrate wash (pH 3) and the monolayers returned to the incubator. Plaques were counted after 2 days and results are expressed as a percentage of the number of plaques obtained on an untreated sample. Values presented are the means from triplicate dishes. Error bars represent SD.

 
To test whether these molecules could mediate cell fusion, we co-expressed them together with wild-type gHL and gB in 293T cells, as described by Harman et al. (2002), and scored the number of nuclei that were recruited into polykaryocytes after overlaying the transfectants with Vero cells. Under these conditions, no background syncytia were detected in untransfected controls and only polykaryocytes containing more than one nucleus were scored as positive. Table 1(a) shows that while gDCD8CD8 was as efficient as wild-type gD at mediating fusion, the gDDAF molecule was completely non-functional in this assay. These results suggest that, although no specific sequences in the TM or cytoplasmic tail domains of gD are required for fusion to occur, there appears to be an absolute requirement for the molecule to be anchored in both leaflets of the lipid bilayer.


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Table 1. (a) Ability of chimeric gD molecules to mediate cell fusion

293T cells expressing chimeric gD molecules and wild-type gB, gH and gL were overlaid with Vero cells for 24 h and the number of nuclei recruited into polykaryocytes was scored.

 

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(b) Complementation of a gD-null virus by gDCD8CD8 and gDDAF

293T cells were transfected with plasmids expressing chimeric gD molecules. After 24 h, cells were infected with 10 p.f.u. per cell of a gD-null virus, STZgD-, and after a 1 h adsorption period, residual input inoculum was inactivated with a citrate wash (pH 3). At 24 h post-infection, cells were harvested and sonicated. Yields of infectious virus were determined by plaque assay on gD-positive cell monolayers.

 
To find whether the modified forms of gD could rescue a gD-null virus, a complementation assay was performed in which cells were transfected with the relevant expression constructs and superinfected with a gD-negative mutant, STZgD- (Rodger et al., 2001). The yield of infectious virions was determined by plaque assay on a gD-expressing helper cell line, gD+ (Rodger et al., 2001). Replacement of the gD TM and cytoplasmic domains with either analogous regions from CD8, or with the gpi-anchor sequence from DAF, had no effect on the ability of these molecules to complement the infectivity of a gD-negative virus. As shown in Table 1(b), both chimeras were as efficient as wild-type gD at rescuing infectivity. However, such rescue assays do not discriminate between the ability of pseudotyped progeny virions to enter cells at equivalent rates to wild-type HSV. As the virions are present for the 48 h duration of a plaque assay and because a gpi-anchored form of gD was non-functional in the fusion assay, it remained a possibility that virions containing this molecule may be less competent than particles containing wild-type gD at entering cells. Therefore, we measured the rates at which the progeny virions from complementation experiments entered cells over a time-period of 90 min (Fig. 1b). In agreement with the results of cell fusion assays, a gD molecule containing the CD8 TM and cytoplasmic tail domains conferred entry kinetics that were equivalent to those mediated by the wild-type molecule, while virions containing gDDAF entered cells more slowly. These data confirm that there is a correlation between the phenotypes of mutated glycoprotein molecules in transient cell fusion assays and their ability to mediate virus entry (as has been reported previously by Harman et al., 2002), and that there are no requirements for specific sequences in the TM domain and tail of gD for either process to take place. They also imply that receptor binding by gD is not the only event required for efficient fusion to occur, as both chimeric molecules contain the receptor-binding regions of gD and yet the gDDAF molecule is compromised in its ability to mediate fusion.

However, a further possible interpretation of these findings is that pseudotyped virions containing gpi-linked gD enter cells more slowly because they incorporate less gD into their envelopes. To address this issue, we constructed a recombinant virus expressing gpi-linked gD so that we could determine whether purified virions contained wild-type amounts of the gDDAF molecule.

The open reading frame encoding gDDAF was isolated from plasmid pCDNA3gDDAF as a HindIII–XbaI fragment and ligated into the vector pINGHinc11gD (Whiteley et al., 1999). This contains HSV-1 sequences flanking the gD gene from nt 136 449 to 140 555, according to the numbering of nucleotides in the HSV-1 genome (McGeoch et al., 1988); the resulting construct was called pINGHincgDgpi. BHK cells were co-transfected, using the method of Chen & Okayama (1987), with pINGHincgDgpi and with DNA extracted from cells infected with a gD-negative derivative of HSV-1 strain SC16, SCgDdelZ (Whiteley et al., 1999). At 5 days after transfection, small plaques were observed and these were harvested and used to grow stocks of the recombinant virus, SCgDgpi. Stocks of this virus were assayed on a gD-helper cell line and extracellular virions were purified from infected BHK cells on Ficoll gradients, as described by Rodger et al. (2001). Numbers of virus particles were estimated by comparison with latex particles of known concentrations using negatively stained preparations, as described by Watson et al. (1963). The particle to infectivity ratio of SCgDgpi was estimated to be approximately 1000 : 1, whereas a preparation of purified wild-type SC16 virions had a ratio of 40 : 1.

The gD content of these virions was compared with that of wild-type virions by Western blotting serial twofold dilutions of equivalent numbers of virus particles using an anti-gD monoclonal antibody, LP14 (Minson et al., 1986). Parallel samples were immunoblotted with an antibody that recognizes the tegument protein VP16 as an internal control for loading equivalence; results are shown in Fig. 2. It is notable that the electrophoretic characteristics of gpi-linked gD are slightly different from the wild-type molecule and it appeared to migrate as a more diffuse species. The amounts of gD and VP16 in both wild-type and SCgDgpi virions were, therefore, semi-quantified by densitometric analysis of the Western blot using a ChemiImager 4000 (Flowgen). These data, which are also presented in Fig. 2, show that there are no significant differences between the amounts of gpi-linked gD and the amounts of wild-type gD that are incorporated into the virion envelope during assembly.



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Fig. 2. Analysis of the gD content of purified SCgDgpi virions. Serial twofold dilutions of wild-type virions (SC16) or virions containing gpi-linked gD (SCgDgpi) were immunoblotted for VP16 and gD. The first track in each set of dilutions corresponds to a lysate of 109 virus particles. The relative intensity of these signals was quantified by densitometric scanning. Values are presented as the ratios of the signal obtained for SCgDgpi samples to that of the signal in the wild-type track containing an equivalent number of virus particles.

 
The finding that a gpi-anchored gD molecule is compromised in cell fusion and virus entry is similar to observations on influenza virus haemagglutinin (HA) and human immunodeficiency virus type 1 (HIV-1) gp160, both of which are unable to mediate fusion when anchored to the membrane by gpi sequences (Kemble et al., 1994; Weiss & White, 1993) and which can only induce a hemifusion state. Although, it is perhaps unwise to draw too close a parallel between HIV and influenza virus-mediated fusion and that induced by HSV-1, because the latter requires a combination of four proteins (gB, gD and gHL), it remains a possibility that gB, gHL and gDDAF cause hemifusion efficiently but that completion of fusion is slow.

This is not the first report of a gpi-anchored alphaherpesvirus gD molecule: Liang et al. (1995) produced a stably transfected cell line that expressed a gpi-anchored form of bovine herpes virus type 1 (BHV-1) gD. In agreement with our findings, they noted that gpi-anchored BHV-1 gD rescued the infectivity of a gD-negative virus. They also reported that a gD-null virus could enter cells expressing this hybrid molecule, implying that gDDAF, unlike wild-type gD, could act in trans to mediate the entry of virions lacking gD. However, these studies did not address the issue of whether gpi-anchored BHV-1 gD was less efficient than wild-type gD at mediating cell–cell fusion nor whether virions containing this chimera could enter cells at rates similar to those with wild-type gD in their envelopes; whether this is a common property of alphaherpesvirus gD molecules is, at present, unclear.

A further possible implication of the observations described in this report concerns the mechanism by which HSV glycoproteins assemble into virions during morphogenesis. It is believed that the virus acquires its envelope glycoproteins at a post-endoplasmic reticulum compartment (either the trans-Golgi network or an early endosome) (Enquist et al., 1998; Harley et al., 2001; Skepper et al., 2001; Whiteley et al., 1999), although the signals that direct incorporation of at least ten membrane proteins into the particle are not well understood. The results presented here show that gD assembles efficiently into virions in the absence of its authentic TM and cytoplasmic tail domains and that it is also incorporated when anchored in membranes by a gpi sequence. The gpi-anchor region of DAF has been shown to target the ectodomains of heterologous molecules to membrane microdomains, known as lipid rafts, which are enriched in cholesterol and sphingolipids (Friedrichson & Kurzchalia, 1998). And, as a gDDAF hybrid molecule is incorporated into virus particles, it is possible that such membrane microdomains may represent a site of HSV-1 glycoprotein accumulation during budding, as has been shown to be the case for the envelope proteins of measles and influenza viruses (Manie et al., 2000; Scheiffele et al., 1999).


   ACKNOWLEDGEMENTS
 
This work was supported by the Wellcome Trust, UK, and by an MRC Co-operative Group Award.


   REFERENCES
Top
ABSTRACT
MAIN TEXT
REFERENCES
 
Balan, P., Davis-Poynter, N., Bell, S., Atkinson, H., Browne, H. & Minson, T. (1994). An analysis of the in vitro and in vivo phenotypes of mutants of herpes simplex virus type 1 lacking glycoproteins gG, gE, gI or the putative gJ. J Gen Virol 75, 1245–1258.[Abstract]

Browne, H., Bruun, B. & Minson, T. (2001). Plasma membrane requirements for cell fusion induced by herpes simplex virus type 1 glycoproteins gB, gD, gH and gL. J Gen Virol 82, 1419–1422.[Abstract/Free Full Text]

Cai, W., Gu, B. & Person, S. (1988). Role of glycoprotein B of herpes simplex virus type 1 in viral entry and cell fusion. J Virol 62, 2596–2604.[Medline]

Campadelli-Fiume, G., Cocchi, F., Menotti, L. & Lopez, M. (2000). The novel receptors that mediate the entry of herpes simplex viruses and animal alphaherpesviruses into cells. Rev Med Virol 10, 305–319.[CrossRef][Medline]

Carfi, A., Willis, S. H., Whitbeck, J. C., Krummenacher, C., Cohen, G. H., Eisenberg, R. J. & Wiley, D. C. (2001). Herpes simplex virus glycoprotein D bound to the human receptor HveA. Mol Cell 8, 169–179.[Medline]

Chen, C. & Okayama, H. (1987). High-efficiency transformation of mammalian cells by plasmid DNA. Mol Cell Biol 7, 2745–2752.[Medline]

Chiang, H.-Y., Cohen, G. H. & Eisenberg, R. J. (1994). Identification of functional regions of herpes simplex virus glycoprotein gD by using linker-insertion mutagenesis. J Virol 68, 2529–2543.[Abstract]

Cocchi, F., Menotti, L., Mirandola, P., Lopez, M. & Campadelli-Fiume, G. (1998). The ectodomain of a novel member of the immunoglobulin subfamily related to the poliovirus receptor has the attributes of a bona fide receptor for herpes simplex virus types 1 and 2 in human cells. J Virol 72, 9992–10002.[Abstract/Free Full Text]

Davis-Poynter, N., Bell, S., Minson, T. & Browne, H. (1994). Analysis of the contributions of herpes simplex virus type 1 membrane proteins to the induction of cell–cell fusion. J Virol 68, 7586–7590.[Abstract]

Enquist, L. W., Husak, P. J., Banfield, B. W. & Smith, G. A. (1998). Infection and spread of alphaherpesviruses in the nervous system. Adv Virus Res 51, 237–247.[Medline]

Feenstra, V., Hodaie, M. & Johnson, D. C. (1990). Deletions in herpes simplex virus glycoprotein D define nonessential and essential domains. J Virol 64, 2096–2102.[Medline]

Forrester, A., Farrell, H., Wilkinson, G., Kaye, J., Davis-Poynter, N. & Minson, T. (1992). Construction and properties of a mutant of herpes simplex virus type 1 with glycoprotein H coding sequences deleted. J Virol 66, 341–348.[Abstract]

Friedrichson, T. & Kurzchalia, T. V. (1998). Microdomains of GPI-anchored proteins in living cells revealed by crosslinking. Nature 394, 802–805.[CrossRef][Medline]

Haanes, E. J., Nelson, C. M., Soule, C. L. & Goodman, J. L. (1994). The UL45 gene product is required for herpes simplex virus type 1 glycoprotein B-induced fusion. J Virol 68, 5825–5834.[Abstract]

Harley, C. A., Dasgupta, A. & Wilson, D. W. (2001). Characterization of herpes simplex virus-containing organelles by subcellular fractionation: role for organelle acidification in assembly of infectious particles. J Virol 75, 1236–1251.[Abstract/Free Full Text]

Harman, A., Browne, H. & Minson, T. (2002). The transmembrane domain and cytoplasmic tail of herpes simplex virus type 1 glycoprotein H play a role in membrane fusion. J Virol 76, 10708–10716.[Abstract/Free Full Text]

Kemble, G. W., Danieli, T. & White, J. M. (1994). Lipid-anchored influenza hemagglutinin promotes hemifusion, not complete fusion. Cell 76, 383–391.[Medline]

Liang, X., Pyne, C., Li, Y., Babiuk, L. A. & Kowalski, J. (1995). Delineation of the essential function of bovine herpesvirus 1 gD: an indication for the modulatory role of gD in virus entry. Virology 207, 429–441.[CrossRef][Medline]

Ligas, M. W. & Johnson, D. C. (1988). A herpes simplex virus mutant in which glycoprotein D sequences are replaced by {beta}-galactosidase sequences binds to but is unable to penetrate into cells. J Virol 62, 1486–1494.[Medline]

Lisanti, M. P., Caras, I. W., Davitz, M. A. & Rodriguez-Boulan, E. (1989). A glycophospholipid membrane anchor acts as an apical targeting signal in polarized epithelial cells. J Cell Biol 109, 2145–2156.[Abstract]

Manie, S. N., Debreyne, S., Vincent, S. & Gerlier, D. (2000). Measles virus structural components are enriched into lipid raft microdomains: a potential cellular location for virus assembly. J Virol 74, 305–311.[Abstract/Free Full Text]

McGeoch, D. J., Dalrymple, M. A., Davison, A. J., Dolan, A., Frame, M. C., NcNab, D., Perry, L. J., Scott, J. E. & Taylor, P. (1988). The complete DNA sequence of the long unique region on the genome of herpes simplex virus type 1. J Gen Virol 69, 1531–1574.[Abstract]

Minson, A. C., Hodgman, T. C., Digard, P., Hancock, D. C., Bell, S. E. & Buckmaster, E. A. (1986). An analysis of the biological properties of monoclonal antibodies against glycoprotein D of herpes simplex virus and identification of amino acid substitutions that confer resistance to neutralization. J Gen Virol 67, 1001–1013.[Abstract]

Montgomery, R. I., Warner, M. S., Lum, B. J. & Spear, P. G. (1996). Herpes simplex virus-1 entry into cells mediated by a novel member of the TNF/NGF receptor family. Cell 87, 427–436.[Medline]

Muggeridge, M. I. (2000). Characterization of cell–cell fusion mediated by herpes simplex virus 2 glycoproteins gB, gD, gH and gL in transfected cells. J Gen Virol 81, 2017–2027.[Abstract/Free Full Text]

Muggeridge, M. I., Isola, V. J., Byrn, R. A., Tucker, T. J., Minson, A. C., Glorioso, J. C., Cohen, G. H. & Eisenberg, R. J. (1988). Antigenic analysis of a major neutralization site of herpes simplex virus glycoprotein D, using deletion mutants and monoclonal antibody-resistant mutants. J Virol 62, 3274–3280.[Medline]

Pertel, P. E., Fridberg, A., Parish, M. L. & Spear, P. G. (2001). Cell fusion induced by herpes simplex virus glycoproteins gB, gD, and gH-gL requires a gD receptor but not necessarily heparan sulfate. Virology 279, 313–324.[CrossRef][Medline]

Rodger, G., Boname, J., Bell, S. & Minson, T. (2001). Assembly and organization of glycoproteins B, C, D, and H in herpes virus type 1 particles lacking individual glycoproteins: no evidence for the formation of a complex of these molecules. J Virol 75, 710–716.[Abstract/Free Full Text]

Scheiffele, P., Rietveld, A., Wilk, T. & Simons, K. (1999). Influenza viruses select ordered lipid domains during budding from the plasma membrane. J Biol Chem 274, 2038–2044.[Abstract/Free Full Text]

Shukla, D., Liu, J., Blaiklock, P. & 7 other authors (1999). A novel role for 3-O-sulfated heparan sulfate in herpes simplex virus 1 entry. Cell 99, 13–22.[Medline]

Skepper, J. N., Whiteley, A., Browne, H. & Minson, A. (2001). Herpes simplex virus nucleocapsids mature to progeny virions by an envelopment -> deenvelopment -> reenvelopment pathway. J Virol 75, 5697–5702.[Abstract/Free Full Text]

Spear, P. G., Eisenberg, R. J. & Cohen, G. H. (2000). Three classes of cell surface receptors for alphaherpesvirus entry. Virology 275, 1–8.[CrossRef][Medline]

Turner, A., Bruun, B., Minson, A. & Browne, H. (1998). Glycoproteins gB, gD, and gHgL of herpes simplex virus type 1 are necessary and sufficient to mediate membrane fusion in a Cos cell transfection system. J Virol 72, 873–875.[Abstract/Free Full Text]

Watson, D. H., Russell, W. C. & Wildy, P. W. (1963). Electron microscope particle counts on herpes virus using the phosphotungstate negative staining technique. Virology 19, 250–260.[Medline]

Weiss, C. D. & White, J. M. (1993). Characterization of stable Chinese hamster ovary cells expressing wild-type, secreted, and glycosylphosphatidylinositol-anchored human immunodeficiency virus type 1 envelope glycoprotein. J Virol 67, 7060–7066.[Abstract]

Whiteley, A., Bruun, B., Minson, T. & Browne, H. (1999). Effects of targeting herpes simplex virus type 1 gD to the endoplasmic reticulum and trans-Golgi network. J Virol 73, 9515–9520.[Abstract/Free Full Text]

Zurzolo, C., Lisanti, M. P., Caras, I. W., Nitsch, L. & Rodriguez-Boulan, E. (1993). Glycosylphosphatidylinositol-anchored proteins are preferentially targeted to the basolateral surface in Fischer rat thyroid epithelial cells. J Cell Biol 121, 1031–1039.[Abstract]

Received 12 December 2002; accepted 16 January 2003.