Infection of cultured human adrenal cells by different strains of HIV

Ashley Barboza, Blesilia A. Castro, Mary Whalen, Chris C.D. Moore*, Jeffrey S. Parkint, Walter L. Miller*, Francisco Gonzalez-Scaranot and Jay A. Levy

Objective: To determine whether human adrenal cells can be infected by HIV.

Methods: Cultured human fetal adrenal cells and the SW13 human adrenocortical carcinoma cell line were inoculated with several HIV-1 and HIV-2 strains. Virus replication was detected by viral core antigen enzyme-linked immunosorbent and reverse transcriptase assays. CD4 expression was measured by Northern blot and polymerase chain reaction procedures.

Results: HIV infection of these adrenal cells was detected and was most evident after cocultivation of the inoculated cells with peripheral blood mononuclear cells. Infection does not involve the CD4 molecule, which is not expressed by these adrenal cells. The relative level of HIV replication depended on the viral strain used. Virus production occurred best in cells that maintained evidence of adrenal cell function. Infection did not appear to disturb steroidogenesis measured in the cells.

Conclusions: These observations indicate that human adrenal cells are susceptible to HIV infection, and provide further evidence of the polytropic nature of the virus.

AIDS 1992, 6:1437-1443

Keywords: Human adrenal cells, HIV infection, CD4 expression, steroidogenesis.

Introduction

Individuals infected with HIV often present with symp- toms suggesting adrenal insufficiency [1-6]. Many have weight loss, diarrhea, fatigue, and dizziness. Cor- tisol response to synthetic ACTH in some patients is reduced and focal adrenal necrosis and hemorrhage have been reported in some infected individuals at au- topsy [1,4-6]. Cytomegalovirus (CMV) is often found in adrenal glands at autopsy [1,4-6] and detected in tissue by in situ hybridization (J. Nelson, personal communication). We investigated the possibility that adrenal cells could also be directly infected by HIV. Our results indicate that certain strains of HIV-1 and HIV-2 can productively infect fetal adrenal cells and the SW13 adrenocortical carcinoma cell line.

Materials and methods

Cells and media

Peripheral blood mononuclear cells (PBMC) from HIV-seronegative individuals were separated by Ficoll- Hypaque from blood provided by Irwin Memorial Blood Bank (San Francisco, California, USA). These cells were stimulated with phytohemagglutinin (PHA; 3 µg/ml) for 3 days, washed, and then used for virus growth, as described previously [7].

Fetal adrenals were obtained at 16 and 21 weeks’ ges- tation from the International Institute for the Advance- ment of Medicine (Essington, Pennsylvania, USA). Cells were isolated by a modification of the proce- dure described by DiBlasio et al. [8]. The capsule

From the Departments of Medicine and *Pediatrics, Cancer Research Institute, University of California, School of Medicine, San Francisco, California and the tDepartments of Neurology and Microbiology, University of Pennsylvania, School of Medicine, Philadelphia, Pennsylvania, USA.

Sponsorship: Supported by the State of California Universitywide Task Force on AIDS, 86SF024 (to W.L.M.), and USPHS grants PO1-AI24286 and NS27405.

Requests for reprints to: Dr Jay A. Levy, Professor of Medicine, Department of Medicine, Cancer Research Institute, School of Medicine, San Francisco, CA 94143-0128, USA.

Date of receipt: 10 June 1992; revised: 25 August 1992; accepted: 3 September 1992.

of the tissue was carefully dissected away and the remaining tissue minced with a scalpel and forceps, and incubated in M-199/Eagle’s Balanced Salt Solution (EBSS) medium containing 1 mg/ml collagenase (Wor- thington Biochem, Freehold, New Jersey, USA) at 37℃ for 30 min in a shaking water bath. The cells were then filtered through a nylon filter mesh (Tetko Inc., Mon- teray Park, California, USA) in a Sweaney filter (Fisher, Pittsburgh, Pennsylvania, USA) and spun at 1500 r.p.m. for 5 min.

Medium for growth of the fetal adrenal cells consisted of 44% M-199/EBSS and Ham’s F12 medium, heat- inactivated (56℃, 30 min) fetal calf serum (10%), 1% glutamine (2mM) and antibiotics (100 IU/ml peni- cillin, 100 µg/ml streptomycin). The cell pellet noted above was resuspended in 1 ml of this fetal adrenal cell medium (FAD) containing 20 µg/ml DNase (Sigma, St Louis, Missouri, USA). After 1 min at 30℃, 9ml of FAD medium was added to the suspension and the cells were plated in T25 flasks. After 24h, the cells remaining in suspension in the flasks were removed and replated in another T25 flask. The initial flask by cell morphology primarily contained fibroblasts, which were maintained in Dulbecco’s modified Ea- gle’s medium (DMEM) with 10% heat-inactivated FCS. The second flask contained almost pure fetal adrenal cortical cells, which required FAD medium for growth. This cell culture was also tested for macrophages by esterase staining; none was detected.

Fetal adrenal medullary cells were isolated from the adrenal medulla of fetuses (13-15 weeks’ gestation) as described above, except that cells were grown in 30 mm dishes (Falcon, Lincoln Park, New Jersey, USA) without replating after 24 h [9]. The FAD medium was used to maintain the medullary cells in culture. The cell line SW13 is a human adrenal cortex carcinoma cell line [10] and was purchased from the American was grown in Leibowitz’s L15 medium supplemented with 10% heated FCS.

Virus

The HIV-1 strains SF2, SF33, SF128A and SF170, and the HIV-2 strains UC1 and UC3 were recovered from PBMC of infected individuals as described previously [7,11-13]. HIV-1SF2mc and HIV-1SF33mc are infectious viruses obtained from cells transfected with the molec- ular clones of the SF2 and SF33 isolates. Virus stocks were grown in culture to high titer in the PBMC of seronegative individuals. Supernatants with reverse transcriptase (RT) activity [14] >106 c.p.m./ml were frozen in 1 ml aliquots at - 70℃ for later use.

Infection of fetal adrenal cells

After 5 days of initial culture, the fetal adrenal cells were treated with 0.5% trypsin for 30 sec, washed and replated in 35 mm tissue culture dishes (Corn- ing, Corning, New York, USA) at a concentration of 2 × 106 cells/dish. After 24 h, the plated cells were in-

cubated with 1 ml diethylaminoethyl dextran (DEAE- D) (25 µg/ml) for 30 min to enhance virus infection [15]. The dextran was removed, the cells washed, and virus added to the cultures for 1h. In all ex- periments 0.5 ml of virus (106 c.p.m. of RT activ- ity/ml) was used. Following absorption, 2ml FAD medium was added to the plates and cultures were left for 2 days before a medium change. When cells were confluent (approximately 5 days), they were trypsinized (0.05%), washed, and replated in fresh 35 mm dishes. Trypsinization was performed to en- sure elimination of the initial virus added [16]. Af- ter 2 days, a second trypsinization was performed and the cells were plated in duplicate dishes. Thus, one set of the fetal adrenal cells could be cultured alone and the other cocultured with PHA-stimulated PBMC from HIV-seronegative individuals. In the latter cultures, PBMC were removed after 3 days and grown separately in RPMI-1640 medium containing 10% heat- inactivated FCS and 5% interleukin-2 (Electronucleon- ics, Silver Spring, Maryland, USA) [7]. The fibroblast- containing cultures derived from the fetal adrenals were treated in a similar manner.

The SW13 cell line was trypsinized and plated at 2 × 106 cells/35 mm plate. After 24h, the cells were treated with DEAE-D as described above, and then in- oculated with virus. Subsequent trypsinizations and virus detection procedures were conducted as de- scribed for fetal adrenal cells.

Virus detection

The presence of virus in culture supernatants was de- termined through either p25 core antigen enzyme- linked immunosorbent assay (ELISA; Coulter Im- munology, Hialeah, Florida, USA) or by standard RT assay [14]. Expression of HIV antigens in the adrenal cells was measured by an indirect immunofluores- cence assay (IFA), as described previously [17].

CD4 mRNA measurement

The presence of CD4 mRNA was detected by North- ern blot of polyadenylated RNA [15] and by a poly- merase chain reaction (PCR). In the first procedure, total cellular RNA from approximately 5 x 106 cells was extracted using the guanidium isothyocyanate method. A prepacked oligo dT column was used for poly (A) RNA purification (Pharmacia, Piscat- away, New Jersey, USA). The poly (A) RNA concen- tration was determined and the entire sample run on a 1% formaldehyde gel in MOPS buffer (20 mM MOPS, 5mM NaOAc, 1 mM EDTA, pH7.0), before being transferred to Nytran in 5 x SSC. The Nytran membrane was prehybridized in 50% formamide/5X Dernhardt’s/0.1% sodium dodecylsulfate (SDS)/200 µg denatured salmon-sperm DNA/5X SSPE at 42℃ for 2 h and then hybridized overnight with a probe consisting of 1.4, 0.55, and 0.35 Kb SsiI and EcoRI fragments of PT4b [18] labeled by the random primer method (Boehringer-Mannheim, Indianapolis, Indiana, USA) to a specific activity >106 c.p.m./µg DNA [19]. The

membrane was hybridized overnight, and washed with 0.5 × SSPE/0.1% SDS at 42℃ for 30 min.

In the second procedure, reverse transcription of poly(A+) RNA and subsequent PCR amplification of the cDNA was performed according to published pro- tocols [20], using enzymes purchased from Perkin Elmer Cetus (San Jose, California, USA). For the ex- perimental samples, 2 µg poly(A+) RNA was reverse transcribed and amplified. The positive control con- sisted of 100 ng poly(A+) RNA from the CEM T- cell line. The negative control contained 100 ng RNA from a human B-cell line, 721.174. This cell line cannot be infected with HIV-1 [21] (K. Stefano and F. Gonzalez-Scarano, unpublished data). The primer pairs have been described previously [20,22]. The pos- itive sense primer was end-labeled to a specific activ- ity of 5 x 106 c.p.m./ug with 32P-labeled y-ATP (NEN Research Products, Boston, Massachusetts, USA) and T4 polynucleotide kinase (Boehringer-Mannheim) ac- cording to the manufacturer’s instructions. The sam- ples were subjected to 35 rounds of amplification at the following cycle conditions: denaturation at 94℃ for 1 min, annealing at 50℃ for 1 min, extension at 72℃ for 2 min. The reaction products were analyzed on a 6% polyacrylamide gel and visualized by direct autoradiography of the dried gels. B-actin primers were used to control for variations in RNA content in an identical but separate reaction. Using dilutions of CEM RNA, the reaction was determined to be capa- ble of giving a signal above background (721.174 cells) when 100-1000 pg of CEM RNA was used for reverse transcription.

Steroid assays

Cultures were tested for the production of corti- sol and dehydroepiandrosterone sulfate (DHAS) using standard radioimmunoassays (RIA) [8]. Antibodies to cortisol were purchased from Diagnostic Products Inc. (Los Angeles, California, USA) and antibodies to DHAS from Radioimmunoassay Systems Inc. (Carson, Califor- nia, USA).

RNase protection assay for P450scc

RNA from control and infected human fetal adrenal cells was hybridized to a 409-base 32p riboprobe transcribed from the T3 promoter of a pBluescript construction containing a 334 bp PCR-amplified frag- ment of the human P450scc gene, which was amplified with oligonucleotides 5’scc1 and 3’scc2 and encom- passes 310 bp of exon 1 and 24 bp of intron 1 [23]. RNA preparation, riboprobe synthesis, and conditions for hybridization, electrophoresis, and autoradiogra- phy were performed as described by Moore et al. [24]. The assay detected P450scc mRNA in 0.5 µg total RNA from JEG-3 choriocarcinoma cells, or in 0.01 µg total RNA from intact human fetal adrenals.

Results

Culture of cells from fetal adrenal tissue

The T25 flasks receiving cells in suspension after 24 h contained almost exclusively epithelioid cells that had morphologic properties of adrenocortical cells (see Materials and methods) (Fig. 1a). These adrenal cells were sensitive to trypsinization and grew well in FAD medium. Cells in T25 flasks that adhered in the first 24 h had fibroblast morphology. Over time, these lat- ter cultures contained a high percentage of fibroblasts, although some epithelioid cells were still present. The fetal medullary cells plated in 30 mm dishes did not form a confluent monolayer, but grew in aggregates on the surface of the dish. Within 3-5 days, filaments resembling neurites extended from the clumps (Fig. 1b), as described previously [9].

Virus infection and replication in cultured adrenal cells

In the first set of experiments, five different HIV-1 strains were added to fetal adrenal or SW13 cells and virus replication measured by RT assays (Tables 1 and 2). Here, virus was detected only after cocul- tivation with normal PBMC. Fetal adrenal medullary cells appeared to be more susceptible to HIV than fetal cortical cells, since all four HIV-1 strains tested replicated in the medullary cells. In contrast, only HIV-1SF33 replicated in the SW13 line (Table 2). HIV-1SF33 and HIV-1SF128A replicated reproducibly in adrenal cortical cells. The first strain has a wide cellu- lar host range, but is not macrophage-tropic [13]; the second grows to high titer in macrophages [12].

In other experiments, virus replication in fetal adrenal cells was measured by the p25 antigen ELISA; this pro- cedure is more sensitive than the RT assay for detect- ing low-level virus production in culture. These stud- ies indicated that a large number of HIV-1 and HIV-2 strains can infect fetal adrenal cells (Table 3), but that virus production is low. The p25 antigen levels in cul- tures not cocultivated with PBMC ranged from 20 to 80 pg/ml, but were persistent, even after two or three trypsinizations (Fig. 2a).

In comparative studies, HIV-1SF128A and HIV-1SF33 grew to higher levels in fetal adrenal cells than HIV-1SF2. The p25 antigen levels after three cell pas- sages reached >700 pg/ml, compared with 43 pg/ml with HIV-1SF2 (data not shown). Supernatants from fetal adrenal cell cultures cocultured with PBMC showed much higher levels of p25 antigen (Fig. 2b). Moreover, in contrast to the fluids obtained from adrenal cells cultured alone, these supernatants con- tained sufficient virus to be detected by the RT assay (≥5000 c.p.m./ml). Inoculation of this fluid onto fresh PBMC also produced productive infection. Sensitivity to virus infection was most evident in early passaged cells. The cells had greatly reduced susceptibility to

Fig. 1. (a) Fetal adrenocortical cells (16-21 weeks' gestation) cultured for 1 week. (b) Fetal adrenal medullary cells showing neurite formation after 3-5 days in culture.

(a)

(b)

Table 1. Infection of fetal adrenal cells.
Virus	Cells
Virus	Cortical	Medullary
HIV-1SF2	0/3	3/4
HIV-1SF33	2/4	3/4
HIV-1SF128A	3/5	3/4
HIV-1SF162	0/1	1/1

All cultures were tested for the presence of virus by reverse transcrip- tase (RT) assay [14]; a sample was considered positive if the culture fluid had ≥ 10 000 c.p.m./ml of RT above background (1000 c.p.m./ml). Figures represent number of cultures positive/number of attempts. In all cases, virus was detected only after coculture of the cells with peripheral blood mononuclear cells from seronegative donors.

HIV after four to five passages and 1 month in cul- ture.

In related experiments, adherent cells derived from fe- tal adrenals within 24h of plating in T25 flasks were inoculated with HIV-1. In general, these cells were less susceptible to virus infection than the epithe- lioid cultures, and demonstrated lower levels of p25 antigen in the supernatants of the cells not cocul- tivated with PBMC. Nevertheless, it was not possible to determine whether virus replication was restricted

Table 2. Infection of the SW13 human adrenal cell line.
Virus	Alone	+ PBMC
HIV-1SF2	0/2	0/2
HIV-1SF33	0/2	2/2
HIV-15F128A	0/1	0/1
HIV-2UC3	0/1	0/1

See legend to Table 1. Virus was detectable only in HIV-15F33-infected cul- tures mixed with normal phytohemagglutinin-stimulated peripheral blood mononuclear cells (PBMC; see text).

to the epithelial-like cells present or the fibroblasts that predominated in the culture. HIV antigens could not be detected by IFA in any of the inoculated adrenal cell cultures; these results probably reflect the low-le- vel virus replication in these cells.

Steroidogenic capacity of infected cultured fetal adrenal cells

P450scc mRNA, which encodes the first step in steroid hormone synthesis, is detectable only in the adrenals and other steroidogenic tissues [24,25]. There is abun- dant P450scc mRNA in uncultured human fetal adrenal tissue, as shown by the very strong signals after 6 or 20 h of autoradiographic exposure of a specific RNase protection assay (Fig. 3). After 5 days of culture, the

p25 Ag Level (pg/ml) ~

DAYS

Fig. 2. Replication of HIV-1 strains in fetal adrenal cells. (a) p25 antigen levels in cultures of fetal adrenal cells infected with HIV-1SF128A or HIV-1SF33. Arrow indicates the time of the second trypsinization. (b) p25 antigen levels in normal peripheral blood mononuclear cells cocultivated with the adrenal cell cultures 7 days after inoculation of the above viruses and following two trypsinizations. SF128; SF33.

p25 Ag Level (pg/ml) 5

1000

800

600

400

200

DAYS

Table 3. Infection of fetal adrenocortical cells.
Virus	Alone	+ PBMC
HIV-15F2	1/3	3/3
HIV-1SF2C	1/2	2/2
HIV-1SF13 HIV-1SF33	1/1 2/4 2/3 1/1	1/1
HIV-1SF13 HIV-1SF33		4/4 3/3 1/1
HIV-1SF128A HIV-1SF162		4/4 3/3 1/1
HIV-1SF170	1/2	2/2
HIV-2UC1	0/1	1/1
HIV-2UC3	0/1	1/1

All culture fluids were tested for the presence of virus production by the p25 antigen assay using the Coulter enzyme-linked immunosorbent as- say. Figures represent number of positive cultures/number of attempts. A sample was considered positive if it had > 10pg/ml of p25 antigen. Virus, detection was enhanced by cocultivation of the adrenal cells with normal peripheral blood mononuclear cells (PBMC).

abundance of P450scc mRNA declined from the level seen in the intact adrenal tissue (compared with the 6- h exposure). After 3 weeks of culture, P450scc mRNA

Fig. 3. RNase protection assay of P450scc mRNA. Each lane shows the hybridization products of 10 µg total cellular RNA with the 409 base pair (bp) 32P riboprobe of human P450scc. All lanes were run on a single gel, but for clarity the figure is shown as a composite of various autoradiographic exposures. The left lane (a), designated 'Adrenal', contains RNA from an initial 5-day cul- ture of human fetal adrenal cells (i.e., a monolayer in a secondary flask grown in fetal adrenal medium); the autoradiographic expo- sure was 6 h. The next six (b-g) lanes contain RNA from primary adrenocortical or fibroblast-containing cultures from human fetal adrenals infected with the three strains of HIV-1 indicated. The next two lanes (h,i) are controls of adrenocortical or fibroblast cultures from human fetal adrenals without HIV infection. These eight lanes (b-i) of adrenocortical or fibroblast-containing cells, with or without HIV infection, show a 2-week autoradiographic exposure. Note that the abundance of P450scc mRNA is sharply decreased in the 4-week HIV-infected and control cultured adrenal cells compared with the initial 5-day culture (a). The lanes (j and I) labeled fetal adrenal 6 and 20 h are 6- and 20-h expo- sures of a single lane containing 1 µg RNA from an intact human fetal adrenal; this latter is shown for contrast with the 2-week exposure of the HIV-infected and control cells. JEG-3 (k) is 10 µg total RNA from a steroidogenic human choriocarcinoma cell line known to have P450scc mRNA that was detected in these exper- iments on a 20-h exposure. tRNA (m) is a control hybridization with 10 mg of calf thymus tRNA; the probe (n) is the 409 bp probe without RNase digestion. The molecular size markers are end- labeled Hpall fragments of pUC18, with sizes shown in nu- cleotides. The sizes of the 409 bp probe and the 310 bp protected fragment are indicated at the left.

Adrenal Fibrob

Fetal adrenal (6 hr)

Felal adrenal (20 hr)

HIV strain

Adrenal

SF128A

SF3A

Fibroblasts

SF2

SF128A

SF3A

SF2

Adrenal

JEG-3

IRNA

Probe

Markers

501

489

404

409

353

310

242

190

was still detectable, but at a substantially lower level (note 2-week autoradiographic exposure). P450scc mRNA is not expressed in fibroblasts or in the adrenal medulla, so that cultures containing a high percent- age of fibroblast-like cells had much lower levels of P450scc mRNA. Virus infection did not appear to affect the expression of this mRNA; whether some enhance- ment in expression occurred (as suggested in Fig. 1) is not clear. P450scc mRNA could no longer be de- tected in cells that had been cultured for more than 6 weeks. Nevertheless, these cells remained infectable by HIV, although, as noted above, at lower efficiency. In addition, after 6 weeks in culture cortisol and DHAS production was not detected in the adrenal cells (data not shown). These results could reflect loss of activity

of the adrenal cortical cells, or dominance of fibrob- lasts after long-term culture, or both.

Expression of CD4 mRNA and protein in cultured adrenal cells

No expression of CD4 protein was detected on fetal adrenal cells or SW13 cells by immunofluo- rescence testing using Leu 3a antibodies (Becton Dickinson, San Jose, California, USA). Moreover, when the cultured cells were extracted and examined for CD4 mRNA, none was detected with Northern blot or PCR analyses (Figs 4 and 5). PCR was used because of its increased sensitivity; this method permitted detec- tion of CD4 mRNA in 100-1000 pg of poly(A+) RNA from CEM cells. The PCR analysis was performed on the same extracted RNA used for the measurement of the P450scc mRNA present. Thus, cells with morpho- logic and biochemical features of fetal adrenal cells did not appear to express CD4 protein or message.

Discussion

These cell culture studies were undertaken to deter- mine whether the adrenal gland can be infected by HIV-1 in vivo. Such an infection might help to explain the adrenal necrosis and disorders of adrenal steroido- genesis observed in some infected individuals [1-6]. While HIV has not yet been detected in adrenal tis- sue by in situ hybridization (J. Nelson, personal com- munication), the present studies suggest that adrenal cells can be infected by certain strains of HIV-1 and HIV-2. The receptor for HIV entry of adrenal cells is unknown, since CD4 could not be demonstrated by Leu 3a antibody studies, nor by the presence of CD4 protein or mRNA in the cultured cells. Although viral infection of CD4+ cells often leads to high titers of the infecting virus, the adrenal cell infection, like those of many other CD4+ cells [15], is limited. Only low levels of p25 antigen were found in the cell cul- ture supernatant after infection, and viral replication was best detected after cocultivation with appropriate target cells, such as PBMC.

The data indicate that the infected cells came from the adrenal cortex or medulla. No macrophages were detected in the inoculated cultures and some of the strains that replicated are not macrophage-tropic (HIV-1SF33, HIV-1SF170). The morphology of the cul- tured cells is consistent with adrenal cells, rather than fibroblasts (Fig. 1). Furthermore, the cells from the adrenal cortex contained P450scc mRNA, characteris- tic of adrenocortical cells [24] (Fig. 5), and the highest sensitivity to HIV infection correlated with the expres- sion of the steroidogenic enzymes. Another feature not observed with fibroblasts is detection of HIV-1 in the culture fluid by p25 antigen assay; human PBMC must be added to the cultured infected fibroblasts to detect the virus [15]. Moreover, HIV-1SF2, which does not in- fect fibroblasts [15], replicates in adrenocortical and

medullary cells (Tables 1 and 3). Furthermore, with medullary cells, it appears unlikely that any contam- inating fibroblasts or macrophages were present in the clumps of epithelial cells that produced neurites in culture. Finally, the successful infection of adreno- cortical cells cultured for over 2 months and found to be devoid of P450scc activity is also consistent with our ability to infect the SW13 human adrenal epi- thelial carcinoma cell line, which also fails to produce steroids.

Fig. 4. CD4 mRNA in uninfected adrenal cells. Approximately 5 × 106 cells were extracted and polyadenylated RNA trans- ferred to nitran membranes and hybridized overnight with a CD4 probe. The amounts of poly(A+) RNA subjected to elec- trophoresis were 17 µg for SW13, 3.6 µg for fetal adrenal cells (FAC), and 2.2 µg for the Sup-T1 established cell line. This cell line served as a control for CD4 mRNA expression.

SW 13 FAD

SUPT 1

Fig. 5. RNA polymerase chain reaction (PCR) detection of CD4 in adrenal tissues. PCR amplification of mRNA was performed as indicated in Materials and methods. The amplified products of reactions using either 32P end-labeled CD4 or b-actin primers were resolved on a 6% polyacrylamide gel with tri-buffered EDTA. Samples were visualized by direct autoradiography for 30 min at room temperature. POS, reaction using mRNA ex- tracted from CEM cells (100 ng); NEG, reaction using mRNA ex- tracted from 721.174 cells (100 ng); RNA from the non-infectable cell line generated a small signal, presumably from contaminating genomic DNA. Lanes 1-9 are with aliquots of the RNA samples shown in Fig. 3, starting from the left; lane 1 is 5-day-old adrenal cell culture; lanes 2-9 are 3-week-old fetal adrenal control cells. Note that the negative experimental samples contained 20 times as much RNA as the negative cell line, 721.174.

5 Q Z 1 2 3 4 5 6 7 8 9

CD4 -

B-ACTIN -

The loss of steroidogenic competency of the cultured adrenal cells after 4-6 weeks in culture is not surpris- ing. DiBlasio et al. [8] showed that primary cultures

of human fetal adrenal cells maintained under con- ditions that optimize steroidogenesis lost 90-95% of their mRNA for P450scc (the rate-limiting enzyme in steroidogenesis) after 12 days. The protracted cul- ture period used in these experiments further reduced steroidogenic capacity.

The heterogeneity of HIV-1 and HIV-2 strains was also revealed by the relative ability of different viruses to infect and replicate in human fetal adrenal cells and the SW13 human adrenal cell line.

HIV-1SF33 and HIV-1SF128 were particularly capable of infecting cultured fetal adrenal cells. These studies sug- gest that certain HIV strains may selectively infect adrenal cells, influencing the function of this tissue, even if high levels of virus replication do not occur. Moreover, further studies with adrenal cell lines pro- ducing hormones should determine whether the virus affects basic cell function.

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