Vol. 64, No. 1

JOURNAL OF VIROLOGY, Jan. 1990, p. 215-221

0022-538X/90/010215-07$02.00/0 Copyright ©) 1990, American Society for Microbiology

Failure of Human Immunodeficiency Virus Entry and Infection in CD4-Positive Human Brain and Skin Cells BRUCE CHESEBRO,* RICHARD BULLER, JOHN PORTIS, AND KATHY WEHRLY

National Institutes of Health, National Institute of Allergy and Infectious Diseases, Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories, Hamilton, Montana 59840 Received 15 May 1989/Accepted 14 September 1989

CD4 molecules on human cells function as a major receptor for human immunodeficiency virus (HIV); however, certain CD4-negative cell types may also be susceptible to infection. Therefore, we attempted to quantitate the relationship between HIV infection and CD4 expression on human cell lines before and after introduction of the CD4 gene by using a retrovirus vector. Prior to introduction of the CD4 expression vector, low levels of HIV infection were detected by a sensitive focal immunoassay on all three cell types studied. With several HIV strains in clones of human cervical carcinoma (HeLa) cells expressing different levels of CD4, HIV titer increased with increasing CD4 expression. In contrast, in squamous cell carcinoma cells (SCL1) and astroglial cells (U87MG), even high levels of CD4 expression failed to augment HIV infection. The CD4 protein expressed in these two cell lines had the expected molecular weight and was capable of binding HIV virions. However, in contrast to CD4-positive HeLa cells, CD4-positive U87MG and SCL1 cells were unable to form syncytia when cultured with cells expressing HIV envelope protein. Thus, the inability of HIV to infect these cells appeared to be due to lack of fusion between HIV virion envelope proteins and CD4-positive cell membranes. This block in infectivity was overcome when cells were infected with HIV which was pseudotyped with the envelope protein of amphotropic murine leukemia virus. Thus, in addition to CD4, other cell surface molecules appear to be required for successful HIV entry into and infection of these two human cell lines. Numerous lines of evidence indicate that the cell surface T4 (CD4) protein encoded by the CD4 gene is a receptor for human immunodeficiency virus (HIV) (10, 16). However, not all CD4-positive cells in the blood of acquired immunodeficiency syndrome (AIDS) patients are infected with HIV, and one possible explanation is that not all CD4-positive cells are permissive to HIV infection. Furthermore, there have been several reports of HIV infection in vivo and in vitro of cell types which do not express CD4 (6, 8, 9, 11, 22, 28, 29, 31). Thus, it seems likely that both cell type and receptor availability might influence HIV infection. We recently developed an accurate quantitative focal immunoassay for HIV infectivity based on creating plasticadherent target cells susceptible to HIV infection by insertion of the CD4 gene in an expression vector (7). Foci of HIV infection can then be quantitated by immunological techniques. With the availability of this new assay, we wanted to accurately compare the level of sensitivity to infection by HIV with the level of CD4 expression in different types of human cell lines. The results indicated that even in the absence of CD4 expression, all three cell types showed low but significant levels of infection by most HIV strains. After introduction of a CD4 expression vector, HIV infection correlated well with CD4 expression in HeLa cells. In contrast, CD4 expression did not consistently increase infection of astroglioma or squamous cell carcinoma cells. CD4 expressed in these cells could bind HIV, but HIV envelope protein was unable to induce syncytium formation, suggesting that HIV virions were unable to enter these cells. This block to HIV entry was overcome when HIV particles pseudotyped with amphotropic murine leukemia virus (MuLV) envelope protein were used for infection. *

MATERIALS AND METHODS

Viruses and cells. HIV stocks were obtained as described previously (7). In addition, the Alabama strain (2) was obtained from T. Folks, National Institute of Allergy of Infectious Diseases, Bethesda, Md. Viruses were propagated by infection of A3.01 cells. U87MG human astroglioma cells (24) were obtained from the American Type Culture Collection, Rockville, Md. SCL1 human squamous cell carcinoma cells (4) were obtained from L. Roberts, Department of Dermatology, University of Utah School of Medicine, Salt Lake City, Utah. HeLa human cervical carcinoma cells (12) were obtained from D. Lodmeil, Rocky Mountain Laboratories. Generation of CD4-positive cell clones. PA12 cells producing a replication-defective retroviral vector encoding the human CD4 gene and neomycin resistance (Neor) gene (17) were obtained from D. Littman, University of California at San Francisco, and were used to produce HeLa cells expressing CD4 as described previously (7). These PA12 cells were found in some cases to produce an undesired replication-competent helper murine retrovirus with amphotropic MuLV envelope. This replication-competent virus was eliminated by two successive cycles of limiting dilution cloning on T2 cells (18) and PA317 cells (20) as described by Miller et al. (21). After the final infection, PA317 clones were selected by growth in the neomycin analog G418. The clones obtained were tested for replication-competent MuLV by infection of NIH 3T3 cells and analysis of release of reverse transcriptase after one or two passages. The final clones used were negative for MuLV and produced CD4 and Neor-expressing defective retrovirus particles at a concentration of 1 x 104 to 5 x 104 Neor CFU/ml. These supematant fluids were used to infect U87MG and SCL1 cells, and neomycin-resistant clones were selected and screened for CD4 expression as described previously (7).

Corresponding author. 215

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J. VIROL.

CHESEBRO ET AL.

FIA. Cells (5 x 104) were seeded in 35-mm wells, and 1 day later wells were treated for 20 min with 8 ,ug of DEAE-dextran per ml, rinsed once, and infected with 0.5 ml of serial dilutions of virus for 90 min at 37°C. Then, 2 ml of medium plus 10% serum was added. Cells were cultured for 4 more days and then fixed with methanol and processed for focal immunoassay (FIA) as described previously (7). Briefly, wells were rinsed with phosphate-buffered saline (PBS), incubated for 20 min with 0.15 ml of mouse monoclonal antibody specific for HIV p249ag or a 1:300 dilution of human AIDS patient serum with antibody against HIV. Wells were then washed twice with buffer containing 0.01 M Tris hydrochloride (pH 7.5)-0.15 M NaCl-0.001 M EDTA (TNE) and incubated for 30 min with horseradish peroxidase-conjugated antibodies reactive with either mouse or human immunoglobulins. Wells were washed again twice with TNE and then reacted with aminoethylcarbazol and H202 as described by Nexo (23). Foci of infected cells were counted with a dissecting microscope at a magnification of 20 to 40x. Foci consisted of 2 to 20 grouped cells with stained cytoplasm and clear nuclei. Similar results were obtained with either the monoclonal antibody or the human anti-HIV serum. FACS analysis. To prepare cells for fluorescence-activated cell sorter (FACS) analysis, adherent cells were removed

from plastic tissue culture flasks by trypsinization for 8 min at 37°C. Fetal calf serum was added to 25% to block trypsin activity. The cells were then filtered through nylon mesh to remove clumps, counted, and distributed to a 96-well tray (5 x 105 cells per well) in 50 ,ul of RPMI 1640 medium containing 0.01 M NaN3, washed twice with RPMI-azide, and then incubated with 5 RI of commercially obtained monoclonal antibodies OKT4A and OKT8 (Ortho Diagnostics, Raritan, N.J.) in 50 ,u of RPMI-azide for 1 h at 4°C. After two washes with RPMI-azide, cells were incubated in 150 ,ul of a 1:180 dilution of fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse immunoglobulin in RPMI-azide for 1 h at 4°C. Following two washes with RPMI-azide, the cells were suspended in 1 ml of PBS containing 1% formaldehyde. The stained and fixed cells were analyzed on a FACSTAR cell sorter (Becton Dickinson, Mountain View, Calif.) with an argon ion laser emitting 488-nm light at 200 mW. Fluorescence was measured with logarithmic gain, full-scale fluorescence being 4 log units. Cells were also analyzed with monoclonal antibodies OKT4 and anti-Leu-3a, which gave results similar to OKT4A, and controls were done with no first antibody, which gave results identical to those with OKT8 (data not shown). In some experiments, cells were detached from plastic dishes by scraping in PBS with 0.002 M EDTA without trypsin. Similar FACS results were obtained, although cell clumping caused more technical difficulties. Northern (RNA) blot analysis. mRNA was isolated from tissue culture cells by lysis in 4 M guanidinium isothiocyanate followed by CsCl centrifugation and poly(A) selection on an oligo(dT) column. Samples (3 pRg) of poly(A)-selected RNA were electrophoresed on a 1% agarose-6% formaldehyde gel, transferred to nitrocellulose, hybridized, and washed as described previously (5). Immunoprecipitation. Cells were labeled for 7 h with 200 ,Ci of [35S]methionine per ml in methionine-free minimal essential medium (MEM) containing 1% dialyzed fetal calf serum. After three washes with phosphate-buffered balanced-salt solution, cells were lysed for 1 min with 2 ml of ice-cold lysis buffer (0.1% sodium dodecyl sulfate [SDS], 0.5% sodium deoxycholate, 1% Nonidet P-40 in 0.05 M Tris

[pH 7.4], 0.15 M NaCl, 0.001 M EDTA). Nuclei were pelleted for 30 s in a Beckman Microfuge B, and the supernatant was saved. For immunoprecipitation of CD4, lysate containing 100,000 trichloroacetic acid-precipitable counts per minute (volume ranging from 180 to 800 ,ul) was incubated overnight at 4°C with 2 ,ug of OKT4A antibody. Immune complexes were recovered by addition of 50 p.l of a 10% suspension of protein A-Sepharose CL-4B (Pharmacia, Uppsala, Sweden). After 30 min on ice, the beads were washed seven times with 1 ml of iced lysis buffer and eluted by boiling in 40 pul of sample buffer (15). Immunoprecipitates were resolved by 10% SDS-polyacrylamide gel electrophoresis (PAGE). Gels were treated with Entensify (NEN Research Products, Boston, Mass.) prior to autoradiography. Virus binding. Virus binding was done as described before (16, 19). Cells (2 x 105) were incubated for 30 min at 37°C in round-bottomed microtiter wells with 200 ,ul of supernatant from HIV-producing A3.01 cells. HIV strains Zaire-2 (Z-2) and IIIB were used, and fluid from uninfected cells was the negative control. Cells were then washed twice with medium containing 0.01 M NaN3 and incubated for 60 min at 4°C with 50 ,ul of a 1:50 dilution of human anti-HIV serum in medium with azide. Cells were washed twice, as before, incubated for 45 min at 4°C with 50 ,l of a 1:180 dilution of FITCconjugated sheep anti-human immunoglobulin in medium with azide, washed twice again, and fixed with fresh 1% formaldehyde in PBS. Cells were examined in a double-blind fashion with a Leitz Orthoplan fluorescence microscope and scored on a relative scale for brightness of fluorescence intensity. From 100 to 200 cells were examined, and negative populations had less than one to two positive cells observed. Cell fusion. Syncytium induction was done by seeding flat-bottomed microtiter wells with 1.2 x 104 cells in medium. After incubation at 37°C for 3 h, 2 x 104 H9 cells chronically infected with HIV (strain NL4-3) were added. No H9 cells or uninfected H9 cells were added to control wells. After incubation for 15 h at 37°C, the cells were fixed with methanol, stained with Giemsa, and examined for multinucleated syncytial cells. Production of HIV pseudotyped with amphotropic MuLV envelope protein. A3.01 cells were infected with amphotropic MuLV (strain 4070), and cells were passaged several times to allow all the cells to become infected. Infection was followed by staining cells with a monoclonal antibody reactive with MuLV envelope, kindly provided by L. Evans. Cells were then infected as usual with HIV (NL4-3 strain), and virus stocks were made from culture fluid just before maximal cytopathic effect was evident. RESULTS HIV infection of three human cell lines before and after CD4 expression. For these studies we used HeLa cells (derived from a cervical cell carcinoma), U87MG cells (derived from an astroglioma), and SCL1 cells (derived from a squamous cell carcinoma). CD4 genes were introduced into these cells by infection with a retroviral vector containing the human CD4 gene and the neomycin resistance gene (17). Representative clones were selected by neomycin resistance. Detection of cell surface CD4 by FACS analysis indicated that several clones expressing different amounts of CD4 were obtained from each of the original cell lines (Fig. 1). Five HIV strains (LAV, NY5, Alabama, NL4-3, and IIIB) propagated on A3.01 human T-lymphoma cells were studied. In the HeLa cell series, it was observed that the highest HIV

VOL. 64, 1990

CD4 EXPRESSION AND HIV INFECTION

SCL- 1

U87MG

HeLa N

217

N

N 400100200-

11

60

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1

2

110 3

104 2001

3

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IA 101

100

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103

104

162

163

104

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200-

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161

102

103

2001

6C 400-

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FIG. 1. FACS analysis of cell lines with monoclonal antibodies reactive with CD4 (OKT4A, solid line) or with CD8 (OKT8, dashed line). Plots marked with the letter N represent normal cells not infected with the retroviral vector carrying the CD4 gene.

TABLE 1. Infectivity titer of HIV stocks determined in various cell lines by FIA' Infectivity titer (FFU/ml) HIV

HeLa

strain 6C

LAV NY5 Alabama NL4-3 IIIB

(++)b

360 7,500 4,000 2,500 1,800

SCL1

U87MG

4C(+)

1C ()

N(-)

2 (+++)

1 (++)

N(-)

6 (+++)

5 (++)

3(+)

N(-)

120 5,300 2,000 800 1,000

20 530 86 20 55

4