Expression, Purification, and Characterization of gp160e, the Soluble, Trimeric Ectodomain of the Simian Immunodeficiency Virus Envelope Glycoprotein, gp160*

The envelope glycoprotein, gp160, of simian immunodeficiency virus (SIV) shares ∼25% sequence identity with gp160 from the human immunodeficiency virus, type I, indicating a close structural similarity. As a result of binding to cell surface CD4 and co-receptor (e.g. CCR5 and CXCR4), both SIV and human immunodeficiency virus gp160 mediate viral entry by membrane fusion. We report here the characterization of gp160e, the soluble ectodomain of SIV gp160. The ectodomain has been expressed in both insect cells and Chinese hamster ovary (CHO)-Lec3.2.8.1 cells, deficient in enzymes necessary for synthesizing complex oligosaccharides. Both the primary and a secondary proteolytic cleavage sites between the gp120 and gp41 subunits of gp160 were mutated to prevent cleavage and shedding of gp120. The purified, soluble glycoprotein is shown to be trimeric by chemical cross-linking, gel filtration chromatography, and analytical ultracentrifugation. It forms soluble, tight complexes with soluble CD4 and a number of Fab fragments from neutralizing monoclonal antibodies. Soluble complexes were also produced of enzymatically deglycosylated gp160e and of gp160e variants with deletions in the variable segments.

The interaction of the gp120 polypeptide of gp120/gp41 with CD4 causes a conformational change in the viral glycoprotein, inducing altered antibody reactivity, increased proteolytic susceptibility, and the tendency in some viral strains for gp120 to dissociate from gp41 (14 -16). The conformational change caused by CD4 binding apparently increases the affinity of gp120/gp41 for the chemokine-binding co-receptor (17)(18). Interaction with the chemokine receptor activates the membrane fusion activity of gp120/gp41, probably by triggering a conformational change in gp41. This change is likely to expose the N-terminal fusion peptide and to refold gp41 into a helical hairpin, thereby placing the fusion peptides and transmembrane anchors of gp41 at the same end of a rod-shaped molecule (19 -24).
There is a formal similarity among the SIV and HIV-1 gp160 glycoproteins, the envelope glycoproteins of other retroviruses such as Moloney murine leukemia virus, and HTLV-1, the Gp glycoprotein of filoviruses, and the hemagglutinin (HA) of influenza virus. Each is synthesized as a single chain precursor, which after cleavage results in an N-terminal subunit (gp120, SU, Gp1, HA1) with receptor binding activity and a C-terminal, membrane-anchored subunit (gp41, TM, Gp2, HA2) having a nonpolar, glycine-rich N-terminal fusion peptide followed by heptad repeats characteristic of ␣-helical coiled-coils (reviewed in Refs. 25 and 26). Evidence suggests that these viral glycoproteins are all trimeric (27)(28)(29)(30)(31)(32), although some early studies proposed dimers or tetramers for gp160 (33)(34)(35). In each case, the N-terminal subunit (gp120, SU, Gp1, HA1) is monomeric when prepared in the absence of the transmembrane subunit, but oligomeric in its presence (36,37). The C-terminal subunits are all trimeric when expressed in the absence of the N-terminal polypeptides, and they adopt similar structures dominated by an N-terminal, central triple-stranded ␣-helical coiled-coil and an outer layer of three helices or extended strands packing antiparallel along the central coiled-coil and connected to it by a loop with a reverse (reviewed in Ref. 26).
The three-dimensional structure of an enzymatically deglycosylated fragment of monomeric gp120 from HIV-1, with the variable segments V1, V2, and V3 deleted, along with 52 residues at the N terminus and 19 at the C terminus, has been determined by x-ray crystallography. The crystals contain a * This research was supported by a special National Institutes of Health Program for AIDS, the Innovation Grant Program for Approaches in HIV Vaccines Research (to S. C. H. and D. C. W.), the Medical Research Council (UK), Novel HIV Therapy-IPCP Grant AI43649 (to E. R.), and the Howard Hughes Medical Institute (HHMI). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
ʈ Investigator of the HHMI. ** To whom correspondence should be addressed: Laboratory of Molecular Medicine, The Children's Hospital, Howard Hughes Medical Institute, 320 Longwood Ave., Enders 673, Boston, MA 02215. Tel.: 617-355-7373; Fax: 617-738-0184; E-mail: dcwadmin@crystal.harvard. edu. 1 The abbreviations used are: SIV, simian immunodeficiency virus; HIV, human immunodeficiency virus; HA, hemagglutinin; mAb, monoclonal antibody; PCR, polymerase chain reaction; bp, base pair; kb, kilobase pair; PAGE, polyacrylamide gel electrophoresis; PBS, phos-complex of truncated gp120 with a two-domain fragment of CD4 and an Fab fragment from the monoclonal antibody 17b (38). This structure contains about 60% of the gp120 residues (39,40). It is in the CD4-induced conformation, which has enhanced affinity for the monoclonal antibody 17b (41). The structure of the gp120 trimer has not been determined, although it has been modeled (42), and the conformation of gp120 as it would appear on virus before CD4 binding is currently unknown.
We report the development of expression systems using insect and mammalian cells for production of soluble, oligomeric ectodomain, gp160e, from the SIV envelope glycoprotein. Purified oligomers are shown to bind CD4 and Fab fragments of neutralizing monoclonal antibodies. Experiments show that the molecule is trimeric. These observations should facilitate biochemical and structural characterization of the viral glycoprotein in its native, oligomeric form.

EXPERIMENTAL PROCEDURES
SIV gp160e Expression in CHO Cells-SIV gp160e from the SIV strain Mac32H pJ5 (43) was expressed in CHO-Lec3.2.8.1 cells using the expression construct pSIV-M (44) following a procedure described previously (45). Briefly, 20 g of SalI-linearized pSIV-M DNA was transfected into the Lec3.2.8.1 cells by calcium phosphate precipitation (Stratagene). Transformants were selected in GMEM medium (Life Technologies, Inc.) containing 25 M methionine sulfoximine and assayed for secretion of soluble SIV gp160e by enzyme-linked immunosorbent assays. Immulon plates (Dynatech Laboratories Inc.) were coated with anti-SIV ENV monoclonal antibody KK19 (46) by incubating a 4 g/ml solution overnight at 4°C; the plates were then blocked with 1% bovine serum albumin in PBS at room temperature for 2 h. Cell culture supernatant (50 l) from each transformed clone was added to the plates and incubated overnight at 4°C. The plates were washed with 0.1% bovine serum albumin in PBS and incubated with 0.2 g/ml biotinylated mAb KK41 (47) for 2 h at room temperature. After washing again, the plates were developed with horseradish peroxidase-conjugated streptavidin (Sigma). The highest expressing cells were chosen for rescreening with the same assay. The expression of SIV gp160e was also confirmed by immunoprecipitation. Large scale production of SIV gp160e was carried out following the protocol previously described (45) in 175-cm 2 tissue culture flasks or 850-cm 2 roller bottles (Corning).
Both restriction digestion and DNA sequencing verified all of the expression constructs.
Recombinant baculovirus was generated according to the manufacturer's protocol and amplified in Sf9 insect cells in TNM-FH medium (Sigma). Expression of SIV gp160e was confirmed by a Western blot using sheep antisera against SIV envelope glycoprotein. The optimal amount of virus and postinfection harvest time was determined by small scale tests in 100-ml spinner flasks. Infected Trichoplusia ni (Hi-5) cells were found to secrete significantly more SIV gp160e than Sf9 cells, judging by Western blot. For large scale protein production, 10 liters of T. ni (Hi-5) cells (2 ϫ 10 6 cells/ml) in Ex-Cell 405 medium (JRH Biosciences) were infected at a multiplicity of infection of 2.5. 3 days after infection, the supernatant was harvested by centrifugation and concentrated to 1 liter in a tangential flow filtration system, ProFlux M 12 (Millipore Corp.).
Purification of SIV gp160e-The CHO Lec3.2.8.1 supernatants containing secreted SIV gp160e were harvested by centrifugation or by filtration through a Corning filter (0.22 mm). SIV gp160e was purified by an immunoaffinity chromatography using a mAb 17A11 3  cross-linked at 5 mg/ml to GammaBind Plus Sepharose (Amersham Pharmacia Biotech) with dimethyl pimelimidate (Pierce). The supernatants were passed through the column with a flow rate of about 0.5 ml/min. After extensive washing with PBS, the protein was then eluted with 100 mM glycine (pH 3.0), followed by immediate neutralization with 2 M Tris-HCl (pH 8.0). The fractions were analyzed by SDS-PAGE. The fractions containing SIV gp160e were pooled, concentrated, and further purified by gel filtration chromatography on Superdex 200 or Superose 6 (Amersham Pharmacia Biotech) with a buffer containing 25 mM Tris-HCl (pH 8.0) and 150 mM NaCl. SIV gp160e⌬(V1V2) and gp160e⌬(V1V2V3) proteins were purified following the same procedure. SIV gp160e expressed from insect cells was purified by metal chelate affinity chromatography with ProBond resin (Invitrogen). Concentrated insect cell culture supernatants were immediately changed into 1ϫ column buffer (25 mM sodium phosphate (pH 8.0), 250 mM NaCl) in a ProFlux M 12 flow filtration system to remove small molecules in the medium that interfere with the binding of His-tagged SIV gp160e to the nickel column. After centrifugation at 5000 rpm in a JA-14 rotor (Beckman) for 15 min to remove insoluble materials, imidazole was added to the final concentration of 15 mM to reduce nonspecific binding to the resin. Batch binding was then performed at 4°C for 3 h. After the column was packed (about 5-ml bed volume), the beads were washed by 100 ml of 1ϫ column buffer containing 15 mM imidazole, followed by further washing with 50 ml of 40 mM imidazole in 1ϫ column buffer. The protein was eluted with 300 mM imidazole in 1ϫ column buffer. The fractions were analyzed by SDS-PAGE. The fractions containing SIV gp160e were pooled, concentrated, and further purified by gel filtration chromatography on Superdex 200 or Superose 6 (Amersham Pharmacia Biotech) with a buffer containing 25 mM Tris-HCl and 150 mM NaCl. SIV gp160e⌬(V1V2) and gp160e⌬(V1V2V3) proteins were purified following the same procedure.
N-terminal analyses of purified proteins were carried out by the HHMI biopolymer facility.
Chemical Cross-linking and Analytical Ultracentrifugation-For chemical cross-linking experiments, SIV gp160e protein was dialyzed extensively against PBS. In 20-l reactions, SIV gp160e (1 mg/ml) was incubated with ethylene glycol bis(succinimidylsuccinate) (EGS; Pierce) at concentrations of 0.06, 0.18, 0.55, 1.67, and 5 mM respectively, on ice for 30 min. The reactions were then quenched by adding 5 l of 100 mM Tris-glycine (pH 7.0) and incubated at room temperature for 45 min. The cross-linked products were analyzed by SDS-PAGE. Cross-linked phosphorylase b (Sigma) was used as an SDS-PAGE molecular weight standard.
Analytical ultracentrifugation was performed on a Beckman XL-A analytical ultracentrifuge at 4°C. Experiments were performed at concentrations of 1.6 M, 3.3 M, and 6.5 M protein and centrifuged at a rotor speed of 6000 rpm. Data were fitted to a single-species model. The protein partial specific volume and solvent density were calculated according to Laue et al. (49). Briefly, all glycans in gp160e from CHO-Lec3.2.8.1 cells were found to be (GlcNAc) 2 (Man) 5 4 by mass spectroscopy using previously described methods (45), and assuming that all 25 potential glycosylation sites are occupied, the partial specific volume of gp160e was calculated to be 0.70 ml/g.
Binding of gp160e to CD4 and Antibody Fab Fragments-Four-domain sCD4 expressed from CHO cells, affinity-purified, and sized by Superdex 75 gel filtration was kindly provided by Dr. Yi Xiong. Monoclonal antibodies were purified from cell supernatants of hybridomas growing in roller bottles using a 5-ml GammaBind Plus Sepharose (Amersham Pharmacia Biotech) affinity column. Fab fragments were produced as described (45), from the neutralizing monoclonal antibodies KK9 (46), 17A11 (a kind gift of J. Hoxie), and 9G3. 2 Purified SIV gp160e was incubated at room temperature for 1 h with CD4 or Fab fragments. The complexes were separated from excess unbound CD4 or Fabs by a gel filtration chromatography on Superose 6 (Amersham Pharmacia Biotech). Molecular weights were calculated based on a standard curve plotted from the elution volumes of known proteins. Peak fractions were verified to contain both gp160e and CD4 or Fabs by SDS-PAGE.
Circular Dichroism Spectroscopy-CD spectra were recorded at 25°C using SIV gp160e at a concentration of 0.56 mg/ml in PBS with a circular dichroism spectrometer model 62DS (Aviv). The molar ellipticity [] was monitored as the average of 12 scans with 0.5-nm bandwidth and 1.0-nm wavelength increments from 195 to 260 nm. The spectra were corrected with a base line obtained using buffer alone under the same conditions.

FIG. 1. Constructs for expression of the ectodomain, gp160e, of the SIV envelope glycoprotein in mammalian and insect cells.
Schematic representations for the expression constructs, pSIV-M (44), pNES1⌬V1V2V3, pFBSIV-His1, and pFBSIV⌬V1V2V3-His1, are shown. In all cases, the posttranslational cleavage sites (residues 512 and 523) between gp120 and gp41 have been mutated as shown to prevent cleavage and dissociation of gp120. pNES1⌬V1V2V3 has a shorter tissue plasminogen activator leader sequence than pSIV-M, while both pFBSIV-His1, and pFBSIV⌬V1V2V3-His1 use the honeybee melittin (HM) secretion signal. The cleavage sites after the leader sequences were confirmed by N-terminal sequencing of the expressed products and are indicated by arrows beneath the sequences. The sequences where the V1, V2, and V3 segments were deleted and replaced with a GAG linker (in italics) are shown for pNES1⌬V1V2V3 and pFBSIV⌬V1V2V3-His1. The sequences where the transmembrane segment of gp41 was truncated are also shown for all constructs. Leader sequences are shown in normal type, and SIV gp160 sequences included in the expression constructs are in boldface type and underlined; sequences not included are printed with a strike-through. sequence 5Ј to the coding region and terminating the gene just 5Ј to the transmembrane anchor sequence in gp41 (Fig. 1). Two basic residues at the primary (Lys-523) cleavage site and a secondary (Arg-512) cleavage site between gp120 and gp41 were replaced by glutamic acids, as described previously (44), to prevent cleavage of the gp160e precursor and to avoid shedding of gp120 that had been observed in native constructs. 5 Variants were also expressed with only the V1 and V2 segments deleted (not shown) or with the V1, V2, and V3 segments deleted (Fig. 1). Such deletion variants of HIV-1 gp120 have been shown to retain CD4 binding activity (1, 38 -40).
Purification and Size Determination by Gel Filtration Chromatography-SIV gp160e secreted from CHO Lec3.2.8.1 cells was purified from culture fluid by immunoaffinity chromatography using the monoclonal antibody 17A11 (a gift from J. Hoxie). The protein was eluted from the affinity column by low pH (3.0) and immediately neutralized. Peak fractions were concentrated and further purified by gel filtration chromatography. The purified gp160e eluted from the sizing column (Superose 6) with an apparent molecular weight of approximately 440,000, when compared with standard proteins (Fig. 2A). The relatively sharp gel filtration profile suggests that the molecule is reasonably monodisperse. Polydispersity is approximately 20% measured by dynamic light scattering. Analysis by SDS-PAGE showing a single protein band at an apparent molecular weight of approximately 120,000 indicates a high level of purity ( Fig. 2A). The gp160e monomer molecular weight from SDS-PAGE (120,000) is between one-third and one-fourth of the gp160e oligomer apparent molecular weight (440,000) measured by gel filtration chromatography, indicating that the soluble gp160e molecule is oligomeric.
SIV gp160e secreted from Hi-5 insect cells had been expressed with a C-terminal hexahistidine tag. This gp160e was purified from cell culture fluid by metal chelate chromatography on a nickel resin. The protein was eluted with buffer containing 300 mM imidazole, concentrated, and further purified by gel filtration chromatography. The purified gp160e eluted from the sizing column (Superose 6) with an apparent molecular weight of approximately 440,000 when compared with standard proteins (Fig. 2B). The relatively sharp gel filtration profile again suggests that the molecule is reasonably monodisperse. Polydispersity of this sample was also approximately 20%, as measured by dynamic light scattering. Analysis by SDS-PAGE showing a single prominent protein band at an apparent molecular weight of approximately 120,000 indicates a high level of purity (Fig. 2B). Again, the ratio of the monomer molecular weight to the apparent molecular weight of the secreted molecular species indicates that the molecule is an oligomer.
CD spectra of SIV gp160e recorded at 25°C indicate that the protein is folded and contains a mixture of secondary structures (Fig. 3).
Evidence from Chemical Cross-linking and Analytical Ultracentrifugation That gp160e Is Trimeric-The oligomeric structure of the soluble ectodomain of SIV gp160e was examined by both chemical cross-linking and sedimentation equilibrium by analytical ultracentrifugation. Purified SIV gp160e (1 mg/ml) was incubated for 30 min with the cross-linking reagent EGS at a series of EGS concentrations from 0.06 to 5 mM. The reaction products were analyzed by SDS-PAGE (Fig. 4A). At the lowest concentration of cross-linker, three protein bands are evident, migrating at apparent molecular weights corresponding to one, two, and three gp160e polypeptide chains (see legend to Fig.  4A). As the concentration of cross-linking reagent is increased, the apparent trimer band increasingly dominates. Some faint higher molecular weight bands are observed at higher crosslinker concentrations, but their mobility is too low to represent tetramers, and they presumably represent minor intertrimer cross-linking at high cross-linker concentrations.
The molecular weight of the soluble ectodomain of SIV gp160e was measured by sedimentation equilibrium. Measurements were made at three protein concentrations and one rotor speed. The results from one experiment are shown in Fig. 4B. No systematic deviations were found from a single-species model at the concentrations studied. The partial specific volume of gp160e, required for the buoyancy correction, was calculated from the amino acid composition and the carbohydrate content (see "Experimental Procedures") (49). The resulting measured molecular weight of SIV gp160e of 340,000 is almost exactly 3 times the monomer molecular weight calculated (110,000) from the sequence and carbohydrate content or estimated by SDS -PAGE (120,000).
Complexes of gp160e with CD4 and Fab Fragments of Neutralizing Monoclonal Antibodies-The purified, soluble ectodo-

and insect cells.
The SIV gp160e proteins were purified from supernatant of a CHO cell line that expresses the gp160e by mAb 17A11 affinity chromatography (A) or from supernatant of insect cells infected with recombinant baculoviruses expressing gp160e by metal chelate affinity chromatography (B). The purified proteins were then resolved by gel filtration chromatography using a Superose 6 column. Molecular masses were calculated based on a standard curve plotted using known proteins, which include thyroglobulin (669 kDa), ferritin (440 kDa), catalase (232 kDa), aldolase (158 kDa), and bovine serum albumin (67 kDa). Peak fractions were pooled and analyzed by SDS-PAGE (inset). Similar experiments using an analytical Superdex 200 column gave the same results (not shown). main of the HIV-1 receptor human CD4 (4-domain CD4), expressed as a secreted protein in CHO cells, was incubated with purified SIV gp160e to determine its receptor binding activity. Excess CD4 was removed from the gp160e-CD4 complex by gel filtration chromatography (Fig. 5). Analysis of the peak fractions from the gel filtration column by SDS-PAGE indicates that the pure gp160e binds CD4 (Fig. 5).
SIV gp160e binds 13 mAbs raised against SIV gp160 or against SIV-infected cells (mAb 17A11); the mAbs have been mapped to various epitopes: V1, V2, V3, and V4 on gp120 and others on gp41. 2 Complexes of SIV gp160e with Fab fragments from either of the neutralizing mAbs KK9 (which recognizes the V3 and V4 segments of gp120) or 9G3 (which recognizes gp41) were prepared by incubating gp160e with a molar excess of the Fab and removing the excess Fab by gel filtration chromatography. Analysis of the peak fractions from the gel filtration column indicated that both Fabs bind the gp160e molecule. The change in apparent molecular weight estimated from the elution volume of molecular weight standards is approximately 150,000, suggesting that more than one and probably three Fab fragments bind per gp160e oligomer (Fig. 6).
Deglycosylation of SIV gp160e Expressed in CHO-Lec3.2.8.1 Cells-Endo H digestion at room temperature under native and denaturing conditions was used to remove sugar residues from SIV gp160e. Digestion was complete in 30 min at 25°C, pH 5.5, as judged by the similar increase in mobility on SDS-PAGE of gp160e samples deglycosylated under native and denaturing conditions (Fig. 8A). Deglycosylated SIV gp160e was insoluble.
To prepare soluble, deglycosylated SIV gp160e required that complexes of SIV gp160e and Fab fragments (17A11 or 9G3) be incubated with endoglycosidase H for 4 h. This treatment yielded the same level of deglyscolation as digestion of free gp160e or denatured gp160e, as judged by mobility of the digested gp160e on SDS-PAGE (data not shown). The resulting deglycosylated gp160e-Fab complexes were soluble and monodisperse. These complexes were purified from the excess, unbound Fab by gel filtration chromatography (Fig. 8B). DISCUSSION We have prepared a soluble oligomer of the envelope glycoprotein of SIV in the single chain, precursor state before cleavage into gp120/gp41, in an attempt to capture the native conformation of the envelope glycoprotein before CD4 receptor binding. The expression systems and purification developed provide biochemical quantities of pure protein for use in the study of the functions and structure of the glycoprotein. Following Rhodes and colleagues (44), we have used the tissue plasminogen activator leader sequence to induce secretion of the glycoprotein oligomer from mammalian cells, but we have used a variant cell line, CHO-Lec3.2.8.1 (50), which N-glycosylates with only a single, simple oligosaccharide, to reduce heterogeneity in the product and to permit full enzymatic deglycosylation by Endo H. This strategy was previously shown to The cross-linked products were analyzed by SDS-PAGE in a 3.5% gel. The molecular weight standard for SDS-PAGE was cross-linked phosphorylase b (Sigma), which includes monomer (97 kDa), dimer (194 kDa), trimer (292 kDa), tetramer (389 kDa), pentamer (487 kDa), and hexamer (584 kDa) species. The molecular masses of monomer, dimer, and trimer bands (Fig. 4A) of SIV gp160e were determined from a plot of the logarithm of molecular mass of the standards against relative mobility (not shown). The bands marked monomer, dimer, and trimer in the 0.06 mM EGS lane are apparently 123 kDa, 234 kDa (246 calculated), and 346 kDa (369 calculated), respectively. B, analytical ultracentrifugation was performed on a Beckman XL-A analytical ultracentrifuge at 4°C. The data shown were collected with the protein at concentration of 1.6 M and rotor speed of 6000 rpm. Data sets were fitted to a single species model, and protein partial specific volume and solvent density were calculated according to Laue et al. (49). The molecular mass determined is 340 kDa.
work well for other heavily glycosylated surface protein (45). We have confirmed that the protein purified from the culture fluid of the CHO cells can be completely deglycosylated by enzyme treatment (Fig. 8A), and we have shown that deglycosylated oligomers can be stabilized against aggregation by forming complexes with Fab fragments from neutralizing mAbs (Fig. 8B).
We have also expressed the molecule in insect cells, using the honeybee melittin leader sequence to induce secretion. Both molecules purified from the culture fluids of mammalian and insect cells appear to be in a native conformation judging from their abilities to bind the soluble ectodomain of the viral receptor CD4 (Fig. 5; not shown for insect cells) and to be recognized by a number of conformationally sensitive neutralizing monoclonal antisera 2 (Fig. 6). The gp160e from mammalian cells was purified by immunoaffinity chromatography requiring a low pH elution step, while a hexahistidine tag on the gp160e from insect cells permitted purification by metal chelation chromatography. Both methods produced proteins with the same properties, suggesting that the purified protein has a single conformation that is robust to such treatments.
Following Rhodes and colleagues (44), we eliminated the primary and secondary cleavage sites between the gp120 and gp41 segments by replacing two basic residues with glutamic acids. In experiments with SIV gp160e molecules produced in CHO cells without these mutations, we had observed spontaneous cleavage between gp120 and gp41 and some shedding of gp120 during purification. 6 With the two mutations present, no cleavage between gp120 and gp41 has been observed (Fig. 2,  inset), permitting the production of quantities of a stable oligomeric ectodomain.
The finding that the transmembrane anchor-containing subunits of the lenti-and retrovirus, HIV-1, SIV, MoMuLV, and HTLV-1 are all trimeric (21,(51)(52)(53) and some, but not all, of the earlier attempts to define the oligomeric state of HIV gp160 (27,28) suggested that SIV gp160e was trimeric. We add evidence for a trimeric state from chemical cross-linking and from a direct molecular weight determination on the gp160e. Chemical cross-linking generates products that form three bands on SDS-PAGE, which migrate at apparent molecular weights that are 1, 2, and 3 times that of the gp160e monomer as determined by SDS-PAGE (120,000) (Fig. 4A). This "ladder" is a direct indication of the presence of a trimer of gp160e. Earlier chemical cross-linking experiments on full-length HIV gp160 have been interpreted to favor trimeric or tetrameric states. The  6. Purification of complexes of SIV gp160e with Fab fragments derived from neutralizing monoclonal antibodies by gel filtration chromatography. Purified SIV gp160e was incubated with Fab fragments (as shown 9G3 in A, KK9 in B) generated by papain digestion from either the KK9- (46) or 9G3 2 -neutralizing mAb, which recognize epitopes on gp120 and gp41, respectively. The complexes were separated from excess unbound Fabs by gel filtration chromatography on a Superose 6 column. The apparent molecular mass was calculated based on a standard curve plotted using the elution times of known proteins. The peak fractions were pooled, concentrated, and analyzed by SDS-PAGE in an 8% gel. Lane 1, peak eluted at 13 ml; lane 2, peak eluted at 18 ml.

FIG. 7.
Purification of complex of an SIV gp160e deletion variants with V1, V2, and V3 segments deleted, gp160e⌬(V1V2V3), and Fab fragments. Purified SIV gp160e⌬(V1V2V3) expressed from pFBSIV⌬V1V2V3-His1 was incubated with Fab fragment generated by papain digestion of the neutralizing monoclonal antibody 17A11 (a gift from J. Hoxie), which recognizes an epitope on gp120. The complex was separated from excess unbound Fabs by a gel filtration chromatography on a Superose 6 column. The peak fractions were pooled, concentrated, and analyzed by SDS-PAGE in an 8% gel. Lane 1, peak eluted at 14 ml; lane 2, peak eluted at 18 ml. experiments on the pure ectodomain (Fig. 4A) clearly indicate a trimer.
An independent method of establishing the oligomeric state is to measure the molecular weight of the monomer and the oligomer. The oligomer molecular weight was measured as 340,000 by equilibrium sedimentation, a method that yields a value independent of molecular shape or hydrodynamic radius. The monomer molecular weight measured by mass spectrometry (ϳ100,000) or calculated from the amino acid sequence and carbohydrate content (110,000) is almost exactly one-third of the oligomer molecular weight, again indicating a trimeric structure. The centrifugation data show no indication of a monomer-oligomer equilibrium, demonstrating that the oligomeric species is stable under the conditions studied (Fig. 4B).
The similarity of the sequences and functions of the SIV and HIV envelope glycoproteins suggest that they will have very similar structures. Both bind the same receptor molecules and undergo conformational alterations leading to membrane fusion/viral entry activities. The conformations of the SIV and HIV-1 gp41 ectodomains, determined from molecules expressed without the gp120 subunit are essentially identical (22, 23, 51, 54 -56). The sequence of SIV gp160 is over 50% identical to that of HIV-2, and that of HIV-2 to HIV-1 is about 30%. Molecules related at this level have very similar structures, which normally allow structure/function insights to be drawn from one to the next.
The availability of methods to produce and purify mg quantities of SIV gp160e should improve the possibilities for determining the structure of the precursor state of a lentivirus envelope glycoprotein closely related to HIV. The only structure of a precursor viral glycoprotein currently known is that of HA0 of influenza virus (57). In that case, only 19 residues in the vicinity of the cleavage site differ in structure between the precursor and the cleaved (HA1/HA2) structure. It remains to be seen whether the structure of gp160e will resemble as closely that of proteolytically processed gp120-gp41 complex.