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• W2049135087 abstract "The varicella-zoster virus (VZV) is the etiological agent of two different human pathologies, chickenpox (varicella) and shingles (zoster). This alphaherpesvirus is believed to acquire its lipidic envelope in the trans-Golgi network (TGN). This is consistent with previous data showing that the most abundant VZV envelope glycoprotein gE accumulates at steady-state in this organelle when expressed from cloned cDNA. In the present study, we have investigated the intracellular trafficking of gI, another VZV envelope glycoprotein. In transfected cells, this protein shows a very slow biosynthetic transport to the cell surface where it accumulates. However, upon co-expression of gE, gI experiences a dramatic increase in its exit rate from the endoplasmic reticulum, it accumulates in a sialyltransferase-positive compartment, presumably the TGN, and cycles between this compartment and the cell surface. This differential behavior results from the ability of gE and gI to form a complex in the early stages of the biosynthetic pathway whose intracellular traffic is exclusively determined by the sorting information in the tail of gE. Thus, gI provides the first example of a molecule localized to the TGN by means of its association with another TGN protein. We also show that, during the early stages of VZV infection, both proteins are also found in the TGN of the host cell. This suggests the existence of an intermediate stage during VZV biogenesis in which the envelope glycoproteins, transiently arrested in the TGN, could promote the envelopment of newly synthesized nucleocapsids into this compartment and, therefore, the assembly of infective viruses. The varicella-zoster virus (VZV) is the etiological agent of two different human pathologies, chickenpox (varicella) and shingles (zoster). This alphaherpesvirus is believed to acquire its lipidic envelope in the trans-Golgi network (TGN). This is consistent with previous data showing that the most abundant VZV envelope glycoprotein gE accumulates at steady-state in this organelle when expressed from cloned cDNA. In the present study, we have investigated the intracellular trafficking of gI, another VZV envelope glycoprotein. In transfected cells, this protein shows a very slow biosynthetic transport to the cell surface where it accumulates. However, upon co-expression of gE, gI experiences a dramatic increase in its exit rate from the endoplasmic reticulum, it accumulates in a sialyltransferase-positive compartment, presumably the TGN, and cycles between this compartment and the cell surface. This differential behavior results from the ability of gE and gI to form a complex in the early stages of the biosynthetic pathway whose intracellular traffic is exclusively determined by the sorting information in the tail of gE. Thus, gI provides the first example of a molecule localized to the TGN by means of its association with another TGN protein. We also show that, during the early stages of VZV infection, both proteins are also found in the TGN of the host cell. This suggests the existence of an intermediate stage during VZV biogenesis in which the envelope glycoproteins, transiently arrested in the TGN, could promote the envelopment of newly synthesized nucleocapsids into this compartment and, therefore, the assembly of infective viruses. The trans-Golgi network (TGN) 1The abbreviations used are: TGN, trans-Golgi network; BFA, brefeldin A; BHV, bovine herpesvirus; ER, endoplasmic reticulum; FHV, feline herpesvirus; HSV, herpes simplex virus; PRV, pseudorabies virus; ST, sialyltransferase; VZV, varicella-zoster virus; FITC, fluorescein isothiocyanate; TRITC, tetrahodamine isothiocyanate; MEM, minimum Eagle's medium; VSV-G, vesicular stomatitis virus glycoprotein G. is a tubuloreticular compartment located on the trans-most side of the Golgi complex (1Rambourg A. Clermont Y. Eur. J. Cell Biol. 1990; 51: 189-200PubMed Google Scholar). This organelle houses different proteins that are involved in adding post-translational modifications to polypeptides traveling along the secretory pathway. In addition, this organelle constitutes the main sorting station in the secretory pathway (2Mellman I. Simons K. Cell. 1992; 68: 829-840Abstract Full Text PDF PubMed Scopus (383) Google Scholar, 3Griffiths G. Simons K. Science. 1986; 234: 438-443Crossref PubMed Scopus (764) Google Scholar). The TGN can undergo rapid tubularization and mixing with endosomal compartments in the presence of the fungal metabolite brefeldin A (BFA) (4Klausner R.D. Donaldson J.G. Lippincott-Scwharz J. J. Cell Biol. 1992; 116: 1071-1080Crossref PubMed Scopus (1542) Google Scholar). The TGN has also been used by certain viruses as a membrane donor for their lipidic envelope (envelopment or budding process), as it happens in the case of the varicella-zoster virus (VZV) (5Gershon A.A. Sherman D.L. Zhu Z. Gabel C.A. Ambron R.T. Gershon M.D. J. Virol. 1994; 68: 6372-6390Crossref PubMed Google Scholar, 6Zhu Z. Gershon M.D. Gabel C. Sherman D. Ambron R. Gershin A. Neurology. 1995; 45: S15-S17Crossref PubMed Google Scholar). VZV is a human alphaherpesvirus causing chickenpox (varicella), as a result of the primary infection, and shingles (zoster) upon reactivation of the latent virus (7Weller T.H. N. Engl. J. Med. 1983; 309: 1362-1368Crossref PubMed Scopus (219) Google Scholar, 8Weller T.H. N. Engl. J. Med. 1983; 309: 1434-1440Crossref PubMed Scopus (173) Google Scholar). As it occurs in other alphaherpesviruses, the VZV nucleocapsids are assembled in the nuclei of the infected cells. These nucleocapsids are then released into the periplasmic space by budding through the inner nuclear membrane, thereby acquiring a transient envelope that is lost upon fusion with the outer nuclear membrane. In this way, the nucleocapsids are released in the cytosol, where they acquire a second and definitive envelope. This envelope is derived from the TGN, as initially demonstrated by Gershon et al. (5Gershon A.A. Sherman D.L. Zhu Z. Gabel C.A. Ambron R.T. Gershon M.D. J. Virol. 1994; 68: 6372-6390Crossref PubMed Google Scholar) by examining VZV-infected cells at the ultrastructural level. Mature viruses accumulate finally in an intracellular endosomal compartment (9Gabel C.A. Dubey L. Steinberg S.P. Sherman D. Gershon M.D. Gershon A.A. J. Virol. 1989; 63: 4264-4276Crossref PubMed Google Scholar). As happens in other cases of viruses that undergo intracellular assembly, envelopment of VZV in the TGN requires that the corresponding envelope glycoproteins have to be delivered to this compartment during viral infection (10Zhu Z. Gershon M.D. Hao Y. Ambron R.T. Gabel C.A. Gershon A.A. J. Virol. 1995; 69: 7951-7959Crossref PubMed Google Scholar, 11Zhu Z. Hao Y. Gershon M.D. Ambron R.T. Gershon A.A. J. Virol. 1996; 70: 6563-6575Crossref PubMed Google Scholar, 12Alconada A. Bauer U. Hoflack B. EMBO J. 1996; 15: 6096-6110Crossref PubMed Scopus (136) Google Scholar). This implies that sorting signals must exist within these glycoproteins to ensure their correct targeting, making these molecules very useful tools for analyzing the mechanisms involved in TGN localization. We and others have recently shown that the most abundant envelope glycoprotein of VZV (gpI or gE) accumulates in the TGN when expressed from cloned cDNA and that this accumulation results, at least partially, from its ability to be rapidly retrieved form the cell surface (10Zhu Z. Gershon M.D. Hao Y. Ambron R.T. Gabel C.A. Gershon A.A. J. Virol. 1995; 69: 7951-7959Crossref PubMed Google Scholar, 11Zhu Z. Hao Y. Gershon M.D. Ambron R.T. Gershon A.A. J. Virol. 1996; 70: 6563-6575Crossref PubMed Google Scholar, 12Alconada A. Bauer U. Hoflack B. EMBO J. 1996; 15: 6096-6110Crossref PubMed Scopus (136) Google Scholar). The sorting information in the sequence of gE has been mapped to its cytoplasmic tail, and shown to consist of two tyrosine-containing tetrapeptides related to endocytosis motifs (11Zhu Z. Hao Y. Gershon M.D. Ambron R.T. Gershon A.A. J. Virol. 1996; 70: 6563-6575Crossref PubMed Google Scholar, 12Alconada A. Bauer U. Hoflack B. EMBO J. 1996; 15: 6096-6110Crossref PubMed Scopus (136) Google Scholar) and a more C-terminal acidic cluster that contains casein-kinase II- phosphorylatable residues (11Zhu Z. Hao Y. Gershon M.D. Ambron R.T. Gershon A.A. J. Virol. 1996; 70: 6563-6575Crossref PubMed Google Scholar, 12Alconada A. Bauer U. Hoflack B. EMBO J. 1996; 15: 6096-6110Crossref PubMed Scopus (136) Google Scholar). These signals are similar to those found in other molecules known to be localized in the TGN at steady state, such as TGN38 or furin (13Jones B.G. Thomas L. Molloy S.S. Thulin C.D. Fry M.D. Walsh K.A. Thomas G. EMBO J. 1995; 14: 5869-5883Crossref PubMed Scopus (164) Google Scholar, 14Schäfer W. Stroh A. Bergho¨fer S. Seiler J. Vey M. Kruse M.-L. Kern H.F. Klenk H.-D. Garten W. EMBO J. 1995; 14: 2424-2435Crossref PubMed Scopus (220) Google Scholar, 15Takahashi S. Nakagawa T. Banno T. Watanabe T. Murakami K. Nakayama K. J. Biol. Chem. 1995; 270: 28397-28401Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 16Voorhoes P. Deignan E. van Donselaar E. Humphrey J. Marks M. Peters P.J. Bonifacino J.S. EMBO J. 1995; 14: 4961-4975Crossref PubMed Scopus (187) Google Scholar, 17Ponnambalam S. Rabouille C. Luzio J.P. Nilsson T. Warren G. J. Cell Biol. 1994; 125: 253-268Crossref PubMed Scopus (120) Google Scholar, 18Bos K. Wraight C. Stanley K. EMBO J. 1993; 12: 2219-2228Crossref PubMed Scopus (193) Google Scholar, 19Wong S.H. Hong W. J. Biol. Chem. 1993; 268: 22853-22862Abstract Full Text PDF PubMed Google Scholar). In addition to gE, there are at least five additional glycoproteins in the envelope of VZV (gB, gH, gI, gC, and gL, formerly known as gpII, gpIII, gpIV, gpV, and gpVI, respectively) (20Davidson A.J. Edson C.M. Ellis R.W. Forghani B. Gilden D. Grose C. Keller P.M. Vafai A. Wroblewska Z. Yamanishi K. J. Virol. 1986; 57: 1195-1197Crossref PubMed Google Scholar), whose sequences are apparently devoid of TGN-sorting information. If VZV indeed acquires its final envelope in the TGN, then mechanisms must exist to ensure that all these molecules reach this compartment in order to promote infective VZV formation. In the present article, we have focused our attention on another type I glycoprotein of the viral envelope, the glycoprotein gI (or gpIV). This molecule has been shown to physically interact with gE in VZV (21Yao Z. Jackson W. Forghani B. Grose C. J. Virol. 1993; 67: 305-314Crossref PubMed Google Scholar), as well as in herpes simplex virus (HSV-1) (22Johnson D.C. Feenstra V. J. Virol. 1987; 61: 2208-2216Crossref PubMed Google Scholar), feline herpesvirus (FHV-1) (23De Mijnes J.D.F. van der Horst L.M. van Anken E. Horzinek M.C. Rottier P.J.M. de Groot R.J. J. Virol. 1996; 70: 5466-5475Crossref PubMed Google Scholar), and pseudorabies virus (PRV) (24Whealy M.E. Card J.P. Robbins A.K. Dubin J.R. Rziha H.-J. Enquist L.W. J. Virol. 1993; 67: 3786-3797Crossref PubMed Google Scholar), three other members of the alphaherpesvirinae subfamily. Our results indicate that gI, which is found in the cell surface when expressed alone, accumulates in the TGN when expressed together with gE. This accumulation of gI in the TGN also relies on its rapid internalization from the cell surface. Our data indicate that gE and gI precursors can form a stoichiometric complex in the endoplasmic reticulum (ER), which results in an increased maturation rate of gI. We have also found that, in VZV-infected cells, both gE and gI can be found shortly after infection in a perinuclear compartment that most likely corresponds to the TGN. Monoclonal antibodies SG1 and SG4, against VZV gE and gI, respectively, were obtained from Viro Research Inc. (Rockford, IL). The polyclonal serum against the cytoplasmic tail of furin was generously provided by Dr. W. Garten (University of Marburg, Marburg, Germany). The SA48 HeLa clone stably expressing VSVG-tagged sialyltransferase (ST-VSVG) was a generous gift of Dr. Tommy Nilsson (EMBL, Heidelberg, Germany). All secondary antibodies against the Fc of mouse or rabbit IgGs coupled to FITC or rhodamine were purchased from Dianova (Hamburg, Germany). Construction of the gE expression vector has been previously described (12Alconada A. Bauer U. Hoflack B. EMBO J. 1996; 15: 6096-6110Crossref PubMed Scopus (136) Google Scholar). In order to clone VZV gI, the complete open reading frame was amplified from a lysate of VZV (Dumas strain)-infected cells using the Expand High Fidelity kit (Boehringer Mannheim, Mannheim, Germany). The resulting fragment was digested withXbaI and HindIII and was cloned into the same sites of the eukaryotic expression vector pSFFV6 (25Chen H.J. Remmler J. Delaney J.C. Messner D.J. Lobel P. J. Biol. Chem. 1993; 268: 22338-22346Abstract Full Text PDF PubMed Google Scholar), or downstream the T7 promoter in pGEM1. The gE mutants containing the cytoplasmic tail of the yeast protein Wbp1p with either the C-terminal KKXX or the SSXX signals were constructed by polymerase chain reaction-based amplification using reverse primers in which the corresponding sequences of the wild-type or mutated Wbp1p cytoplasmic tails had been introduced as translational fusions with the sequence of the gE transmembrane domain. The resulting polymerase chain reaction fragments were digested with XbaI and HindIII and cloned into the same sites in the pSFFV6 vector. The sequences of both mutants were verified using the Sanger dideoxy chain termination method. The antibody 1667 against the full-length gE was obtained by cloning a cDNA fragment coding for the mature VZV gE open reading frame with a hexahistidine tag at the C terminus into the NcoI/BamHI sites of the pET15b vector (Novagen, Wiesbaden, Germany). The protein was expressed in BL21 cells and the insoluble fraction (containing most of the recombinant gE) was solubilized in 8 m urea and loaded on a Talon metal-affinity column (CLONTECH, Heidelberg, Germany). After extensive washing, the bound protein was eluted with SDS-loading buffer, and approximately 50 μg were loaded on a 7.5% preparative SDS-polyacrylamide gel. The part of the gel containing the recombinant protein was excised, homogenized using a Teflon-glass homogenizer, mixed with either Freund's complete or incomplete adjuvant, and used to immunize rabbits following standard procedures. To produce the 2679 antibody against the cytoplasmic tail of gI, a fragment comprising amino acids 314–354 of the gI precursor form was cloned into the pGEX-4T-1 vector (Pharmacia, Freiburg, Germany) as a fusion to glutathione S-transferase. The glutathioneS-transferase-gI fusion was expressed in XL-1 Blue cells and purified by affinity chromatography on a glutathione-Sepharose column (Pharmacia, Freiburg, Germany), following the manufacturer's instructions. After elution, the fusion protein was loaded on a preparative 7.5% preparative SDS-polyacrylamide gel. The gel fragment containing the band was excised, homogenized, mixed with Freund's adjuvant, and used to inoculate rabbits following a standard immunization schedule. The serum was affinity-purified by incubation with a nitrocellulose strip onto which the recombinant glutathioneS-transferase-gI had been previously bound (26Harlow E. Lane D. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1988Google Scholar). For the internalization assays, a continuous uptake was performed in which transfected cells seeded on coverslips were washed with prewarmed α-MEM and overlaid with 200 μl of complete α-MEM in which the antibodies had been diluted as specified in the figure legends. After 1-h incubation, the internalization medium was removed and the cells were immediately fixed and processed for immunofluorescence using fluorescein or rhodamine-coupled secondary antibodies. Due to the cell-associated nature of VZV, infections were carried out as described previously (27Defechereux P. Debrus S. Baudoux L. Schoonbroodt S. Merville M.-P. Rentier B. Piette J. J. Gen. Virol. 1996; 77: 1505-1513Crossref PubMed Scopus (14) Google Scholar), by co-culture of VZV (Ellen strain)-infected Vero cells with noninfected either Vero or HeLa cells. For the immunofluorescence experiments, infected and noninfected cells were plated on coverslips at a 1:4 ratio and grown in complete α-MEM for different times as indicated in the figure legends. The cells were subsequently fixed and processed for immunofluorescence. Published procedures were used for vaccinia T7 infection (12Alconada A. Bauer U. Hoflack B. EMBO J. 1996; 15: 6096-6110Crossref PubMed Scopus (136) Google Scholar), metabolic labeling of the cells and immunoprecipitation (28Mauxion F. Le Borgne R. Munier-Lehmann H. Hoflack B. J. Biol. Chem. 1996; 271: 2171-2178Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar), and for calcium-phosphate transient transfection and indirect immunofluorescence (12Alconada A. Bauer U. Hoflack B. EMBO J. 1996; 15: 6096-6110Crossref PubMed Scopus (136) Google Scholar). To address the subcellular localization of the VZV-envelope glycoprotein gI (gpIV), we have cloned the complete gI open reading frame in the mammalian expression vector pSFFV6 (25Chen H.J. Remmler J. Delaney J.C. Messner D.J. Lobel P. J. Biol. Chem. 1993; 268: 22338-22346Abstract Full Text PDF PubMed Google Scholar). We have used this construct to perform transient transfection assays in HeLa cells followed by immunofluorescence using anti-gI-specific antibodies. This experiment revealed that, in every transfected cell, gI was exclusively found at the cell surface (Fig.1 b). As a control, we also performed transient transfections with an analogous construct in which the complete gE (gpI) open reading frame had been inserted into the same expression vector (12Alconada A. Bauer U. Hoflack B. EMBO J. 1996; 15: 6096-6110Crossref PubMed Scopus (136) Google Scholar). In agreement with previous data (12Alconada A. Bauer U. Hoflack B. EMBO J. 1996; 15: 6096-6110Crossref PubMed Scopus (136) Google Scholar), in cells transfected with the gE expression vector, this protein was exclusively localized in the perinuclear region of the cell (Fig.1 c), in a compartment that has been previously identified as the TGN, based on its co-localization at the light microscopy level with the TGN markers TGN38, furin, and sialyltransferase (12Alconada A. Bauer U. Hoflack B. EMBO J. 1996; 15: 6096-6110Crossref PubMed Scopus (136) Google Scholar), at the electron microscopic level with galactosyltransferase, 2A. Alconada, S. Ro¨ttgers, and B. Hoflack, unpublished observations. and by its sensitivity to BFA and nocodazole, two drugs that affect the morphology of this compartment (12Alconada A. Bauer U. Hoflack B. EMBO J. 1996; 15: 6096-6110Crossref PubMed Scopus (136) Google Scholar). We next looked at the localization of gI in HeLa cells that had been simultaneously transfected with gE and gI expression constructs. In these cells, expression of gI was mainly restricted to the perinuclear region of the cell, largely colocalizing with gE, and almost absent from the cell surface (Fig. 1, e and f). We also performed an analogous double-transfection experiment using gI and an unrelated TGN marker (the convertase furin), whose intracellular traffic closely resembles that of gE (13Jones B.G. Thomas L. Molloy S.S. Thulin C.D. Fry M.D. Walsh K.A. Thomas G. EMBO J. 1995; 14: 5869-5883Crossref PubMed Scopus (164) Google Scholar, 14Schäfer W. Stroh A. Bergho¨fer S. Seiler J. Vey M. Kruse M.-L. Kern H.F. Klenk H.-D. Garten W. EMBO J. 1995; 14: 2424-2435Crossref PubMed Scopus (220) Google Scholar, 15Takahashi S. Nakagawa T. Banno T. Watanabe T. Murakami K. Nakayama K. J. Biol. Chem. 1995; 270: 28397-28401Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 16Voorhoes P. Deignan E. van Donselaar E. Humphrey J. Marks M. Peters P.J. Bonifacino J.S. EMBO J. 1995; 14: 4961-4975Crossref PubMed Scopus (187) Google Scholar). In this case, whereas expression of furin was restricted to the perinuclear region of the cell (Fig. 1 g), gI was exclusively detected at the cell surface (Fig. 1 h), therefore excluding the possibility that the perinuclear localization of gI in gE-expressing cells was simply due to an inability of the cell to properly sort gI at the TGN in the presence of another highly expressed molecule in this compartment. It has been previously suggested that gE and gI might share common antigenic determinants (29Vafai A. Wroblewska Z. Mahalingan G. Cabirac G. Wellish M. Cisco M. Gilden D. J. Virol. 1988; 62: 2544-2551Crossref PubMed Google Scholar, 30Vafai A. Jensen K. Kubo R. Virus Res. 1989; 13: 319-336Crossref PubMed Scopus (9) Google Scholar), which could explain the perinuclear signal attributed to gI in cells expressing gE if the antibodies used in this study would recognize any of these shared epitopes. However, this does not seem to be the case, since in cells expressing gI, no signal was detected with the anti-gE antibody (Fig. 1 a) and, conversely, no signal was observed with anti-gI antibodies in cells exclusively transfected with gE (Fig. 1 d). To exclude that the strong cell surface gI-staining observed in cells expressing gI alone or gI and furin could mask any labeling of intracellular compartments, we analyzed the single or double-transfected cells by laser scanning confocal microscopy. As expected, no gI-staining could be detected intracellularly (data not shown). The same distribution was observed when polyclonal antibodies against the cytoplasmic domain of gI were used (data not shown). From all these results, we concluded that the localization of gI in transfected cells can be shifted from the cell-surface to the perinuclear region by the simultaneous co-expression of gE. The colocalization experiments shown above indicate that, when expressed together, gE and gI are localized to the same cellular compartment, but they do not prove that this compartment is indeed the TGN, the organelle where gE accumulates when expressed alone (10Zhu Z. Gershon M.D. Hao Y. Ambron R.T. Gabel C.A. Gershon A.A. J. Virol. 1995; 69: 7951-7959Crossref PubMed Google Scholar, 11Zhu Z. Hao Y. Gershon M.D. Ambron R.T. Gershon A.A. J. Virol. 1996; 70: 6563-6575Crossref PubMed Google Scholar, 12Alconada A. Bauer U. Hoflack B. EMBO J. 1996; 15: 6096-6110Crossref PubMed Scopus (136) Google Scholar). We have previously used the rapid tubularization in response to BFA as a hallmark of the TGN to distinguish it from other membrane-bound compartments clustered in the perinuclear region of the cell (12Alconada A. Bauer U. Hoflack B. EMBO J. 1996; 15: 6096-6110Crossref PubMed Scopus (136) Google Scholar). When HeLa cells expressing gE and gI were treated for 5 min with 10 μg/ml BFA, fixed and decorated with anti-gE and anti-gI antibodies, both molecules were found to colocalize in thin tubules that emanated from the perinuclear region into the cell periphery (Fig.2, a and b), strongly suggesting that the TGN is the compartment where gE and gI accumulate upon co-expression. Another property of certain TGN markers (gE, furin, and TGN38) is their ability to constantly cycle between the TGN and the cell surface (16Voorhoes P. Deignan E. van Donselaar E. Humphrey J. Marks M. Peters P.J. Bonifacino J.S. EMBO J. 1995; 14: 4961-4975Crossref PubMed Scopus (187) Google Scholar,18Bos K. Wraight C. Stanley K. EMBO J. 1993; 12: 2219-2228Crossref PubMed Scopus (193) Google Scholar, 31Molloy S.S. Thomas L. VanSlyke J.K. Stenberg P.E. Thomas G. EMBO J. 1994; 13: 18-33Crossref PubMed Scopus (420) Google Scholar). Since gI is found mainly in the TGN in the presence of gE, we wanted to investigate whether this also involves cycling of gI between these two compartments. To address this question, anti-gE and anti-gI antibody uptake experiments were performed on HeLa cells that had been double-transfected with gE and gI expression constructs. As shown in Fig. 2 c and in agreement with our previous findings (12Alconada A. Bauer U. Hoflack B. EMBO J. 1996; 15: 6096-6110Crossref PubMed Scopus (136) Google Scholar), after 1 h of incubation, the anti-gE antibodies were mainly concentrated in the perinuclear region of the cell, as a result of their internalization bound to the luminal domain of the recycling gE molecules. Interestingly, the anti-gI monoclonal antibody, when incubated with the cells for the same time, was also found in the perinuclear region, colocalizing with the anti-gE antibodies (Fig.2 d). In contrast, when the same experiment was performed on cells transfected exclusively with the pSFFV-gI construct, only cell surface bound anti-gI antibody could be detected (data not shown). These data indicate that gI, when simultaneously co-expressed with gE, cycles between the TGN and the cell surface, and that its accumulation in the TGN most likely depends on its rapid internalization from the cell surface together with gE, suggesting that the distribution of gI when co-expressed with gE is indistinguishable from that observed for gE when expressed alone. The results presented so far suggest that gE and gI are found within a complex in the cell whose traffic and distribution is solely determined by the sorting information in the cytoplasmic tail of gE. To verify this hypothesis, we constructed a modified version of gE in which its cytoplasmic tail had been replaced by that of the yeast protein Wbp1p, a type-I membrane protein that forms part of the ER resident oligosaccharyl-transferase complex (32Silberstein S. Gilmore R. FASEB J. 1996; 10: 849-858Crossref PubMed Scopus (207) Google Scholar). The tail of Wbp1p, which contains a consensus KKXX ER retention motif, has been shown to be sufficient to confer ER localization to reporter molecules both in mammalian and yeast cell systems (33Cosson P. Letorneur F. Science. 1994; 263: 1629-1631Crossref PubMed Scopus (483) Google Scholar, 34Letourneur F. Gaynor E.C. Demolliere C. Duden R. Emr S.D. Riezman H. Cosson P. Cell. 1994; 79: 1199-1207Abstract Full Text PDF PubMed Scopus (671) Google Scholar). When the gE-KKXX and the gI expression constructs were simultaneously transfected into HeLa cells and the localization of both molecules was assessed by indirect immunofluorescence, both the gE-KKXXchimera and gI were found in a cytoplasmic reticular compartment showing all the morphological features of the ER (Fig.3, a and b). As a control, we also constructed a gE-SSXX expression plasmid, in which the two lysines at positions −3 and −4 in the KKXX signal have been replaced by serines. This mutation is known to abolish the ER retention capacity of the KKXX motif (33Cosson P. Letorneur F. Science. 1994; 263: 1629-1631Crossref PubMed Scopus (483) Google Scholar). As expected, in cells co-expressing the gE-SSXX mutant and gI, both molecules were only detected at the cell surface (Fig. 3,c and d). These result confirms our prediction that the intracellular traffic of gI is exclusively determined by the sorting information on the tail of gE, presumably as a reflect of their association in the early secretory pathway. We then asked whether the expression of one given protein could influence the maturation of the other. In order to address this question, gE and gI were expressed either alone or simultaneously in HeLa cells with the help of a T7 RNA-polymerase recombinant vaccinia virus. The cells were metabolically labeled with radioactive methionine, chased for increasing periods of time, and lysed, and the lysates were immunoprecipitated with anti-gE- and anti-gI-specific antibodies. To identify the precursor and mature forms of gE and gI, both molecules were immunoprecipitated from cells lysates that were obtained either immediately after the labeling period or after 6 h of chase. The results showed that gE was initially synthesized as a 70-kDa band that was converted during the chase to a 100-kDa polypeptide, and gI was initially found as a 50-kDa band that matured to yield a fuzzy 65-kDa band (Fig.4 a). These values are in agreement with those found by other groups, either in transfected (21Yao Z. Jackson W. Forghani B. Grose C. J. Virol. 1993; 67: 305-314Crossref PubMed Google Scholar,35Litwin V. Jackson W. Grose C. J. Virol. 1992; 66: 3643-3651Crossref PubMed Google Scholar) or in VZV-infected cells (36Montalvo E.A. Parmley R.T. Grose C. J. Virol. 1985; 53: 761-770Crossref PubMed Google Scholar), for both the precursor and mature forms of gE and gI. When gE was expressed alone, immunoprecipitation with anti-gE antibodies revealed that maturation of the protein occurred rather rapidly, since as early as 20 min after initiation of the chase, almost 50% of the labeled 70-kDa precursor molecule was converted to the mature 100-kDa form (Fig. 4, b andc). When gI was expressed alone and immunoprecipitated with anti-gI antibodies under analogous conditions, its processing occurred very slowly, requiring more than 1 h to convert only 20% of the precursor to the mature form (Fig. 4, b and c). However, when gE and gI were expressed together, processing of gI was considerably enhanced, because 50% of the mature form could be detected after only 40 min of chase (Fig. 4, b andc). Under the same conditions, no difference was observed in the maturation of gE, when compared with the results obtained when this protein was expressed alone (Fig. 4 b). In addition, the anti-gE- and anti-gI-specific antibodies failed to immunoprecipitate any gI and gE, respectively (data not shown). It is worth mentioning that, when both proteins were expressed together, processing of gI occurred almost with identical kinetics as the processing of gE (Fig. 4 c). However, under these conditions, we reproducibly observed a decrease in the amounts of gE and gI that could be immunoprecipitated with their cognate antibodies when compared with the single transfections (Fig. 4 b). Since the expression l" @default.
• W2049135087 created "2016-06-24" @default.
• W2049135087 creator A5058353648 @default.
• W2049135087 creator A5070731126 @default.
• W2049135087 creator A5073574070 @default.
• W2049135087 creator A5088081229 @default.
• W2049135087 creator A5089619185 @default.
• W2049135087 date "1998-05-01" @default.
• W2049135087 modified "2023-09-29" @default.
• W2049135087 title "Intracellular Transport of the Glycoproteins gE and gI of the Varicella-Zoster Virus" @default.
• W2049135087 cites W1275105234 @default.
• W2049135087 cites W1479874716 @default.
• W2049135087 cites W1483463937 @default.
• W2049135087 cites W1543864720 @default.
• W2049135087 cites W1548736406 @default.
• W2049135087 cites W1549377959 @default.
• W2049135087 cites W1557241876 @default.
• W2049135087 cites W1559415385 @default.
• W2049135087 cites W1560841381 @default.
• W2049135087 cites W1569729229 @default.
• W2049135087 cites W1577494405 @default.
• W2049135087 cites W1596215911 @default.
• W2049135087 cites W1599322649 @default.
• W2049135087 cites W1645986795 @default.
• W2049135087 cites W1704822668 @default.
• W2049135087 cites W1738645663 @default.
• W2049135087 cites W1754262074 @default.
• W2049135087 cites W1765288636 @default.
• W2049135087 cites W1841985584 @default.
• W2049135087 cites W1846954693 @default.
• W2049135087 cites W1898273578 @default.
• W2049135087 cites W1933680278 @default.
• W2049135087 cites W1937327614 @default.
• W2049135087 cites W1959236722 @default.
• W2049135087 cites W1973993324 @default.
• W2049135087 cites W1983956190 @default.
• W2049135087 cites W1989586615 @default.
• W2049135087 cites W2005768267 @default.
• W2049135087 cites W2009210302 @default.
• W2049135087 cites W2019424072 @default.
• W2049135087 cites W2028398918 @default.
• W2049135087 cites W2030758635 @default.
• W2049135087 cites W2033432694 @default.
• W2049135087 cites W2049652225 @default.
• W2049135087 cites W2054593368 @default.
• W2049135087 cites W2059428912 @default.
• W2049135087 cites W2071427211 @default.
• W2049135087 cites W2081143076 @default.
• W2049135087 cites W2082545558 @default.
• W2049135087 cites W2099382752 @default.
• W2049135087 cites W2102948276 @default.
• W2049135087 cites W2105961777 @default.
• W2049135087 cites W2108178640 @default.
• W2049135087 cites W2111651943 @default.
• W2049135087 cites W2127043145 @default.
• W2049135087 cites W2132023705 @default.
• W2049135087 cites W2132985208 @default.
• W2049135087 cites W2137739893 @default.
• W2049135087 cites W2139166154 @default.
• W2049135087 cites W2139929655 @default.
• W2049135087 cites W2139930780 @default.
• W2049135087 cites W2140596567 @default.
• W2049135087 cites W2142524238 @default.
• W2049135087 cites W2145717207 @default.
• W2049135087 cites W2146108681 @default.
• W2049135087 cites W2149227633 @default.
• W2049135087 cites W2153936367 @default.
• W2049135087 cites W2156123399 @default.
• W2049135087 cites W2166714339 @default.
• W2049135087 cites W2169191388 @default.
• W2049135087 cites W2184457366 @default.
• W2049135087 cites W2337596941 @default.
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