JOURNAL OF VIROLOGY, Sept. 1991, p. 4777-4785

Vol. 65, No. 9

0022-538X/91/094777-09$02.00/0 Copyright © 1991, American Society for Microbiology

Recombinant CD4-Selected Human Immunodeficiency Virus Type 1 Variants with Reduced gpl20 Affinity for CD4 and Increased Cell Fusion Capacity JANE McKEATING,l* PETER BALFE,2 PAUL CLAPHAM,' AND ROBIN A. WEISS' Chester Beatty Laboratories, The Institute of Cancer Research, Fulham Road, London SW3 6JB,l and Institute of Animal Genetics, University of Edinburgh, Edinburgh EH9 3JN,2 United Kingdom Received 14 March 1991/Accepted 31 May 1991

Variants of molecularly cloned human immunodeficiency virus type 1 (HIV-1) were analyzed following selection for the ability to replicate after exposure to soluble, recombinant CD4 protein (rCD4). Two variants, 4/1 and 16/2, show 8-fold and 16-fold reduced sensitivity to rCD4 neutralization yet remain as sensitive as the parental wild-type (wt) virus to neutralization by rCD4-immunoglobulin G (IgG) chimeric molecules and to inhibition of cellular infection by anti-CD4 antibody. The 4/1 variant is more cytopathic, with faster cell fusion and replication kinetics than the wt virus. The gpl20s derived from the 4/1 and 16/2 variants have 3-fold and 30-fold reduced binding affinities to rCD4, respectively. The 4/1 variant exhibits diminished shedding of virion gpl20 induced by rCD4. The binding of and neutralization by V3 loop antibodies and other anti-gp120 antibodies is reduced for 4/1 but not for 16/2. Sequence analysis revealed a codon change at amino acid residue 435 in the C4 region of the gpl20 of 16/2. This accounts for its rCD4 insensitivity, since the insertion of this mutation in the wt gpl20 yields the same phenotype. The 4/1 variant has a codon change in the V3 region of gpl20 (amino acid 311), which accounts for its reduced sensitivity to some neutralizing antibodies but not to rCD4. The ready selection of rCD4-resistant variants has obvious relevance for rCD4-based therapeutic stratagems.

V3 loop of gpl20 (32). These escape mutants have amino acid changes in gpl20. We therefore sought similar escape mutants for rCD4. We have selected variants derived from molecularly cloned HIV-1 that replicate in vitro in the presence of neutralizing concentrations of rCD4, and we have analyzed the variants to gain further insight into the regions of gp120 important for CD4 binding. That such viruses can be selected in vitro raises the question of whether similar selection of rCD4-resistant mutants may occur in vivo in HIV-infected individuals undergoing rCD4 therapy.

The envelope (env) gene of the human immunodeficiency virus type 1 (HIV-1) encodes the gp160 glycoprotein, which is cleaved in the host cell to produce the gpl20 surface and the gp4l transmembrane glycoproteins present in the mature virion as an oligomeric unit (16, 41, 44). gp120 mediates attachment to susceptible cells, and the gpl20-gp4l complex is involved in subsequent events leading to membrane fusion and viral penetration into the cell. The predominant cell surface receptor for HIV infection is the CD4 antigen; infection of T cells by HIV-1 is blocked by some monoclonal antibodies to CD4 (12, 25) as well as by recombinant, soluble forms of the CD4 molecule (rCD4) (43). The ability of rCD4 to block HIV-1, HIV-2, and simian immunodeficiency virus (SIV) infection (8) and virus-induced cell fusion in vitro has led to phase I clinical trials of this agent in HIV-infected individuals (21, 45). The use of similar CD4 antigen epitopes as receptors by HIV-1, HIV-2, and SIV (42) suggests that env glycoproteins contain one or more conserved regions involved in their binding of CD4. A number of studies have investigated the role of specific regions of gp120 in its interaction with CD4. Lasky et al. (27) demonstrated that the deletion of 12 amino acids (426 to 437) from the C4 region of gp120 abrogated CD4 binding. Minor amino acid changes in this and other regions of gp120 have also been implicated as important for interaction with CD4 (9, 10, 15, 26, 30, 50). More recently, amino acids in the C2, C3, and C4 regions of gpl20 were shown to influence CD4 binding (40), and monoclonal antibodies (MAbs) to a conserved conformational epitope on gpl20 prevented CD4 binding (19). We have previously selected variants of HIV that are resistant to neutralization by monoclonal antibodies to the *

MATERIALS AND METHODS CD4 and antibody neutralization tests. (i) Cells. The following T-cell lines (previously described for infectivity and neutralization assays [33]) were used: C8166 and MT4, human T-cell lines transformed in vitro by human T-cell leukemia virus type I; H9, a subline of HUT 78 cells; SupTl, expressing high levels of CD4 antigen; Molt4 clone 8; Raji-CD4 and KM-CD4, a human B-cell line and a murine T-cell line, respectively, transfected with and expressing human CD4 (31). These cells and HeLa cells were maintained in RPMI 1640 medium supplemented with 10% fetal calf serum and antibiotics. (ii) Virus. H9 cells were washed in serum-free medium and suspended at 106 cells per ml in 25 mM Tris-RPMI containing 25 ,ug of DEAE-dextran per ml and 1 ,ug of the infectious DNA molecular clone pNL4.3 per ml. After 3 h at 37°C, the cells were shocked with 10% dimethyl sulfoxide for 2 min, washed, and plated in culture medium. Cells were monitored for productive viral infection as judged by detectable levels of p24 antigen (as described below). Once the cells were producing virus (day 6 to 7 posttransfection), viral stocks were made. Infectivity determinations were made by per-

Corresponding author. 4777

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McKEATING ET AL.

forming 10-fold serial dilutions of virus (50 ,u1) and incubating with 100 RIJ of C8166 cells at 2 x 105 cells per ml in microtiter plates at 37°C for 7 days. The plates were scored for the presence or absence of syncytia, and the 50% tissue culture infective dose values were determined by using the Karber formula. Viral stocks were also quantified for reverse transcriptase (RT) activity, as previously described (20), and by MT4 plaque assay (17, 33). (Wii) Neutralization tests. HIV (103 50% tissue culture infective doses) in a volume of 40 ,ul was incubated with 10 RI of a dilution of the antibody or rCD4/rCD4-immunoglobulin G (IgG) (14, 46) under test at 37°C for 1 h. The virus-CD4 or virus-antibody mixture was then incubated with 100 pl of C8166 cells at a concentration of 2 x 105 cells per ml per well in a 96-well plate in quadruplicate. The wells were subsequently scored for the presence of syncytia (7), and the final dilution of antibody or rCD4 or rCD4-IgG giving >90% reduction of syncytia was defined as the reciprocal neutralization titer (33). Replication kinetic studies. Virus containing 10,000 RT U/mnl was incubated with 106 target cells at 37°C for 1 h. This represents a multiplicity of infection of approximately 0.01. The cells were then washed twice and suspended at 2 x 105 cells per ml in medium and maintained at 37°C. Virus infection was monitored by scoring syncytium formation and by titrating for p24 antigen in the medium as described elsewhere (34). CD4 and MAb gpl20 binding assay. (i) Preparation of HIV-1 extracts. Virus-containing culture supernatants were inactivated with 1% Empigen-BB detergent, which does not irreversibly denature gpl20/gpl60 (39). The concentrations of gpl20 present in the preparations were estimated by enzyme-linked immunosorbent assay (ELISA) with D7324 (Aalto BioReagents) anti-gpl20 as the capture antibody and pooled HIV-positive human sera or a polyclonal rabbit anti-gpl20 serum, R1/87, to detect bound gpl20, and with recombinant gpl20 from the HTLV-IIIB(IIIB) strain of HIV-1 (BH10 clone) expressed in and purified from CHO cells (Celltech Ltd., Slough, United Kingdom, for the MRC AIDS Directed Programme) as a reference standard, as described previously (36). The binding of monoclonal antibodies and rCD4 to gpl20 was assessed by ELISA, using methods previously described (35, 36). rCD4 was expressed in and purified from CHO cells (14) and was a gift from R. Sweet (SmithKline Beckman Corp., Philadelphia, Pa.). CD4 immunoadhesin (rCD4-IgG) (46) was a gift from R. Ward (Genentech, Inc., South San Francisco, Calif.). (ii) Antibodies. The following MAbs were used for neutralization studies and/or envelope-binding studies: 110.5, specific for amino acids QRGPGRAF (315 to 322) of IIIB V3 loop (23, 32); 536, specific for a region of gpl20 involved in CD4 binding (amino acids 423 to 437) (49); 15e, specific for a conserved conformational epitope on gpl20 that has been implicated in CD4 binding (19); 178.1, specific for amino acids KSIRI (310 to 314) of IIIB V3 loop (51); 136.1, specific for amino acids (S)KLRE(Q) (352 to 357) of gpl20; and MAbs 187.2, 190.1, and 213.1, which map to undefined epitopes B, A, and E, respectively, on gpl20 (51). Goat antisera raised against recombinant gpl60 and a linear peptide (RP135) covering the V3 loop were gifts from S. Putney (Repligen). Human sera QC1, 2, 4, 5, and 6 were anonymous donations from HIV-infected individuals in the London area (33). (iii) rCD4-induced virion gpl20 dissociation. Culture supernatants from H9 cells infected with the wt virus or the

J. VIROL.

selected clones 16/2 and 4/1 (100 ,ul) were incubated with various concentrations of rCD4 for 2 h at 37°C. To analyze the effect of rCD4 on virion-bound gpl20, virions were separated from soluble gpl20 and soluble p24 by gel exclusion chromatography on Sephacryl S1000, and viral antigens were detected by ELISA, as described previously (34, 37,

38). Sequencing of variants. (i) PCR amplification of proviral D)NA. Cellular DNA containing proviral HIV-1 DNA was diluted and amplified in a single-molecule nested polymerase chain reaction (PCR) reaction as previously described (47, 48). At dilutions which generate a high frequency (>80%) of negative PCR reactions, the majority of positive reactions result from single molecules of provirus. (ii) PCR amplification of viral RNA. In order to verify the expression of the proviral sequences obtained, viral RNA was prepared from 200-pI aliquots of cell-free supernatant by the RNAzol method (6). One-third of this material (10 Pd) was incubated for 1 h at 37°C with 10 U of DNase (RNase free; BCL), heat treated for 30 min at 65°C, and then used in a first-strand cDNA synthesis. The cDNA reaction was carried out for 1 h at 42°C in a total volume of 20 Rd, which contained 50 mM KCl, 5 mM MgCl2, 5 mM dithiothreitol, 50 mM Tris-Cl (pH 8), 0.5 mM dATP, 0.5 mM dCTP, 0.5 mM dGTP, 0.5 mM dTTP, 0.025 mg of bovine serum albumin per ml (DNase and RNase free; Pharmacia), 15 U of RNAsin (Promega Corp.), 10 U of avian myeloblastosis virus RT (Promega Corp.) and 200 ng of the antisense primer -7814 (CCA TAG TGC TTC CTG CTG CT). The cDNA product

was diluted and PCR amplified in the same way as the DNA. The absence of contaminating cellular DNA was shown by the failure to obtain any PCR product when the cDNA synthesis reaction was carried out as described but without enzyme RT. (iii) Sequencing PCR products. PCR amplified DNA was sequenced directly by using a modification of the standard Sequenase protocol (3, 52, 54). Both strands of the PCR product were sequenced. Several replicates of each reaction were sequenced which confirmed the clonal nature of the material in all cases, except that of the DNA prepared from variant 4/1-infected Hq cells. Site-directed mutagenesis. An oligonucleotide (GGG CAT ACG TTG CTT TTC C) containing the substitution found in the genotype of variant 16/2 was used to change the sequence of pCDNA9, a plasmid construct consisting of the 3-kb EcoRI-XhoI fragment of pHXB2 (which includes the entire envelope gene) in the pcDNAI phagemid eukaryotic expression vector (Invitrogen), allowing transient expression of the env gene in transfected HeLa cells. Levels of envelope glycoprotein expression were assessed by ELISA as described previously. Site-directed mutagenesis was performed with the Amersham oligonucleotide mutagenesis kit in accordance with the manufacturer's instructions. The presence of the substitution in the product plasmid pCKATY was confirmed by sequencing. HIV-1 envelope sequences were found to be unstable in standard E. coli hosts, so the host DL655-F', a recA derivative of DL538, was used for maintenance of the plasmid constructs (5).

RESULTS Selection of HIV-1 variants. HIV-1 extracellular virus produced from pNL4.3-transfected H9 cells was incubated with 20 pig of rCD4 per ml for 1 h at 37°C. The virus-CD4 mixture was then used to infect MT4 cells in a plaque assay. HIV plaques that appeared within the 7 days of the assay

rCD4 ESCAPE MUTANTS OF HIV-1

VOL. 65, 1991 TABLE 1. rCD4 and antibody neutralization titers for HIV variants 16/2 and 4/1 and wt virus Neutralizing

agenta

rCD4 rCD4-IgG Leu3a

QC1 QC2 QC4 QC5 QC6 110.5 Goat anti-RP135 Goat anti-HTLV-IIIB gpl60 536

a

C81 66 *0-

10000

Variant 16/2

Variant 4/1

100 1,000 800

6 1,000 800

12 1,000 800

160 80 20 640 320

160 80 20 640 320

20 10