AIDS RESEARCH AND HUMAN RETROVIRUSES Volume 6, Number 6, 1990 Mary Ann Liebert, Inc., Publishers

Expression of HIV-1 gpl20 and Human Soluble CD4 by

Recombinant Baculoviruses and their Interaction In Vitro YUKO MORIKAWA,1 HILARY A. OVERTON,1 4 JOHN P. ANTHONY J. WILKINSON,3 R. LEO BRADY,3 SALLY J. and IAN M. JONES1

MOORE,2 LEWIS,3

ABSTRACT The soluble domains of the envelope glycoprotein of HIV-1 (gpl20) and human CD4 (sCD4) have been individually expressed in insect cells using recombinant baculoviruses. Each product is secreted from infected cells and accumulates in the surrounding media to levels of 1-2 mg/liter of 2 x 109 cells. Both molecules have full biological activity, and conditioned media from infected cells have been used to establish a simple assay for gpl20-sCD4 interaction that is highly specific and amenable to mass screening. The crystallization of sCD4 purified from this source is reported.

INTRODUCTION

The

cellular TROPISM of the Human immunodeficiency virus (HIV) for lymphocytes is determined by the interaction of the viral envelope glycoprotein, gp 120, with the cell surface marker CD4. ' -2 Although there is evidence that CD4-independent infection of some cells can occur,3 CD4-dependent viral entry is the predominant route of infection. This entry process has been suggested as a target for chemotherapeutic or immunologie antiviral therapy.4 The association of gpl20 with CD4 exhibits a high KD (~10~9M)5 and, a priori, the only satisfactory antiviral drugs will be those that are able to block this specific interaction. Similarly, only anti-idiotypic anti-CD4, or direct anti-gpl20 antibodies that can efficiently compete against the interaction of HI V gp 120 and CD4 would be suitable for therapeutic use. For both of these potential areas of therapy, a knowledge of the three-dimensional structure of the binding surfaces of either or both of these molecules will be of considerable benefit. To facilitate crystallographic studies there is a need for large quantities of CD4 and gpl20. Moreover, if meaningful data are to be obtained using recombinant products, it is important that the expression system used produce biologically functional molecules that mimic the natural viral or lymphocyte products. Recombinant baculoviruses have been successfully employed to produce a variety of eukaryotic proteins, often in good yield and with demonstrable functional activity.6'7 In this communication we document the use

'NERC Institute of Virology, Mansfield Road, Oxford 0X1 3SR England, institute of Cancer Research, Chester-Beatty Laboratories, Fulham Road, London SW36JB England, department of Chemistry, University of York, York YOl 5DD England. 4Present address: Department of Biology, Roche Products Ltd, Welwyn Garden City, Herts AL7 3AY

England.

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MORIKAWA ET AL. of recombinant baculoviruses to produce the gpl20 molecule of HIV-1 (LAV) and the soluble domain of human CD4. We show that both molecules have biological activity and we have established a simple assay for their interaction in vitro. The initial attempts to crystallize each product for structural studies is also reported.

MATERIALS AND METHODS Plasmic constructions and production

of recombinant baculoviruses

DNA clones encoding HIV-1 BRU and human CD4 were the generous gifts of Dr. S. Wain-Hobson (Paris). DNA manipulations were carried out according to standard procedures described by Maniatis et al.8 Oligonucleotides were purchased from British Biotechnology Ltd. To generate a clone encoding gpl20, we used the oligonucleotide 5'-GCTCCTATTAAGCTTATCTTTTTTC-3 and procedures described elsewhere9 to direct the introduction of an in-frame stop codon directly following the codon for arginine at position 516 of the HIV-1 env amino acid sequence. Arginine is the C-terminal residue in gpl20 after cleavage of gpl60 to give gpl20 and gp41.10 The oligonucleotide also introduced a HindlH site into the sequence, which facilitated the identification of mutants and the subsequent manipulation of the gpl20 coding sequence. To confirm that no other mutations had been introduced inadvertently, we sequenced the mutated fragment and reconstructed the truncated gene via a series of cloning steps. Subsequently, a Sspl—HindlH fragment encoding the truncated env gene was removed and inserted into the BamHI site of the baculovirus expression vector pAc YM1. ' ' To generate a clone encoding the soluble extracellular domain of CD4, we used the oligonucleotide 5'-GCTGCACCTAGGTGGACC-3' to (1) replace the proline at CD4 amino acid position 368 (numbering is taken from the corrected CD4 sequence12 with a termination codon; and (2) to introduce an overlapping Sty 1 site. After sequencing the mutated region, the truncated gene was reconstructed as before. Subsequently, an EcoRI-Styl fragment encoding the truncated CD4 gene was removed and, inserted into the BamHI site of pAcYM 1. The generation of recombinant baculoviruses was done as described elsewhere7 using wild-type baculovirus DNA as the cotransfectant and by selecting for polyhedrin-negative plaques. '

Cell culture, virus

growth,

and protein

analysis

The culture of Spodoptera frugiperda (Sf9) cells and viral infections were undertaken as described previously.7 Proteins were analysed on 10% sodium dodecyl sulfate (SDS) polyacrylamide gels and stained with Coomassie blue. Western blots were done as reported elsewhere13 except that transfers were carried out in a semidry blot apparatus for 2 h at 0.15 A and blots were developed with enzyme conjugates rather than using iodinated protein A.

Quantitation of gp!20 and sCD4 gpl20 and sCD4 were quantitated by a sandwich enzyme-linked immunosorbant assay (ELISA) similar to previously described.14 In each case, dilutions of antigen were captured to a solid phase using D7324 (Aalto Bio-reagents, Dublin) for gpl20 or OKT4 (Ortho Diagnostics) for sCD4. Following capture, antigen was detected using a rabbit polyvalent serum (R 1/87 for gpl20—a gift from Rod Daniels or CBL 23 for sCD4—a gift from Jane Armes) followed by an antirabbit alkaline phosphatase conjugate. Test results were compared with standard curves prepared with purified gpl20 (Celltech) or sCD4 (Smith Kline and Beecham).12 that

gpl20 and sCD4 interactions in vitro The assay for gpl20-CD4 binding is a development of the antibody capture assay for gpl20 previously described (Ref. 14 and Ref. 26). gpl20-sCD4 complexes are allowed to form in solution in vitro and are trapped in the solid phase by an anti-gpl20 antibody (D7324). Any complex bound is detected using the 766

EXPRESSION OF HIV-1

gpl20 AND HUMAN SOLUBLE CD4

OKT4 followed by an antimouse alkaline phosphatase conjugate. In our with coated D7324 at 10 p-g/ml in 0.1 M NaHC03, pH 9.0, for between 1 and 16 h experiments, plates at 4°C. Unbound antibody was washed away with Tris-buffered saline (TBS, i.e., 10 mM, Tris-HCl', pH 7.5, 144 mM NaCl) and the plates blocked for 30 min with 2% dried milk powder in TBS. Plates were then incubated with mixtures of sCD4 and gp 120 containing infected cell supernatant fluid for 2 h at 20°C and then rinsed with TBS. Bound complexes were probed with OKT4 ( 1 p.g/ml in TBS 2% milk powder) for 1 h at 20°C and, after washing, for an additional hour with rabbit antimouse IgG-alkaline phosphatase conjugate. Finally, the plate was washed and enzyme activity was detected using the sensitive AMPAK system1415 with a substrate incubation time of 20 min and a development time of 10 min. In a variation of this assay, sCD4 from Chinese hamster ovary cells was bound to the solid phase by OKT4 and complex formation with gpl20 detected using D7324 followed by an antisheep alkaline phosphatase conjugate and AMPAK. anti-CD4 monoclonal

antibody

were

Purification and crystallization The soluble portion of human CD4 was purified by affinity chromatography using the murine monoclonal RFT4 (a gift of G. Janossy). The monoclonal was coupled to CNBr-activated Sepharose 4B (Pharmacia) using the manufacturer's recommended conditions. The column was equilibrated with 20 mM Tris-HCl, 140 mM NaCl, pH 7.5. Cell culture supernatants, four days after infection with the recombinant baculovirus, were passed over the column which was then washed with 20 mM Tris-HCl, 500 mM NaCl, pH 7.5. The bound sCD4 was eluted with 100 mM glycine, pH 2.5, fractions being immediately neutralized by the addition of 1 M Tris-HCl, pH 8.0. The purified sCD4 was then concentrated to 10 mg/ml in 50 mM sodium phosphate, pH 7.0 using a Centricon 30 (Amicon). Purified recombinant gpl20 was obtained from American Biotechnologies Inc. (Boston). This protein was expressed in insect cells using the recombinant baculovirus described here, and was purified by chromatography on an anti-gpl20 monoclonal antibody column. The gpl20 was concentrated to 10 mg/ml as described for sCD4 above. Complexes of gpl20 and sCD4 were obtained by incubating equimolar ratios of two proteins at 4°C for at least 24 h. Samples of sCD4, gpl20, or their complex were subjected to crystallization trials using the hanging-drop method.!6 A wide variety of precipitation conditions were examined, at 4°C and 20°C.

RESULTS Production

of baculovirus gpl20 (gp!20bac)

Recombinant plaques containing the truncated env gene described in Materials and Methods were screened for the production of gpl20 using Western blotting. Sf9 cells were infected with recombinant virus at a multiplicity of infection (MOI) of 5-10, harvested at various times postinfection (PI), and the whole cell and cell supernatant fractions examined for the presence of antigen using a rabbit poly valet anti-en v serum (a gift of R. Daniels). The results are shown in Figure 1. gpl20 is detected 24 h postinfection and rises to an intracellular peak at 48 h postinfection. Peak levels in the supernatant of infected cells occurred slightly later (72 h PI), reflecting the additional time needed for secretion of the intracellular pool. At 72 h PI we estimate that approximately 50-70% of the gpl20 produced is present extracellularly, a level corresponding to 1-2 p-g/ml of culture based on semiquantitative ELISA.14 The gp 120bac is glycosylated and has an apparent molecular weight (MW) of 110, somewhat smaller than that observed for mammalian gpl20 (gpl20mamm). The difference in MW is due to the altered carbohydrate structures associated with insect cell-derived

glycoproteins.6

To test the biological activity of gpl20bac we harvested culture supernatants at 2 days PI and assayed their ability to bind to mammalian-soluble CD4 (sCD4mamm Ref. 17) captured to the solid phase by monoclonal antibody OKT4. Figure 2 shows efficient binding of gpl20bac to sCD4mamm that was concentration dependent. Binding at 50% was obtained at 0.5 p-g/ml (4.1 nm), suggesting a specific activity close to that observed for mammalian gpl20(2-3nM;Ref. 5). 767

MORIKAWA ET AL.

FIG. 1. Western blot analysis of gpl20 expression in infected insect cells. Numbers above the tracks refer to days postinfection and CELLS and SNAT refer to whole cell and supernatant fraction, respectively. Numbers on the left side of the figure are molecular weights in kilodaltons and are taken from prestained markers run at the same time (Sigma-SDS 7B). The position of secreted gpl20 is indicated.

0-1

[gp120]

pg/ml

FIG. 2. Binding of gp 120bac to CD4 (sCD4mamm). Mammalian sCD4 was captured to a solid phase using OKT4 and incubated with varying concentrations of gpl20 previously quantitated by ELISA. gpl20 binding was detected using a sheep anti-gpl20 serum followed by an antisheep alkaline phosphatase conjugate and AMPAK. Omission of sCD4 from the assay led only to background absorbance.

768

EXPRESSION OF HIV-1

gpl20 AND HUMAN SOLUBLE CD4

We have observed a gradual breakdown of gpl20bac to products at 70 and 50 kD upon prolonged storage at 4°C similar to that reported for gpl20mamm.18 Thus for all subsequent experiments, gp 120bac was harvested at 2 days PI and stored frozen at -70°C until required.

Production

of baculovirus-soluble CD4 (sCD4bac)

Baculovirus expression of sCD4 has been reported previously,19 but there are no data on the ability of gp 120bac to bind to sCD4bac as opposed to sCD4mamm (above). This is clearly of some importance if báculo virus-produced products are to be used interchangeably with their mammalian counterparts. Therefore, to examine this question we constructed a sCD4 recombinant virus as outlined in Materials and Methods. Our sCD4bac is similar to the construct already described19 except that it is truncated two amino acids earlier at the carboxy terminus. sCD4bac production was assayed by Western blotting using a rabbit anti-CD4 polyvalent serum in a similar fashion to that described for gp 120bac. Figure 3 shows the production of sCD4 in both whole cell and supernatent fractions of Sf9 cells infected with the sCD4 recombinant virus. A product of this virus, with an apparent MW of 45-50, reacts specifically with a rabbit antiserum raised against sCD4mamm. The observed MW for this protein is similar to that previously reported for sCD4bac " and the kinetics of production were very similar to those of gpl20bac with an extracellular peak of production at 3 days PI corresponding to 1 p-g/ml sCD4 based on ELISA. It has been shown previously that baculovirus-produced sCD4 has full biological activity19 and we have also shown that our own sCD4bac binds to gpl20mamm as efficiently as sCD4mamm (data not shown). However, there are no published data on the ability of gpl20bac to bind to ~

8004^.

*CD4bac binding to gpl20bac To test for gpl20bac-sCD4 interaction an ELISA type assay was devised to make use of commercially available antibodies and clarified supernatants from recombinant báculo virus-infected cells. Supernatants from Sf9 cells infected with either gpl20 or sCD4 recombinant viruses were collected two days PI, clarified by low-speed centrifugation, mixed, and allowed to react for 1 h at 20°C in microtiter wells previously coated with D7324 (anti-gpl20). In one experiment, the concentration of gpl20bac was held

12 3 4

S'NAT

CELLS

FIG. 3. Western blot analysis of sCD4 production by recombinant baculovirus. CELLS and SNAT refer to whole cell and supernatant fractions, respectively. Numbers on the left of the figure are molecular weights taken from prestained markers run at the same time. The position of sCD4 is indicated. Some slight distortion of bands in the SNAT panel is due to large amounts of serum albumen on the gel.

769

MORIKAWA ET AL. constant while CD4bac was varied; in a second experiment, the concentration of sCD4bac was constant and the concentration of gpl20bac varied. Following incubation, the plate was washed and probed with OKT4 and finally an antimouse alkaline phosphatase conjugate. The results of this assay are shown in Figure 4. Complex formation occurred under these assay conditions and was observed to depend on the concentration of both gpl20bac and sCD4bac as expected. Omission of either reagent from the assay resulted in only background absorbance.

Crystallization of sCD4 Our results above indicated that recombinant baculoviruses expressed reasonable levels of gp 120 and sCD4 with apparently full biological activity. It seemed reasonable, therefore, to examine whether either baculovirus-derived molecule was suitable for crystallization to enable structural studies to be undertaken. Accordingly, gp 120bac and sCD4bac purified by immune affinity chromatography as outlined in Materials and Methods were tested in a variety of vapor diffusion experiments for crystal growth. Small (0.05 X 0.05 X 0.05 mm) crystals ofsCD4 were obtained by this technique (Fig. 5). These crystals grow within four days in 5 pi drops suspended above 1 ml reservoirs containing 10-15% (w/w) PEG 200 (Sigma), in 1.05 M trisodium citrate, pH 8.3. The crystals grow reproducibly, and can be obtained with PEG 400 and 600 as well. Crystals sufficiently large for x-ray diffraction analysis have yet to be obtained and, despite extensive manipulation of crystallization parameters, no crystals have yet been obtained of either gpl20 or of its complex with sCD4.

DISCUSSION Recombinant baculoviruses have been used to produce gp 120 and sCD4 in insect cells. Following infection with the relevant recombinant virus, each product is secreted into the culture medium and accumulates to levels of above 1-2 mgs/liter by the third day of infection. When analyzed by SDS-PAGE and Western

[gp120] (as percent

or

[sCD4]

of total assay volume)

FIG. 4. Interaction of gpl20bac and sCD4bac. Supernatants from infected cells were taken at 2 days postinfection and mixed in the proportions shown in a total assay volume of 0.1 ml (o). Concentration of sCD4 supernatant varied while gp 120 supernatent was constant at 50% of the assay volume ( O ). Concentration of gp 120 supernatant varied with sCD4 supernatant held at 50% of the assay volume. The assay was developed as described in Materials and Methods.

770

EXPRESSION OF HIV-1

FIG. 5.

gpl20 AND HUMAN SOLUBLE CD4

Crystals of recombinant baculovirus-derived human soluble CD4 shown from

12.5% (v/v) PEG 200. The crystals are approximately 0.1

x

0.1

x

0.05

1.2 M trisodium citrate pH 8.3,

mm.

blotting each product exhibits the expected molecular weight, although gpl20 produced in this system is somewhat smaller than gpl20 expressed in mammalian cells.5 This molecular weight difference reflects the altered carbohydrate structures associated with insect cell-derived glycoproteins, these being high in mannose, lacking sialic acid, and less processed than those produced by a mammalian cell background.6 Notwithstanding this difference, gpl20bac was found to have excellent CD4 binding characteristics with 50% binding occurring at 4-5 nM, only slightly higher than the 2-3 nM reported for mammalian gpl20.5 We consider this small difference in CD4 affinity to more probably reflect inaccuracies in gpl20 quantitation than true loss of activity. Our quantitation ELISA use a mammalian gp 120 source as standard (a gift ofP. Stephens, Celltech, Slough, England) and a polyvalent serum as detecting antibody. As some of this antibody binding may be influenced by carbohydrate structure, a small error is associated with each quantitation of gpl20bac with respect to gpl20mamm. We therefore consider it probable that gpl20bac has essentially full CD4 binding ability. A number of studies have suggested an essential role for gp 120 carbohydrate in CD4 binding (2°-22) and we have also observed that a soluble deglycosylated gpl20 expressed in E. coli fails to bind to sCD4 (Morikawa et al.25). Thus, although associated sugar is required for gp 120 function, it is clear from our data that the exact type of carbohydrate added can vary without substantial loss of activity. Consistent with an earlier report,19 we also find that sCD4 produced in this system is biologically active. By contrast with gpl20, carbohydrate appears to play no role in the function of CD4, as a short region of the molecule (the VI domain) which contains the gpl20 binding site23 has full gpl20 binding activity when expressed in E. coli.24 As expected therefore, we have shown that gpl20bac and sCD4bac are able to associate together. Moreover, a gpl20-CD4 binding assay utilizing commercial antibodies and purified components (Ref. 26) was shown to be highly effective using crude culture supernatants from infected cells. We consider that this assay has many of the properties required for a simple mass screening assay for receptor blocking agents. It is relatively inexpensive to establish, economical of reagent use (100 ml of culture supernatant should allow 10,000 assays), highly specific, and amenable to standard mechanized ELISA formats. 771

MORIKAWA ET AL. In addition to their use in crude culture supernatants, we have assessed these molecules as reagents for structural studies following immune purification. In particular, the reduced sugar content of gpl20bac might make this an attractive candidate for trial crystallization. However, despite considerable manipulation of parameters, no crystals of gp 120 or gp 120-sCD4 complex have yet been obtained. By contrast, small crystals of sCD4bac were obtained relatively easily using standard précipitants. These crystals are not yet suitable for diffraction analysis, but attempts to improve upon them and to further reduce the sugar content of gpl20bac while maintaining biological activity are in progress.

ACKNOWLEDGMENTS We thank David Bishop and Guy Dodson for advice and encouragement. We also thank Paul Stephens (Celltech), Jame Armes and Mark Marsch (Chester-Beatty), Rod Daniels (NIMR), and Harvey Holmes (NIBSC) for many reagents. This work was supported by and reagents are available through the UK Medical Research Councils AIDS Directed Programme.

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