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Self-association of truncated forms of HIV-1 gp120
Virus Research 49 (1997) 163 – 172
Self-association of truncated forms of HIV-1 gp120 Etienne Malvoisin a,*, Marie Paule Kie´ny b, Fabian Wild a a
INSERM Unit 404, Immunity and Vaccination, Institut Pasteur de Lyon, A6enue Tony Garnier, 69365 Lyon Cedex 07, France b Transge`ne, 11 rue de Molsheim, 67082 Strasbourg Cedex, France Received 27 November 1996; accepted 14 February 1997
Abstract HIV-1 gp120 and truncated forms were expressed in HeLa T4 cells by vaccinia recombinant viruses. The truncated gp120 molecules consisted of N-terminal overlapping envelope proteins of 204, 287 and 393 amino acids respectively. Immunoprecipitation with specific monoclonal antibodies and SDS – PAGE analyses of HIV-1 gp120 revealed bands corresponding to low amounts of secreted and cell-bound stable dimers. In contrast, the truncated forms of gp120 expressed larger amounts of SDS–stable putative dimers and the amounts observed were inversely proportional to their size. The shortest gp120 mutant (204 aa) was found to be secreted almost exclusively as a dimer. The processing of gp120 and its truncated forms was further investigated in the presence of inhibitors of N-glycosylation. Monomers and dimers migrated on gels with the same relative changes, confirming that the protein with the higher molecular weight is a multimer of the smaller one. The putative dimeric form of the truncated gp120s could be stabilized by chemical cross-linking. Finally, the possible existence of an association domain in the N-terminal 204 amino acids (aa) of gp120 is discussed. © 1997 Elsevier Science B.V. Keywords: Envelope protein; N-glycosulation; Truncated forms of HIV-1 gp120
1. Introduction The initial step in HIV-1 infection is the interaction of the viral gp120 with its cellular receptor, CD4. The virion glycoprotein oligomeric form is most probably a tetramer when associated with the transmembrane glycoprotein gp41 (Schawaller
* Corresponding author. Tel: +33 472722553; fax: + 33 472722567; e-mail: [email protected]
et al., 1989; Earl et al., 1990). However, a trimeric structure has also been proposed (Weiss et al., 1990). The domains responsible for oligomerization are highly conserved among HIV isolates since heterooligomers are formed between HIV-1 and HIV-2 or SIV envelope proteins (Doms et al., 1990). The transmembrane gp41 can oligomerize independently of gp120 (Pinter et al., 1989) and the N-terminal domain of gp41 (residues 550 to 561) has been implicated in this process (Poumbourios et al., 1995).
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The HIV-1 gp120 dissociated from gp41 is generaly considered as a monomer. However, its monomeric status may be due to its instability during ultracentrifugation or SDS – PAGE analyses. Nevertherless, analysis of gp120 by sucrose density gradient centrifugation and chemical cross-linking suggest that gp120 can exist as a dimer (Owens and Compans, 1990). Studies with the F-MuLV retrovirus indicate that its extracellular glycoprotein remains in an oligomeric form following dissociation from the transmembrane subunit (Tucker et al., 1991). Gp120 by itself, independently of its association with gp41 has many biological properties. For example, gp120 has been demonstrated to be cytotoxic for neurons and has been proposed to be responsible for neuronal loss observed in the brain of AIDS patients (Toggas et al., 1994). HIV gp120 also has the capacity to interact with complement proteins and to enhance the production of cytokines, adhesion molecules and nitric oxide in many cell types (Clouse et al., 1991; Pietraforte et al., 1994; Su¨sal et al., 1994; Shrikant et al., 1996). Often, the biological properties of molecules depend of their quaternary structure. Truncated forms of gp120 may be potential HIV vaccines (Jeffs et al., 1996). When used as an immunogen, the epitopes exposed on the surface of molecules may be conformational when associated as a complex and thus be different to those exposed at the surface of monomers. In addition, the truncated forms of gp120 might be folded in an abnormal way relative to the wild-type protein. For that reason, we have examined the capacity of truncated forms of gp120 to associate with each other.
2. Results The vaccinia recombinant viruses expressing HIV-1 envelope protein used in this study are described in Fig. 1. HeLa T4 cells were infected with VVTG1132, a recombinant vaccinia virus which encodes HIV-1 LAI gp120. Cells were metabolically labelled for 5 h with TRAN 35S (70% [35S]methionine, 15% [35S]cysteine, ICN). Aliquots of the culture medium containing 35Sgp1201132, were immunoprecipitated with MAbs
ICR38.1a (ADP388) or GP68 (ADP3055) which are both specific for the CD4-binding region of gp120 (Fig. 2A) (McKeating et al., 1992; Schutten et al., 1993). In parallel we analyzed 35S-gp1605156 and 35S-gp1608140 which are soluble and non cleavable forms of gp160 expressed by the recombinant vaccinia viruses VVTG5156 and VVTG8140 respectively. Similar constructs expressing soluble and non cleavable forms of gp160 have been shown to form oligomers (Malvoisin and Wild, 1994). In non reducing conditions, the VVTG1132 expressed gp (lanes 1 and 4) gave a minor high Mr band of \ 200. This may correspond to a dimeric form of gp120, since it is not detected in the reduced sample (0.5% 2-mercaptoethanol, final concentration). The gp120 only
Fig. 1. HIV-1 env proteins expressed by recombinant vaccinia viruses. (A) Full-length and carboxy terminal deletion mutants of HIV-1 env protein (clone BH8 of HIV-1 IIIB) (Earl et al., 1991a). The epitopes recognized by the MAbs 4ATC6, 110H(NII) and ICR38.1a are indicated. (B) Soluble chimeric envelope glycoproteins containing gp41LAI and gp120MN or gp120CAR4071. In each case the transmembrane region was removed, the cleavage sites mutated and the intracytoplasmic domain of gp41 truncated at aa 29 (Kieny et al., 1988). The following nomenclature has been used: VVTG5156: gp120MN/gp41LAl (gp1605156). VVTG8140: gp120CAR4071 /gp41LAI (gp1608140). VVTG1132 expressing gp120LAI.
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Fig. 2. Expression of gp120 and C-terminally truncated forms. (A) Hela T4 cells were infected with VVTG1132, VVTG5156 or VVTG8140 and metabolically labelled with 35S-methionine/cysteine (Tran35S). A fraction of the culture medium containing 35 S-gp1201132 (lanes 1, 4), 35S-gp1605156 (lanes 2, 5) or 35S-gp1608140 (lanes 3, 6) was immunoprecipitated with MAb ICR38.1 a (lanes 1– 3) or MAb GP68 (lanes 4–6). The complexes were analyzed under non-reducing (left panels) and reducing (right panel) conditions. (B) Hela T4 cells were infected with vPE20 (lanes 1 and 4), vPE21 (lanes 2 and 5) or vPE22 (lanes 3 and 6) and metabolically labelled with Tran 35S. The culture media containing secreted proteins (left panels) and the cell extracts (right panels) were immunoprecipitated with MAb 110H(NII) (anti-gp120/V3 loop) (lanes 1 – 3) or with MAb 4A7C6 (anti-gp120/N-terminus) (lanes 4 – 6). The complexes were analyzed under non-reducing conditions. The right hand panels show the same autoradiographs after 70 and 35 h exposure respectively. The position of cellular or secreted dimeric vPE22-expressed proteins are indicated by an arrow. In A and B, the cells were infected with the vaccinia virus recombinant (5 PFU/cell) and 18 h later incubated in a medium lacking methionine and cysteine but containing 50 mCi/ml of Tran 35S for a further 5 h. The proteins were separated on a 7% SDS – PAGE. The lane labelled M contains molecular weight markers.
expressed a low amount of SDS – stable dimer, whereas gp1605156 and gp1608140 expressed higher proportions. In an attempt to localize a putative association domain in gp120, we infected HeLa T4 cells with recombinant vaccinia vectors ex-
pressing C-terminally truncated gp120 molecules namely vPE20, vPE21 and vPE22 which code for N-terminal overlapping envelope proteins of 204, 287 and 393 amino acids respectively (Earl et al., 1991a). Infected cells were metabolically labelled
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with TRAN 35S and the secreted and cell-associated proteins were immunoprecipitated with MAbs 110H (NII — anti-V3 loop) (a gift of Dr F. Traincard, Hybridolab, Institut Pasteur, Paris) or 4A7C6 (ADP360 — anti-N terminus). As shown in Fig. 2B, vPE20, vPE21 and vPE22 expressed proteins which may correspond to dimeric forms of secreted and cell-associated proteins. These bands migrated as monomeric forms after reduction with ME (2-mercaptoethanol) (not shown). The protein secreted from vPE22-infected cells existed almost exclusively as a putative dimer (indicated by an arrow in Fig. 2B, non-reducing conditions), whereas the protein secreted from vPE20-infected cells was present as monomer (predominant) and dimer, suggesting that the shortest gp120 mutant is more stable. A slow migrating minor band from vPE22 infected cells may represent a trimer or tetramer. MAb 110H(NII) did not recognize the proteins synthesized by vPE21 or vPE22 since these constructions produce glycoproteins lacking the V3 loop. Inhibition of glycan processing causes a modification in the Mr of the glycoproteins. Gp120 is heavily N-glycosylated and will thus be modified by the presence of inhibitors of oligosaccharide processing. Gp120 synthesis was studied in cells infected with vPE17 or VVTG1132 in the presence or absence of N-butyldeoxynojirimycin (NBDNJ), an inhibitor of ER glucosidase I (SC-48334, a gift of R.A. Marks, Searle) (Karlsson et al., 1993). Ninety min prior to labelling, the infected cells were treated with NBDNJ and incubated for a further 5 h. The proteins were immunoprecipitated from cell lysates and/or culture medium with MAb I 11, 2 (Pasquali et al., 1990). The vPE20, vPE21 or vPE22 proteins synthesized in the presence of NB-DNJ had a decreased electrophoretic mobility (Fig. 3A). Monomeric and oligomeric forms were affected similarly. The mobility of both cell-associated and secreted gp120 was decreased with NB-DNJ (Fig. 3B). The proteins migrated as monomers after reduction with ME (Fig. 3A and Fig. 3B). Other inhibitors of oligosaccharide trimming enzymes were studied: 1-deoxynojirimycin (DNJ) and castanospermine (CSP), ER glucosi-
dases I and II inhibitors, 1-deoxymannojirimycin (DMJ), Golgi mannosidase I inhibitor, swainsonine (SW), Golgi mannosidase II inhibitor. The proteins were immunoprecipitated from cell lysates (Fig. 4A) and culture media (Fig. 4B) with MAb I 11, 2. In cells treated with ER glucosidase inhibitors, CSP or DNJ, the resulting proteins displayed slower electrophoretic mobilities in SDS-PAGE than the controls. In contrast, treatment with SW did not affect the apparent molecular mass of the proteins. The different glycosylation inhibitors modified the oligosaccharide processing of the monomeric and higher molecular mass forms of gp120 and truncated variants to the same degree. Under reducing conditions all gp120 related products migrated as monomers. These observations suggest that the higher-molecular weight bands may represent dimers of the envelope protein. In vPE21-infected cells, the glycosylation inhibitors affected to the same extent the electrophoretic mobility of monomer, dimer and the protein with the highest Mr suggesting that this band may represent a larger form. To further investigate the association of truncated forms of gp120, HeLa T4 cells were infected with the different VV recombinants and metabolically labelled with TRAN 35S. Culture media and/ or cell lysates were treated for 30 min at room temperature with increasing concentrations of the non reversible cross-linking agent EGS. After quenching the cross-linking reaction, the proteins were immunoprecipitated with MAb I 11, 2 and analyzed by SDS–PAGE in non-reducing and reducing conditions. As shown in Fig. 5A, high Mr bands are resistant to reducing conditions indicating that high Mr structures were stabilized by the cross-linking. The apparent molecular weights of the predominantly cross-linked species suggest that they represent dimers. In vPE21-infected cells, the higher-molecular weight species stabilized by chemical cross-linking were in the order of trimeric or tetrameric forms. Using different types of crosslinking agents we have failed to stabilize the putative dimeric gp120 expressed by VVTG1132 (not shown). As an alternative, we studied the oligomeric nature of the envelope protein of HIV-2 ROD. Cells were infected with
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Fig. 3. Effects of N-butyldeoxynojirimycin (NB-DNJ) on the processing of gp120 and C-terminally truncated forms expressed by recombinant vaccinia vectors in HeLa T4 cells. (A) Cells infected with vPE17 (lanes 1, 2), vPE20 (lanes 3, 4), vPE21 (lanes 5, 6) or vPE22 (lanes 7, 8) were incubated in the presence (lanes 2, 4, 6, 8) or in the absence (lanes 1, 3, 5, 7) of NB-DNJ. 35S-gp120 and truncated forms were immunoprecipitated from cell Iysates with MAb I 11, 2. (B) Cells infected with VVTG1132 in the presence (lanes 2, 4) or absence (lanes 1, 3) of NB-DNJ. Culture media and cell Iysates were immunoprecipitated with MAb 110H(NII). In Fig. 3A and Fig. 3B, the cells were infected with the vaccinia virus recombinant (5 PFU/cell) and 18 h later incubated in medium lacking methionine and cysteine but containing 50 mCi/ml of Tran 35S for a further 5 h. The inhibitor (1 mg/ml) was added 90 min before the labelling. The proteins were separated on a 7% SDS – PAGE under reducing and non reducing conditions. The lane labelled M contains molecular weight markers.
recombinant vaccinia virus VVTG2180 which express the HIV-2 ROD envelope protein. After labelling with TRAN 35S, culture medium containing secreted 35S-gp1202180 was incubated with different concentrations of EGS and immunoprecipitated with an HIV-2 positive human serum (gift of Dr J. Andre´, Institut Pasteur de Lyon) and analyzed by SDS-PAGE (Fig. 5B). EGS stabilized a dimeric form of gp120HIV-2 ROD. Increasing concentrations of EGS were accompanied by a concomitant decrease in the migration rate. It has been shown previously that cross-linked proteins have modified migration rates (Doms, 1990; Malvoisin and Wild, 1993). It
is possible that the failure to stabilize dimeric full-length gp120HIV-1 is due to the fact that the amino groups of lysine residues present on two adjacent chains are not accessible to the crosslinking reagent.
3. Discussion The amino-terminal region of gp120 contains an hydrophobic domain which is hyperconserved among the HIV-1 isolates and highly conserved among the primate immunodeficiency viruses (Helseth et al., 1991; Ivey-Hoyle et al., 1991).
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Fig. 4. Effects of inhibitors of N-linked glycosylation on the processing of gp120 and C-terminally truncated forms expressed by recombinant vaccinia vectors in HeLa T4 cells. The cells were infected with the indicated vaccinia virus recombinant, labelled with Tran 35S and incubated in the presence or in the absence of the inhibitor of N-linked glycosylation as described in The inhibitors used were 1-deoxynojirimycin (DNJ) (400 mg/ml), swainsonine (SW) (150 mg/ml), castanospermine (CSP) (400 mg/ml) or 1-deoxymannojirimycin (DMJ) (400 mg/ml). A parallel control culture (con) was radiolabelled in the absence of glycosylation inhibitor. The proteins were immunoprecipitated with MAb I 11, 2. In A, cellular, in B cellular and secreted proteins were analyzed under reducing and non reducing conditions.
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Fig. 5. Cross-linking of C-terminally truncated forms of gp120 and cross-linking of gp120HIV-2 ROD expressed by recombinant vaccinia vectors in HeLa T4 cells. (A) 35S-truncated forms of gp120 were treated with different concentrations of EGS (Ethylene glycolbis-succinimidylsuccinate): Lane 1, control; lane 2, 1.5 mM; lane 3, 0.5 mM; lane 4, 4.5 mM EGS. Proteins were immunoprecipitated with MAb I 11, 2 and separated on a 7% SDS – PAGE under reducing and non reducing conditions. (B) 35 S-gp120HIV-2 ROD was treated with different concentrations of EGS: Lane 1, control; lane 2, 3 mM; lane 3, 1 mM; lane 4, 0.34 mM EGS. Proteins were immunoprecipitated with an HIV-2 positive human serum and separated on a 7% SDS – PAGE under reducing and non reducing conditions. The position of dimeric (2×) gp120HIV-2 ROD is indicated.
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High conservation of sequences between virus isolates often reveals functional regions. This hydrophobic domain may play a role in the association of gp120 molecules. Deletion of the NH2-terminus of gp120 HIV-1 has been shown to affect the folding of the truncated molecules (Jeffs et al., 1996). Owens and Compans (1990), using recombinant vaccinia vectors for expression, demonstrated that the HIV-1 gp160 precursors which acquire aberrant intermolecular disulfide bonds are not proteolytically processed. In our study, both the secreted and cell-associated proteins were processed, even in the presence of inhibitors of oligosaccharide processing. Only properly folded and assembled proteins are transported to the Golgi complex. Therefore, it is unlikely that the dimeric forms we observe are aggregates of erroneously folded products but represent SDS– stable dimers. If aggregates were present, they would not enter the gel in non reducing conditions and would require a high concentration of ME to be reduced (in this study we used 0.5% ME, final concentration). In addition, during our preliminary experiments, we added to the cell lysate and the supernatant 10 mM iodoacetamide to prevent eventual disulfide bond formation. However, this was not necessary since iodoacetamide-treated and untreated samples gave the same protein patterns (not shown). The truncated forms of gp120 could be correctly folded in a way that prevents them from being retained in the reticulum endosplamic, but their assembly and transport may be different by comparison with the wild-type protein. The presence of SDS – stable dimers which migrate as monomers in reducing conditions is not necessarily indicative of the presence of interchain disulfide bonds: the existence of SDS–stable dimers is a well known phenomenon and, when SDS – resistance is observed, there is a high probability that the interaction represents a biologically relevant structure (Gething et al., 1989; Doms, 1990). Their destabilization in reducing conditions is problably due to the rupture of the intrachain disulfide bonds. The smallest protein which contains only one con-
served region of gp120 (C1) shows the highest proportion of dimers. This may be an artefact of deleting increasing lengths of the C-terminus or, on the contrary, reflect the presence of an association domain in that region. Gp120 contains intrachain disulfide bonds (Leonard et al., 1990) and there are also intrachain interactions between the V1/V2 and C4 domains (Freed and Martin, 1994), and between C1, C2 and C5 (Moore et al., 1994). In the shortest gp120 truncated form, the affinity may be increased because the protein is free of the constraints imposed by the other domains. The precursor gp160 oligomerizes in the ER and the cleavage is a late event which occurs after exit from the trans-Golgi network (Earl et al., 1991b; Merkle et al., 1991; Kantanen et al., 1995). After dissociation from gp41, a change in the folding of gp120 may occur and the folding of truncated forms of gp120 may be more similar to their structure in the full-length gp160 molecule. Finally our results suggest that gp120 can associate with itself even after dissociation from the gp41 subunit. We do not known if the interactions observed with truncated forms of gp120 are relevant to the full-length molecule. Further confirmation is needed, especially by deleting the region present in the N-terminus of gp120 which are thought to play a role in the association. The presence of an association domain in gp120 may modify our approach in the design of derived-gp120 based subunit vaccine.
Acknowledgements We thank Dr J.F. Pasquali, Dr F. Traincard and Dr H.C. Holmes of the ADP reagent programme for MAbs and reagents. We would also like to thank Drs B. Moss and P. Earl for providing recombinant vaccinia viruses (vPE17, vPE22, vPE21, vPE20), R.A. Marks for the gift of NB-DNJ and Bernadette Maret for editorial assistance. These studies were supported by a grant from SIDACTION and Etienne Malvoisin held a SIDACTION fellowship.
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Self-association of truncated forms of HIV-1 gp120 Etienne Malvoisin a,*, Marie Paule Kie´ny b, Fabian Wild a a
INSERM Unit 404, Immunity and Vaccination, Institut Pasteur de Lyon, A6enue Tony Garnier, 69365 Lyon Cedex 07, France b Transge`ne, 11 rue de Molsheim, 67082 Strasbourg Cedex, France Received 27 November 1996; accepted 14 February 1997
Abstract HIV-1 gp120 and truncated forms were expressed in HeLa T4 cells by vaccinia recombinant viruses. The truncated gp120 molecules consisted of N-terminal overlapping envelope proteins of 204, 287 and 393 amino acids respectively. Immunoprecipitation with specific monoclonal antibodies and SDS – PAGE analyses of HIV-1 gp120 revealed bands corresponding to low amounts of secreted and cell-bound stable dimers. In contrast, the truncated forms of gp120 expressed larger amounts of SDS–stable putative dimers and the amounts observed were inversely proportional to their size. The shortest gp120 mutant (204 aa) was found to be secreted almost exclusively as a dimer. The processing of gp120 and its truncated forms was further investigated in the presence of inhibitors of N-glycosylation. Monomers and dimers migrated on gels with the same relative changes, confirming that the protein with the higher molecular weight is a multimer of the smaller one. The putative dimeric form of the truncated gp120s could be stabilized by chemical cross-linking. Finally, the possible existence of an association domain in the N-terminal 204 amino acids (aa) of gp120 is discussed. © 1997 Elsevier Science B.V. Keywords: Envelope protein; N-glycosulation; Truncated forms of HIV-1 gp120
1. Introduction The initial step in HIV-1 infection is the interaction of the viral gp120 with its cellular receptor, CD4. The virion glycoprotein oligomeric form is most probably a tetramer when associated with the transmembrane glycoprotein gp41 (Schawaller
* Corresponding author. Tel: +33 472722553; fax: + 33 472722567; e-mail: [email protected]
et al., 1989; Earl et al., 1990). However, a trimeric structure has also been proposed (Weiss et al., 1990). The domains responsible for oligomerization are highly conserved among HIV isolates since heterooligomers are formed between HIV-1 and HIV-2 or SIV envelope proteins (Doms et al., 1990). The transmembrane gp41 can oligomerize independently of gp120 (Pinter et al., 1989) and the N-terminal domain of gp41 (residues 550 to 561) has been implicated in this process (Poumbourios et al., 1995).
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The HIV-1 gp120 dissociated from gp41 is generaly considered as a monomer. However, its monomeric status may be due to its instability during ultracentrifugation or SDS – PAGE analyses. Nevertherless, analysis of gp120 by sucrose density gradient centrifugation and chemical cross-linking suggest that gp120 can exist as a dimer (Owens and Compans, 1990). Studies with the F-MuLV retrovirus indicate that its extracellular glycoprotein remains in an oligomeric form following dissociation from the transmembrane subunit (Tucker et al., 1991). Gp120 by itself, independently of its association with gp41 has many biological properties. For example, gp120 has been demonstrated to be cytotoxic for neurons and has been proposed to be responsible for neuronal loss observed in the brain of AIDS patients (Toggas et al., 1994). HIV gp120 also has the capacity to interact with complement proteins and to enhance the production of cytokines, adhesion molecules and nitric oxide in many cell types (Clouse et al., 1991; Pietraforte et al., 1994; Su¨sal et al., 1994; Shrikant et al., 1996). Often, the biological properties of molecules depend of their quaternary structure. Truncated forms of gp120 may be potential HIV vaccines (Jeffs et al., 1996). When used as an immunogen, the epitopes exposed on the surface of molecules may be conformational when associated as a complex and thus be different to those exposed at the surface of monomers. In addition, the truncated forms of gp120 might be folded in an abnormal way relative to the wild-type protein. For that reason, we have examined the capacity of truncated forms of gp120 to associate with each other.
2. Results The vaccinia recombinant viruses expressing HIV-1 envelope protein used in this study are described in Fig. 1. HeLa T4 cells were infected with VVTG1132, a recombinant vaccinia virus which encodes HIV-1 LAI gp120. Cells were metabolically labelled for 5 h with TRAN 35S (70% [35S]methionine, 15% [35S]cysteine, ICN). Aliquots of the culture medium containing 35Sgp1201132, were immunoprecipitated with MAbs
ICR38.1a (ADP388) or GP68 (ADP3055) which are both specific for the CD4-binding region of gp120 (Fig. 2A) (McKeating et al., 1992; Schutten et al., 1993). In parallel we analyzed 35S-gp1605156 and 35S-gp1608140 which are soluble and non cleavable forms of gp160 expressed by the recombinant vaccinia viruses VVTG5156 and VVTG8140 respectively. Similar constructs expressing soluble and non cleavable forms of gp160 have been shown to form oligomers (Malvoisin and Wild, 1994). In non reducing conditions, the VVTG1132 expressed gp (lanes 1 and 4) gave a minor high Mr band of \ 200. This may correspond to a dimeric form of gp120, since it is not detected in the reduced sample (0.5% 2-mercaptoethanol, final concentration). The gp120 only
Fig. 1. HIV-1 env proteins expressed by recombinant vaccinia viruses. (A) Full-length and carboxy terminal deletion mutants of HIV-1 env protein (clone BH8 of HIV-1 IIIB) (Earl et al., 1991a). The epitopes recognized by the MAbs 4ATC6, 110H(NII) and ICR38.1a are indicated. (B) Soluble chimeric envelope glycoproteins containing gp41LAI and gp120MN or gp120CAR4071. In each case the transmembrane region was removed, the cleavage sites mutated and the intracytoplasmic domain of gp41 truncated at aa 29 (Kieny et al., 1988). The following nomenclature has been used: VVTG5156: gp120MN/gp41LAl (gp1605156). VVTG8140: gp120CAR4071 /gp41LAI (gp1608140). VVTG1132 expressing gp120LAI.
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Fig. 2. Expression of gp120 and C-terminally truncated forms. (A) Hela T4 cells were infected with VVTG1132, VVTG5156 or VVTG8140 and metabolically labelled with 35S-methionine/cysteine (Tran35S). A fraction of the culture medium containing 35 S-gp1201132 (lanes 1, 4), 35S-gp1605156 (lanes 2, 5) or 35S-gp1608140 (lanes 3, 6) was immunoprecipitated with MAb ICR38.1 a (lanes 1– 3) or MAb GP68 (lanes 4–6). The complexes were analyzed under non-reducing (left panels) and reducing (right panel) conditions. (B) Hela T4 cells were infected with vPE20 (lanes 1 and 4), vPE21 (lanes 2 and 5) or vPE22 (lanes 3 and 6) and metabolically labelled with Tran 35S. The culture media containing secreted proteins (left panels) and the cell extracts (right panels) were immunoprecipitated with MAb 110H(NII) (anti-gp120/V3 loop) (lanes 1 – 3) or with MAb 4A7C6 (anti-gp120/N-terminus) (lanes 4 – 6). The complexes were analyzed under non-reducing conditions. The right hand panels show the same autoradiographs after 70 and 35 h exposure respectively. The position of cellular or secreted dimeric vPE22-expressed proteins are indicated by an arrow. In A and B, the cells were infected with the vaccinia virus recombinant (5 PFU/cell) and 18 h later incubated in a medium lacking methionine and cysteine but containing 50 mCi/ml of Tran 35S for a further 5 h. The proteins were separated on a 7% SDS – PAGE. The lane labelled M contains molecular weight markers.
expressed a low amount of SDS – stable dimer, whereas gp1605156 and gp1608140 expressed higher proportions. In an attempt to localize a putative association domain in gp120, we infected HeLa T4 cells with recombinant vaccinia vectors ex-
pressing C-terminally truncated gp120 molecules namely vPE20, vPE21 and vPE22 which code for N-terminal overlapping envelope proteins of 204, 287 and 393 amino acids respectively (Earl et al., 1991a). Infected cells were metabolically labelled
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with TRAN 35S and the secreted and cell-associated proteins were immunoprecipitated with MAbs 110H (NII — anti-V3 loop) (a gift of Dr F. Traincard, Hybridolab, Institut Pasteur, Paris) or 4A7C6 (ADP360 — anti-N terminus). As shown in Fig. 2B, vPE20, vPE21 and vPE22 expressed proteins which may correspond to dimeric forms of secreted and cell-associated proteins. These bands migrated as monomeric forms after reduction with ME (2-mercaptoethanol) (not shown). The protein secreted from vPE22-infected cells existed almost exclusively as a putative dimer (indicated by an arrow in Fig. 2B, non-reducing conditions), whereas the protein secreted from vPE20-infected cells was present as monomer (predominant) and dimer, suggesting that the shortest gp120 mutant is more stable. A slow migrating minor band from vPE22 infected cells may represent a trimer or tetramer. MAb 110H(NII) did not recognize the proteins synthesized by vPE21 or vPE22 since these constructions produce glycoproteins lacking the V3 loop. Inhibition of glycan processing causes a modification in the Mr of the glycoproteins. Gp120 is heavily N-glycosylated and will thus be modified by the presence of inhibitors of oligosaccharide processing. Gp120 synthesis was studied in cells infected with vPE17 or VVTG1132 in the presence or absence of N-butyldeoxynojirimycin (NBDNJ), an inhibitor of ER glucosidase I (SC-48334, a gift of R.A. Marks, Searle) (Karlsson et al., 1993). Ninety min prior to labelling, the infected cells were treated with NBDNJ and incubated for a further 5 h. The proteins were immunoprecipitated from cell lysates and/or culture medium with MAb I 11, 2 (Pasquali et al., 1990). The vPE20, vPE21 or vPE22 proteins synthesized in the presence of NB-DNJ had a decreased electrophoretic mobility (Fig. 3A). Monomeric and oligomeric forms were affected similarly. The mobility of both cell-associated and secreted gp120 was decreased with NB-DNJ (Fig. 3B). The proteins migrated as monomers after reduction with ME (Fig. 3A and Fig. 3B). Other inhibitors of oligosaccharide trimming enzymes were studied: 1-deoxynojirimycin (DNJ) and castanospermine (CSP), ER glucosi-
dases I and II inhibitors, 1-deoxymannojirimycin (DMJ), Golgi mannosidase I inhibitor, swainsonine (SW), Golgi mannosidase II inhibitor. The proteins were immunoprecipitated from cell lysates (Fig. 4A) and culture media (Fig. 4B) with MAb I 11, 2. In cells treated with ER glucosidase inhibitors, CSP or DNJ, the resulting proteins displayed slower electrophoretic mobilities in SDS-PAGE than the controls. In contrast, treatment with SW did not affect the apparent molecular mass of the proteins. The different glycosylation inhibitors modified the oligosaccharide processing of the monomeric and higher molecular mass forms of gp120 and truncated variants to the same degree. Under reducing conditions all gp120 related products migrated as monomers. These observations suggest that the higher-molecular weight bands may represent dimers of the envelope protein. In vPE21-infected cells, the glycosylation inhibitors affected to the same extent the electrophoretic mobility of monomer, dimer and the protein with the highest Mr suggesting that this band may represent a larger form. To further investigate the association of truncated forms of gp120, HeLa T4 cells were infected with the different VV recombinants and metabolically labelled with TRAN 35S. Culture media and/ or cell lysates were treated for 30 min at room temperature with increasing concentrations of the non reversible cross-linking agent EGS. After quenching the cross-linking reaction, the proteins were immunoprecipitated with MAb I 11, 2 and analyzed by SDS–PAGE in non-reducing and reducing conditions. As shown in Fig. 5A, high Mr bands are resistant to reducing conditions indicating that high Mr structures were stabilized by the cross-linking. The apparent molecular weights of the predominantly cross-linked species suggest that they represent dimers. In vPE21-infected cells, the higher-molecular weight species stabilized by chemical cross-linking were in the order of trimeric or tetrameric forms. Using different types of crosslinking agents we have failed to stabilize the putative dimeric gp120 expressed by VVTG1132 (not shown). As an alternative, we studied the oligomeric nature of the envelope protein of HIV-2 ROD. Cells were infected with
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Fig. 3. Effects of N-butyldeoxynojirimycin (NB-DNJ) on the processing of gp120 and C-terminally truncated forms expressed by recombinant vaccinia vectors in HeLa T4 cells. (A) Cells infected with vPE17 (lanes 1, 2), vPE20 (lanes 3, 4), vPE21 (lanes 5, 6) or vPE22 (lanes 7, 8) were incubated in the presence (lanes 2, 4, 6, 8) or in the absence (lanes 1, 3, 5, 7) of NB-DNJ. 35S-gp120 and truncated forms were immunoprecipitated from cell Iysates with MAb I 11, 2. (B) Cells infected with VVTG1132 in the presence (lanes 2, 4) or absence (lanes 1, 3) of NB-DNJ. Culture media and cell Iysates were immunoprecipitated with MAb 110H(NII). In Fig. 3A and Fig. 3B, the cells were infected with the vaccinia virus recombinant (5 PFU/cell) and 18 h later incubated in medium lacking methionine and cysteine but containing 50 mCi/ml of Tran 35S for a further 5 h. The inhibitor (1 mg/ml) was added 90 min before the labelling. The proteins were separated on a 7% SDS – PAGE under reducing and non reducing conditions. The lane labelled M contains molecular weight markers.
recombinant vaccinia virus VVTG2180 which express the HIV-2 ROD envelope protein. After labelling with TRAN 35S, culture medium containing secreted 35S-gp1202180 was incubated with different concentrations of EGS and immunoprecipitated with an HIV-2 positive human serum (gift of Dr J. Andre´, Institut Pasteur de Lyon) and analyzed by SDS-PAGE (Fig. 5B). EGS stabilized a dimeric form of gp120HIV-2 ROD. Increasing concentrations of EGS were accompanied by a concomitant decrease in the migration rate. It has been shown previously that cross-linked proteins have modified migration rates (Doms, 1990; Malvoisin and Wild, 1993). It
is possible that the failure to stabilize dimeric full-length gp120HIV-1 is due to the fact that the amino groups of lysine residues present on two adjacent chains are not accessible to the crosslinking reagent.
3. Discussion The amino-terminal region of gp120 contains an hydrophobic domain which is hyperconserved among the HIV-1 isolates and highly conserved among the primate immunodeficiency viruses (Helseth et al., 1991; Ivey-Hoyle et al., 1991).
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Fig. 4. Effects of inhibitors of N-linked glycosylation on the processing of gp120 and C-terminally truncated forms expressed by recombinant vaccinia vectors in HeLa T4 cells. The cells were infected with the indicated vaccinia virus recombinant, labelled with Tran 35S and incubated in the presence or in the absence of the inhibitor of N-linked glycosylation as described in The inhibitors used were 1-deoxynojirimycin (DNJ) (400 mg/ml), swainsonine (SW) (150 mg/ml), castanospermine (CSP) (400 mg/ml) or 1-deoxymannojirimycin (DMJ) (400 mg/ml). A parallel control culture (con) was radiolabelled in the absence of glycosylation inhibitor. The proteins were immunoprecipitated with MAb I 11, 2. In A, cellular, in B cellular and secreted proteins were analyzed under reducing and non reducing conditions.
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Fig. 5. Cross-linking of C-terminally truncated forms of gp120 and cross-linking of gp120HIV-2 ROD expressed by recombinant vaccinia vectors in HeLa T4 cells. (A) 35S-truncated forms of gp120 were treated with different concentrations of EGS (Ethylene glycolbis-succinimidylsuccinate): Lane 1, control; lane 2, 1.5 mM; lane 3, 0.5 mM; lane 4, 4.5 mM EGS. Proteins were immunoprecipitated with MAb I 11, 2 and separated on a 7% SDS – PAGE under reducing and non reducing conditions. (B) 35 S-gp120HIV-2 ROD was treated with different concentrations of EGS: Lane 1, control; lane 2, 3 mM; lane 3, 1 mM; lane 4, 0.34 mM EGS. Proteins were immunoprecipitated with an HIV-2 positive human serum and separated on a 7% SDS – PAGE under reducing and non reducing conditions. The position of dimeric (2×) gp120HIV-2 ROD is indicated.
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High conservation of sequences between virus isolates often reveals functional regions. This hydrophobic domain may play a role in the association of gp120 molecules. Deletion of the NH2-terminus of gp120 HIV-1 has been shown to affect the folding of the truncated molecules (Jeffs et al., 1996). Owens and Compans (1990), using recombinant vaccinia vectors for expression, demonstrated that the HIV-1 gp160 precursors which acquire aberrant intermolecular disulfide bonds are not proteolytically processed. In our study, both the secreted and cell-associated proteins were processed, even in the presence of inhibitors of oligosaccharide processing. Only properly folded and assembled proteins are transported to the Golgi complex. Therefore, it is unlikely that the dimeric forms we observe are aggregates of erroneously folded products but represent SDS– stable dimers. If aggregates were present, they would not enter the gel in non reducing conditions and would require a high concentration of ME to be reduced (in this study we used 0.5% ME, final concentration). In addition, during our preliminary experiments, we added to the cell lysate and the supernatant 10 mM iodoacetamide to prevent eventual disulfide bond formation. However, this was not necessary since iodoacetamide-treated and untreated samples gave the same protein patterns (not shown). The truncated forms of gp120 could be correctly folded in a way that prevents them from being retained in the reticulum endosplamic, but their assembly and transport may be different by comparison with the wild-type protein. The presence of SDS – stable dimers which migrate as monomers in reducing conditions is not necessarily indicative of the presence of interchain disulfide bonds: the existence of SDS–stable dimers is a well known phenomenon and, when SDS – resistance is observed, there is a high probability that the interaction represents a biologically relevant structure (Gething et al., 1989; Doms, 1990). Their destabilization in reducing conditions is problably due to the rupture of the intrachain disulfide bonds. The smallest protein which contains only one con-
served region of gp120 (C1) shows the highest proportion of dimers. This may be an artefact of deleting increasing lengths of the C-terminus or, on the contrary, reflect the presence of an association domain in that region. Gp120 contains intrachain disulfide bonds (Leonard et al., 1990) and there are also intrachain interactions between the V1/V2 and C4 domains (Freed and Martin, 1994), and between C1, C2 and C5 (Moore et al., 1994). In the shortest gp120 truncated form, the affinity may be increased because the protein is free of the constraints imposed by the other domains. The precursor gp160 oligomerizes in the ER and the cleavage is a late event which occurs after exit from the trans-Golgi network (Earl et al., 1991b; Merkle et al., 1991; Kantanen et al., 1995). After dissociation from gp41, a change in the folding of gp120 may occur and the folding of truncated forms of gp120 may be more similar to their structure in the full-length gp160 molecule. Finally our results suggest that gp120 can associate with itself even after dissociation from the gp41 subunit. We do not known if the interactions observed with truncated forms of gp120 are relevant to the full-length molecule. Further confirmation is needed, especially by deleting the region present in the N-terminus of gp120 which are thought to play a role in the association. The presence of an association domain in gp120 may modify our approach in the design of derived-gp120 based subunit vaccine.
Acknowledgements We thank Dr J.F. Pasquali, Dr F. Traincard and Dr H.C. Holmes of the ADP reagent programme for MAbs and reagents. We would also like to thank Drs B. Moss and P. Earl for providing recombinant vaccinia viruses (vPE17, vPE22, vPE21, vPE20), R.A. Marks for the gift of NB-DNJ and Bernadette Maret for editorial assistance. These studies were supported by a grant from SIDACTION and Etienne Malvoisin held a SIDACTION fellowship.
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