In Vitro Infection of Primary and Retrovirus-Infected Human ... - NCBI

JOURNAL OF VIROLOGY, May 1996, p. 2774–2780 0022-538X/96/$04.0010 Copyright q 1996, American Society for Microbiology

Vol. 70, No. 5

In Vitro Infection of Primary and Retrovirus-Infected Human Leukocytes by Human Foamy Virus JUDY A. MIKOVITS,1* PAUL M. HOFFMAN,2 AXEL RETHWILM,3

AND

FRANCIS W. RUSCETTI4

Biological Carcinogenesis Development Program, SAIC-Frederick,1 and Laboratory of Leukocyte Biology, Division of Basic Sciences,4 NCI-Frederick Cancer Research and Development Center, Frederick, Maryland 21702-1201; Veterans Administration Medical Center and Department of Neurology, University of Maryland, Baltimore, Maryland 212012; and Institute fur Virologie und Immunobiologie, Universitat Wurzburg, 97078 Wurzburg, Germany3 Received 16 August 1995/Accepted 26 January 1996

The infectivity of human foamy virus (HFV) was examined in primary and cultured human leukocytes. Cell-free infectious viral stocks of HFV were prepared from the human kidney cell line 293 transfected with an infectious molecular clone of HFV. HFV productively infects a variety of human myeloid and lymphoid cell lines. In addition, primary cell cultures enriched for human CD41, monocytes and brain-derived microglial cells, were readily infected by HFV. Interestingly, while infected primary CD41 lymphocytes and microglial cells showed marked cytopathology characteristic of foamy virus, HFV-infected monocyte-derived macrophages failed to show any cytopathology. In addition, marked cytotoxicity due to HFV infection was seen in both human T-cell leukemia virus type 1- and human immunodeficiency virus type 1-infected T-cell lines and in human immunodeficiency virus type 1-infected monocytoid cell lines. Thus, HFV infection produces differential cytopathology in a wide host range of primary human leukocytes and hematopoietic cell lines. we examined whether dual retroviral infections could occur in these cell types.

Foamy viruses (FV) comprise the third group of complex human and primate exogenous retroviruses which include the oncovirus (human T-cell leukemia viruses [HTLVs] and simian T-cell leukemia viruses) and lentivirus (human immunodeficiency viruses [HIVs] and simian immunodeficiency viruses) families (10, 30, 32). Human FV (HFV) has several genomic and structural similarities with HIV type 1 HIV-1 and HTLV type 1 HTLV-1, most notably, complex alternate splicing of the genome (3, 10, 28, 32) and the encoding of a sequence-specific activator of viral transcription, Bel-1 (15, 23, 34, 35). However, there are major differences between HFV and other complex retroviruses, including the absence of a Rev/Rex posttranscriptional function (3, 32, 34) and the rapid nuclear localization of Gag precursor in the nucleus (39). FV cause persistent and apparently nonpathogenic infections in their natural hosts (30, 32, 38). While readily isolated from a wide range of tissues, including peripheral blood cells and the brain (11, 13, 31), from many animal species, there exists only one human isolate, which was obtained in 1971 from an explant culture of a nasopharyngeal carcinoma originating in Kenya (1). Recent studies suggest that infections in humans are rare and that FV do not spread in the human population (30, 38, 42, 43). While FV have been shown to replicate well in fibroblasts and poorly in epithelial cells (21, 22, 49), viral replication in other cell types, particularly those of the hematopoietic lineages, is not well characterized. Previous reports demonstrated in vitro infection of a small number of malignant cell lines (24, 49), but little is known about HFV infection in primary human cells. In addition, a high viral load has been found in the brain (13, 30, 31) and HFV transgenes are preferentially expressed in the brains of mice (2, 4). Therefore, HFV infection of primary leukocyte and microglial cells was studied. In addition,

MATERIALS AND METHODS Plasmids. An infectious molecular clone for HFV, pHSRV, was constructed from viral DNA and cDNA clones as previously described (33). HFV p59 cat (2777/14), a plasmid in which expression is directed by the complete 59 long terminal repeat (LTR), and the bel1 expression vector were prepared as previously described (35). Expression vectors for HIV-1 Tat and HTLV-1 Tax were kindly provided by Dave Derse (National Cancer Institute, Frederick, Md.) (7, 8). Detection of HFV nucleic acids. RNAs and DNAs were prepared by using RNA/DNA Stat reagents (Tel Test, Inc., Friendswood, Tex.) according to the manufacturer’s instructions. To detect HFV DNA, DNA was amplified by the following env primer sequences: sense primer (nucleotides 8331 to 8356 of the provirus), 59 ATC TCA AAT ATC AGA TAT TAA TGA TG; and antisense primer (nucleotides 8711 to 8686 of the provirus), 59 CAA TTA TTC AAG TAA ATA TGT TTA GG. The cycle conditions for 35 cycles were 958C for 15 s, 558C for 60 s, and 748C for 90 s. cDNA synthesis with random hexamer primers and reverse transcriptase PCR (RT-PCR) were performed as previously described (25). The primer pairs for RT-PCR, 59 primer in the LTR (59 GAG CTC TTC ACT ACT CGC TGC) and 39 primer in gag (59 GCT CGT TGT ATT ACC TCA TAT C), cross a splice junction (32), thus controlling for contamination by input viral DNA. Amplification was achieved by 35 cycles of 958C for 30 s, 608C for 60 s, and 728C for 120 s, as previously described (25). RT-PCRs for HIV using the SK38 and SK39 primer pair (25) and for HTLV-1 using HTex1-2–HT7875, which crosses gag exons 1 and 2 (8) were performed as previously described (25). Cell lines. Human 293 cells were maintained in Dulbecco’s modified Eagle medium supplemented with 10% fetal bovine serum (Inovar), 100 U of penicillin per ml, 100 mg of streptomycin per ml, and 2 mM glutamine. Human myeloid (KG-1, KG-1a, THP-1, and U937) and lymphoid (HUT 78, CEM, and Raji) cell lines were obtained from the American Type Culture Collection and maintained in RPMI 1640 medium supplemented as was Dulbecco’s modified Eagle medium. The HTLV-1- and HIV-1-infected cell lines (HUT 78/BP-1, THP-1/ADA, MT-2, and 81-66) were maintained and characterized as previously described (26, 37). All cell lines were negative for mycoplasma. Leukocyte and microglia purification. Peripheral blood mononuclear cells (PBMC) were prepared by Ficoll-Hypaque centrifugation, with subsequent centrifugal elutriation to obtain purified lymphocyte (92%) and monocyte (95%) populations, as determined by differential staining as previously described (44). T cells were further separated into CD41 and CD81 subsets (.95% pure) by using magnetic-bead technology according to the manufacturer’s (Miltenyi Biotec, Auburn, Calif.) instructions. T cells were then activated with phytohemagglutinin (1 mg/ml) for 48 h before infection. Microglial cells were obtained, maintained, and infected as previously described (12). Immunofluoresence microscopy. Immunofluoresence microscopy was per-

* Corresponding author. Mailing address: NCI-FCRDC, P.O. Box B, Bldg. 567, Rm. 253, Frederick, MD 21702-1201. Phone: (301) 8465610. Fax: (301) 846-7034. 2774

VOL. 70, 1996

FOAMY VIRUS INFECTION OF PRIMARY HUMAN LEUKOCYTES

TABLE 1. Effects of HFV replication in human cell lines and primary cells Cell target

Cell lines KG-1a KG-1 U937 THP-1 HUT 78 Raji CEM Primary cellsc PBMC CD41 T cell CD81 T cell Monocyte Microglia

Cell type

Promyeloblast Myeloblast Promonocyte Monocyte T cell B cell Immature T cell

HFV RT (103 cpm/ml)a

Cytopathologyb

230

No effect No effect No effect No effect Delayed cell death No effect No effect

28 32 156 21 380 140 6 28 10

Cell death Cell death No effect No effect Cell death

a Cells were infected with HFV stocks at an MOI of 0.1. Supernatants were analyzed for RT activity, with the results for day 6 shown, except the results for microglial cells from day 3 are shown. 6, #15% of twice the background. b Cellular pathology was examined by light microscopy of infected cells biweekly for 1 month after infection. Cell death was measured by trypan blue exclusion. Infected HUT 78 cells died after three or four passages (15 to 20 days). c Cells were enriched (.95%) as described in Materials and Methods.

2775

DE81 filter paper (Whatman). Filters were then treated with 10% trichloroacetic acid to determine acid-insoluble counts. All data are the means (in counts per minute) of triplicate samples. Samples were run in concurrent assays using Mg21 to control for crossover RT counts resulting from HIV and HTLV-1 infection (usually less than 400 cpm), with these counts per minute subtracted from reported Mn21 values. A poly(dA)-oligo(dT) template/primer, in addition to poly(rA)-oligo(dT), was used to control for cellular DNA polymerase activity. This value was never greater than 30% of the counts per minute observed with poly(rA)-oligo(dT), which was expected as FV RTs have been shown to inefficiently use poly(dA)-oligo(dT) (20). Peak activities were also assayed at numerous cation and primer concentrations to optimize the assay. Preparation of virus stocks. Virus stocks were obtained by transfection of BHK-21 or 293 cells with the full-length plasmid clone of HFV by the calcium phosphate method (5). Supernatants and cells were collected at various times. Cells were lysed by three cycles of freezing and thawing to release cell-associated virus. After centrifugation, cell lysates and supernatants with high-level activities by RT assay were combined, filtered (pore size, 0.45 mm), and used directly in infections or stored at 2708C for future use. Fifty thousand counts per minute of RT corresponded to a titer of 106, as measured by the expression of b-galactosidase from an HFV LTR–b-galactosidase indicator BHK-21 cell line (40, 49). Infections. Virus stocks adjusted to 50,000 or 5,000 cpm/ml with serum-free RPMI 1640 medium were used to infect suspension cells as follows. Cells (107) in log-phase growth were infected with 5,000 or 50,000 cpm of HFV RT in 1 ml of serum-free RPMI 1640 containing 2 mg of Polybrene for 1 to 2 h at 378C in a shaking water bath. Adherent monocyte monolayers or glial cell cultures were infected with the same viral titers, except in 0.5 ml for 2 h at 378C. All HFVexposed cells were washed twice with serum-free medium to remove unadsorbed virus.

RESULTS formed on acetone-fixed glass coverslips on which cultures of microglial cells exposed to HFV, heat-inactivated HFV, or medium alone had been grown. Antibodies to c-fms (Oncor, Gaithersburg, Md.) and CD16 (Leu-M3; Becton Dickinson) were used to detect microglial cells. Antibodies to HFV gag, bel1, and bel2 were previously described (2). Optimal staining with anti-HFV antibodies was obtained with a 1:50 dilution. Fluorescein isothiocyanate-conjugated donkey anti-mouse immunoglobulin G and donkey anti-rabbit immunoglobulin G (Jackson Immuno Research Laboratories, West Grove, Pa.) were used for indirect staining. RT assay. RT assays were performed as previously described (20, 27). Briefly, virus was disrupted in a 50 mM Tris buffer containing 0.15 mg of dithiothreitol per ml, and 0.5% Triton X-100 and mixed with a 0.8 mM (final concentration) Mn21 cocktail of 2 U of poly(rA) z oligo(dT)12-18 per ml, 3 mg of dithiothreitol per ml, 3 M KCl, 1 M Tris (pH 7.8), 10% Triton X-100, and [3H]TTP (Amersham). Samples were incubated 378C for 30 min and adsorbed for 15 min onto

Generation of infectious HFV stocks. Transfection of the human kidney cell line 293 with the infectious molecular clone pHSRV resulted in high levels of virus expression. Consistent with studies in which HFV has been shown to be tightly cell associated, considerably higher levels of RT activity were found in cell lysates at later time points (93,500 3 102 cpm/ml at 10 days versus 7,800 3 102 cpm/ml in the supernatant at 5 days after transfection). Total cell-associated virus was 5- to 10-fold higher than total released virus. Cells were monitored up to 28 days (subcultured at 4-day intervals) after transfection (at which time no supernatant RT activity could be detected), with no apparent cytopathicity. Ultrastructural analysis showed

FIG. 1. Detection of HFV proviral DNA by PCR. Cells were infected with HFV stocks at MOI of 0.1 or 0.1, and DNAs were prepared on day 7 as described in Materials and Methods. PCR analysis of HFV proviral DNA was performed, as described in Materials and Methods, with primers from env sequences. The ethidium bromide-stained polyacrylamide gel shows a 330-bp product. Lanes: 1, infected BHK-21; 2, infected KG-1a; 3, mock-infected KG-1a; 4, infected KG-1a (MOI 5 0.01); 5, infected 81-66; 6, mock-infected 81-66; 7, infected 81-66 (MOI 5 0.01); 8, infected KG-1; 9, mock-infected KG-1; 10, infected KG-1 (MOI 5 0.01); 11, infected CD4 T cells; 12, infected CD4 T cells (MOI 5 0.01); 13, mock-infected CD4 T cells; 14, uninfected CD4 T cells; 15, HFV plasmid; 16, H2O; and MW, molecular weight standards.

2776

MIKOVITS ET AL.

FIG. 2. HFV infection of primary human CD41 T cells. CD4-enriched human T cells were prepared and infected with 50,000 cpm of RT as described in Materials and Methods. Supernatants were assayed for RT activity (■) at various time points, and cell viabilities (F) were determined. Data are representative of three independent experiments.

intact viral particles with characteristic HFV morphology (13, 47) (data not shown). In addition, the rapid infection and cytopathology of BHK-21 with this virus stock shows that transfection of the molecular clone leads to infectious particles (data not shown). Effects of HFV replication in human cell lines. Virus stocks were used to infect human myeloid and lymphoid cell lines at multiplicities of infection (MOI) of 0.1 and 0.01. Infections

J. VIROL.

were monitored by measurements of RT activity in cell supernatants as well as RT-PCR at 24 h and 3-day intervals thereafter. Cell lines which were negative by both assays were checked for the presence of proviral sequences by DNA PCR. As shown in Table 1, cell-free HFV can productively infect several human cell lines of myeloid and lymphoid lineage. Virus expression and release into the supernatant could be detected as early as day 2 or 3 after infection, as measured by RT-PCR and RT activity (data not shown). Infection of the early promyeloblast line, KG-1a, showed high levels of productive infection which peaked at day 7 and continued at reduced levels until day 21 without cytopathology. In contrast, the myeloblast line, KG-1, from the same donor was resistant to productive infection by HFV, as demonstrated by the lack of RT activity (Table 1). While no RNA signal was detectable by RT-PCR (data not shown), a signal for HFV DNA was detectable by PCR in KG-1 (Fig. 1), indicating latent infection. Similarly, immature T-cell lines, such as CEM and Molt-4, were susceptible to HFV infection with no effect on cell viability, while the mature T-cell line HUT 78 was productively infected and by day 14 showed reduced viability and eventual cell death. At an MOI of 0.01, HFV was still cytopathic to HUT 78, but 28 days was needed to observe it. Interestingly, like KG-1, the B-cell line Raji was latently infected by HFV (HFV DNA sequences were detected but no RNA expression [data not shown]). Supernatant from infected KG-1a (day 14) and a CD41 T-cell lysate were able to productively infect BHK-21 (data not shown). The monocytoid cell lines U937 and THP-1 were infected by HFV; however, the RT activity in supernatants of infected cells was .10-fold less than that seen after infection of KG-1a or HUT 78 T cells (Table 1). This low-level productive infection could be sustained over several passages in culture without cytotoxicity.

FIG. 3. Detection of HFV RNA in human macrophages and microglial cells by RT-PCR. Each RNA (1 mg) was reverse transcribed as described in Materials and Methods and subjected to RT-PCR, yielding a 700-bp band. Lanes: 1, 293 transfected with HFV; 2, infected MDM (MOI 5 0.1) on day 3; 3, infected MDM (MOI 5 0.01) on day 3; 4, mock-infected MDM (MOI 5 1) on day 3; 5, infected MDM (MOI 5 0.1) on day 7; 6, infected MDM (MOI 5 0.01) on day 7; 7, mock-infected MDM (MOI 5 1) on day 7; 8, infected MDM (MOI 5 0.1) on day 14; 9, infected MDM (MOI 5 0.01) on day 14; 10, mock-infected MDM (MOI 5 1) on day 14; 11, infected MDM (MOI 5 0.1) on day 21; 12, infected MDM (MOI 5 0.01) on day 21; 13, mock-infected MDM (MOI 5 1) on day 21; 14, microglial cells 2 days after exposure to HFV; 15, microglial cells 2 days after exposure to heat-inactivated HFV; 16, MDM 2 days after exposure to HFV; 17, MDM 2 days after exposure to heat-inactivated HFV; 18, H2O; 19, blank; 20, molecular weight standards.

VOL. 70, 1996


2777

FIG. 4. Nuclear expression of Gag-related antigens in microglial cells. Indirect immunofluorescence microscopy of microglial cell cultures exposed to heatinactivated (MOI 5 1) (A and B) and live (MOI 5 0.1) (C and D) HFV. Primary rabbit anti-HFV Gag antibody (1:50) was reacted with fluorescein isothiocyanateconjugated donkey anti-rabbit immunoglobulin. Two hours after exposure, both heat-inactivated (A) and live (C) HFV appeared to be taken up by some microglial cells that express Gag antigen in their cytoplasm (arrows). Twelve hours after exposure, 20% of microglial cells exposed to heat-inactivated HFV (B) and more than 50% of microglial cells exposed to live HFV (D) expressed Gag antigen in their cytoplasm (arrows). Magnification, 3200.

Effects of HFV replication in primary cells. In primary PBMC, as well as in populations enriched for CD41 T cells, high levels of RT expression in supernatants were seen within 2 or 3 days of infection. However, 5 days after infection, cell viability declined rapidly, with a low of 20% at day 7 (Fig. 2). In contrast, infection of CD81 T cells showed lower levels of RT activity and no cytopathicity at similar time points, suggesting that CD81 cells are less susceptible to productive infection by HFV. Primary monocyte-derived macrophages (MDM) were infected by HFV to expression levels similar to those observed in monocytoid cell lines (Table 1). The kinetics of HFV infection of MDM, as determined by RT-PCR, showed a product of the expected size (700 bp) up to 21 days postinfection (Fig. 3). At day 7, the higher but not the lower input virus was positive by RT-PCR (Fig. 3, lanes 5 and 6). On days 14 and 21, both viral inputs gave equivalent RT-PCR signals (Fig. 3, lanes 8, 9, 11, and 12). Similarly, the productive infection of brain microglial cells was comparable to that of MDM by either RT activity (Table 1) or RT-PCR (Fig. 3). Further, MDM treated with heat-inactivated HFV (MOI 5 1) showed no band in the RT-PCR assay (Fig. 3, lanes 4, 7, 10, 13, and 15). HFV Gag antigen could be detected in microglial cells as early as 2 h after exposure to HFV (MOI of 0.1) (Fig. 4C). At 12 h after exposure, more than 50% of microglial cells showed Gag antigen primarily in the cell cytoplasm (Fig. 4D). Nuclear expression of Gag antigen (Fig. 5C) was detected at 48 h after exposure of microglial cells to HFV. At this time, most micro-

glial cells also expressed Gag antigen in the cytoplasm. A similar pattern of expression was observed with antibodies directed against HFV Bel antigens (data not shown). Although no major differences in productive infection between brain microglial cells and MDM were seen by either RT activity (Table 1) or RT-PCR (Fig. 3), massive cytotoxicity

TABLE 2. Effects of HFV infection in retrovirus-infected human cells Cell target

Infection

81-66 MT-2 MT-2 (mock)c CS-2 (B cell) HUT 78/BP-1 THP-1/ADA THP-1/ADA

HTLV-1 HTLV-1 HTLV-1 HTLV-1 HIV-1 HIV-1 Low-level HIV-1

HFV RT (103 cpm/ml)a

Cytopathologyb

50 62

Cell death Cell death No effect Cell death Cell death Cell death No effect

56 NDd 25 30

a Chronically infected cell lines were infected with HFV stocks at an MOI of 0.1, as described in Materials and Methods. Supernatants were analyzed for Mn21 RT activity at 3-day intervals, with the data from day 6 shown. b Cellular pathology was examined by light microscopy for giant cell formation of infected cells every 3 to 5 days for 1 month after infection. Cell death was measured by trypan blue exclusion. Cell death occurred within 7 to 14 days, with an inability to split cells occurring after infection. c Mock infections were done with heat-inactivated virus, as described in Materials and Methods. d ND, not done.

2778

MIKOVITS ET AL.

J. VIROL.

FIG. 5. Cytotoxicity of microglial cultures by HFV infection. Indirect immunofluorescence microscopy utilizing rabbit anti-HFV Gag antibody (1:50 dilution) reacted with fluorescein isothiocyanate-conjugated donkey anti-rabbit immunoglobulin G. Microglial cells exposed to live HFV (MOI 5 0.1) demonstrated Gag expression in the cytoplasm (arrows) at 24 h (A) and in the nucleus (p) and cytoplasm (arrow) at 48 h after exposure (B). (C) At 96 h after live-HFV exposure, cytoplasmic Gag expression (arrows) occurred in the few remaining viable microglial cells, with marked cellular destruction and remaining debris (p). (D) Cytoplasmic Gag expression (arrow) without cytotoxicity occurred in microglial cells at 96 h after exposure to heat-inactivated HFV. Magnification, 3250.

occurred in infected microglial cells within 96 h, but not in MDM. At 48 h after exposure, progressive cellular cytotoxicity of microglial cells became apparent; by 96 h after exposure, only a few Gag-expressing microglial cells appeared to be viable (Fig. 5C). Microglial cells exposed to a lower dose (MOI 5 0.01) of HFV had a similar pattern of Gag expression; however, cytotoxicity was significantly delayed and not maximal until 7 days after exposure (data not shown). Microglial cells exposed to heat-inactivated HFV (MOI 5 1) showed Gag antigen in their cytoplasm by 2 h after exposure, and 20% showed Gag antigen at 12 h after exposure, probably because of phagocytic uptake of virions (Fig. 4B). The number of cells expressing Gag antigen peaked at 24 h after exposure to heat-inactivated HFV and declined to ,50% of the microglial cell population by 96 h (Fig. 5D). Nuclear expression of Gag antigen and cellular cytotoxicity were not seen in microglial cells exposed to heat-inactivated HFV (Fig. 5D). Effects of HFV replication in retrovirus-infected cells. Since it was clear that HFV infects CD41 T cells and monocytes, cell types readily infectible by HTLV-1 and HIV-1, several HTLV-1- and HIV-infected cell lines were coinfected with HFV to examine any increases in cell pathology. When the T-cell line HUT 78 chronically infected with HIV was infected with HFV, the levels of viral RNA (Fig. 6) and RT expression (Table 2) were similar to those of control HUT 78. However,

accelerated cell death, beginning within 2 or 3 days of exposure to HFV and peaking at day 7, occurred. Similar levels of HFV mRNA and RT activity and rapid cell death were observed upon exposure of HTLV-1-infected T-cell line MT-2 to HFV. 81-66, an HTLV-1-infected T-cell line which contains only defective proviruses, is also rapidly killed by HFV infection (Fig. 6; Table 2). Monocyte cell lines productively infected with HIV-1 and producing high levels of HIV were killed by coinfection with HFV, while chronically infected monocytes with low-level expression (26) were not killed by HFV infection (Table 2). Although the level of HFV RT in the supernatant was not significantly higher than that observed in infections of uninfected cell lines, a significant increase in cell-associated virus was possible. However, consistent with previous results (14, 15, 18), HTLV-1 Tax did not appreciably transactivate an HFV LTR-chloramphenicol acetyltransferase construct in transient assays (data not shown), suggesting that increased HFV transcription by retroviral coinfection was not involved in the increased cytopathology. DISCUSSION The ability of HFV to productively infect a variety of human myeloid and lymphoid cell lines and primary cells was shown

VOL. 70, 1996


2779

FIG. 6. RT-PCR of dually infected cell lines. RNAs were isolated from HTLV-1 and HIV-1 cell lines, MT-2 and HUT 78/BP-1, respectively, at 3- to 4-day intervals after infection with live HFV or heat-inactivated HFV, and cDNAs were synthesized as described in Materials and Methods. Of the reaction mixture, 1/20 was used in amplifications with HFV LTR-gag primers (700-bp product; lanes 1 to 8), the housekeeping hypoxanthine phosphoribosyltransferase gene (496 bp; lanes 15 to 19), and the appropriate primers for the coinfected retrovirus, HTex1-2–HT7875 (HTLV-1; 490 bp; lanes 9 and 10) or SK38 and SK39 (HIV; 125 bp; lanes 11 to 14). Lanes: 1, MT-2 infected with heat-inactivated HFV (day 7); 2, MT-2 infected with HFV (day 7); 3, MT-2 infected with HFV (day 10); 4, MT-2 infected with heat-inactivated HFV (day 10); 5 and 11, HUT 78/BP-1 infected with HFV (day 7); 6 and 12, HUT 78/BP-1 infected with heat-inactivated HFV (day 7); 7 and 13, HUT 78/BP-1 infected with HFV (day 10); 8 and 14, HUT 78/BP-1 infected with heat-inactivated HFV (day 10); 9, MT-2 infected with HFV (day 7); 10, MT-2 infected with heat-inactivated HFV (day 7); 15 to 19, hypoxanthine phosphoribosyl transferase primers for samples in lanes 5 to 10; 20, Bethesda Research Laboratories 1,000-bp ladder.

(50). The early blast cell variant (KG-1a) of KG-1 released high levels of RT activity, while the parental CD341 KG-1 myeloblastic cell line capable of myeloid differentiation (16, 17) was not productively infected. However, HFV provirus was present in KG-1, indicating that HFV could latently infect these cells. Latent infection of primate FV due to methylation has previously been described (36, 41). In addition, primary human PBMC, as well as enriched human CD41 T cells, MDM, and brain-derived microglial cells, were readily infected by HFV. CD81 T cells were poorly infectible by HFV. Furthermore, infection of primary activated CD41, not CD81, T cells showed significant characteristic FV cytotoxicity. Similar cytopathology has previously been described for simian PBMC and infected human CD41 T cell lines, such as SubT1 infected by primate FV (24). Marked cytotoxicity was seen in infected microglial cells within 96 h but was not seen in MDM even after 21 days. The nuclear localization of Gag antigens seen in these infected cells is a hallmark of FV infection, distinguishing it from other retroviral infections (39). This striking difference could be due to the different states of differentiation of cells; differential secretion of cytokines, particularly cytotoxic ones; differential production of potentially cytotoxic products of oxidative stress, such as NO, peroxynitrites, hydroxyl radicals, etc. (9); or more cell-associated virus. The ability of FV to infect resting cells has not previously been addressed. In this respect, the ability of HFV to infect .95% of microglial cells is suggestive of the ability of HIV to infect postmitotic cells (6, 19, 45, 46). However, one cannot conclude from these data that HFV integrates into resting cells. In view of the effect of HFV on microglial

cells, it is noteworthy that many simian FV have a brain tropism (13, 31) and that in transgenic mice expressing HFV genes, progressive myopathy and encephalopathy were seen (2, 4). Since these HFV-infectible cell types can also be targets for HIV-1 and HTLV-1 infection, the effects of coinfection by HFV of HIV-1- and HTLV-1-infected cells were investigated. Cytotoxicity after HFV infection was seen in both HTLV-1 (MT-2)- and HIV-1 (HUT 78/BP-1)-infected T-cell lines and in HIV-1 (THP-1 ADA)-infected monocytoid cell lines. In contrast, a previous report showed that coexpression of HIV-1 and a chimpanzee FV in human H-9 T cells can occur without cell cytotoxicity (29). Whether these differences are of viral or cellular origin is not known. However, the levels of neither HFV RT activity in the supernatant nor of HFV transcription significantly increased in these dually infected cells over those of control singly infected cells, suggesting that increased HFV production was not responsible for increased cytopathicity. In addition, even though the HFV transactivator bel1 can transactivate the HIV LTR (14, 15, 18), HFV infection did not greatly increase extracellular production of HIV or HTLV-1. Thus, the mechanism(s) for increased cell death is not readily clear. Despite broad host cell ranges for the immune system, most FV, which are highly cytopathic in vitro, establish an apparent asymptomatic infection in the host and persist for long periods in the presence of neutralizing antibodies (30, 36, 38, 42). Furthermore, HFV is a virus in search of not only a disease (48) but also a host reservoir (30). However, in an immunocompromised host, it is not known what effect the reactivation

2780

MIKOVITS ET AL.

of HFV would have. Thus, it would be of interest to study HFV expression and pathogenesis during simian immunodeficiency virus infections in monkeys as well as in human xenotransplantion. ACKNOWLEDGMENTS We thank Kim Boudreau for her excellent technical assistance, Matthew Gonda and Kunio Nagashima for electron microscopy, the cytapheresis unit of the Biological Response Modifiers Program for their help, and Lin Ridgell for preparation of the manuscript. This work was supported in part by the European Union (BMH1CT93-1142) and the Bayerische Forchungsstifitung. REFERENCES 1. Achong, B., P. Mansell, M. A. Epstein, and P. Clifford. 1971. An unusual virus in cultures from a human nasopharyngeal carcinoma. J. Natl. Cancer Inst. 46:299–307. 2. Aguzzi, A., E. F. Wagner, K.-O. Netzer, K. Bothe, I. Anhauser, and A. Rethwilm. 1993. Human foamy virus proteins accumulate in neurons and induce multi-nucleated giant cells in the brains of transgenic mice. Am. J. Pathol. 142:1061–1072. 3. Baunach, G., B. Maurer, H. Hahn., M. Kranz, and A. Rethwilm. 1993. Functional analysis of human foamy virus accessory reading frames. J. Virol. 67:5411–5418. 4. Bothe, K., A. Aguzzi, H. Lassman, A. Rethwilm, and I. Horak. 1991. Progressive encephalopathy and myopathy in transgenic mice expressing human foamy virus genes. Science 253:555–557. 5. Chen, C., and H. Okayama. 1987. High-efficiency transformation of mammalian cells by plasmid DNA. Mol. Cell. Biol. 7:2745–2752. 6. Collman, R., and N. Nathanson. 1992. Human immunodeficiency virus type-1 infection of macrophages. Semin. Virol. 3:185–202. 7. Derse, D., M. Carvalho, R. Carroll, and B. M. Peterlin. 1991. A minimal lentivirus Tat. J. Virol. 65:7012–7015. 8. Derse, D., J. Mikovits, M. Polianova, B. K. Felber, and F. Ruscetti. 1995. Virions released from cells transfected with a molecular clone of human T-cell leukemia virus type I give rise to primary and secondary infections of T cells. J. Virol. 69:1907–1912. 9. Dugas, B., M. D. Mossalayi, C. Damais, and J. C. Kolb. 1995. Nitric oxide production by human monocytes. Evidence for a role for CD23. Immunol. Today 16:574–580. 10. Flugel, R. M. 1993. The molecular biology of the human spumavirus, p. 193–214. In B. R. Cullen (ed.) Human retroviruses—frontiers in molecular biology. Oxford University Press, Oxford. 11. Herchenroder, O., R. Renne, D. Loncar, E. K. Cobb, K. K. Murphy, J. Schneider, A. Mergia, and P. A. Luciw. 1994. Isolation, cloning and sequencing of simian foamy viruses from chimpanzees (SFVcpz): high homology to human foamy virus (HFV). Virology 201:187–199. 12. Hoffman, P., S. Dhib-Jalbut, J. Mikovits, D. Robbins, A. Wolf, G. Bergey, N. Lohrey, O. Weislow, and F. Ruscetti. 1992. Human T-cell leukemia virus type 1 infection of monocytes and microglial cells in primary cultures. Proc. Natl. Acad. Sci. USA 89:11784–11788. 13. Hooks, J. J., and C. J. Gibbs, Jr. 1975. The foamy viruses. Bacteriol. Rev. 39:169–185. 14. Keller, A., E. D. Garrett, and B. R. Cullen. 1992. The Bel-1 protein of human foamy virus activates human immunodeficiency virus type 1 gene expression via a novel DNA target site. J. Virol. 66:3946–3949. 15. Keller, A., K. M. Partin, M. Lo¨chelt, H. Bannert, R. M. Flu ¨gel, and B. R. Cullen. 1991. Characterization of the transcriptional trans activator of human foamy retrovirus. J. Virol. 65:2589–2594. 16. Koeffler, H., R. Billing, J. Lusis, R. Sparkles, and D. Golde. 1980. An undifferentiated variant derived from the human acute myelogenous leukemia cell line (KG-1). Blood 56:265. 17. Koeffler, H. P., and D. W. Golde. 1978. Acute myelogenous leukemia: a human cell line responsive to colony-stimulating activity. Science 200:1153. 18. Lee, A. H., K. J. Lee, S. Kim, and Y. C. Sung. 1992. Transactivation of human immunodeficiency virus type 1 long terminal repeat-directed gene expression by the human foamy virus bel1 protein requires a novel DNA sequence. J. Virol. 66:3236–3240. 19. Lewis, P., H. Hensel, and M. Emerson. 1992. Human immunodeficiency virus infection of cells arrested in the cell cycle. EMBO J. 11:3053–3058. 20. Liu, W. T., T. Natori, K. S. S. Chang, and A. M. Wu. Reverse transcriptase of foamy virus. Arch. Virol. 55:187–200. 21. Loh, P., and K. Ang. 1981. Replication of human syncytium-forming virus in human cells: effect of certain biological factors and selective chemicals. J. Med. Virol. 7:67–73. 22. Loh, P. C., and F. M. Matsuura. 1984. Human spumavirus replication in human cells. J. Med. Virol. 14:247–254.

J. VIROL. 23. Maurer, B., E. Serfling, V. ter Meulen, and A. Rethwilm. 1991. Transcription factor AP-1 modulates the activity of the human foamy virus long terminal repeat. J. Virol. 65:6353–6357. 24. McClure, M. O., P. D. Bieniasz, T. F. Schulz, I. L. Chrystie, G. Simpson, A. Aguzzi, J. G. Hoad, A. Cunningham, J. Kirkwood, and R. A. Weiss. 1994. Isolation of a new foamy retrovirus from orangutans. J. Virol. 68:7124–7130. 25. Mikovits, J. A., N. Lohrey, R. Schulof, J. Courtless, and F. Ruscetti. 1992. Activation of infectious virus from latent human immunodeficiency virus infection of monocytes in vivo. J. Clin. Invest. 90:1486–1491. 26. Mikovits, J. A., M. Raziuddin, M. Gonda, N. Ruta, N. Lohrey, H.-F. Kung, and F. W. Ruscetti. 1990. Negative regulation of human immune deficiency virus replication in monocytes. Distinctions between restricted and latent expression in THP-1 cells. J. Exp. Med. 171:1705–1720. 27. Miller, J. H. (ed.). 1972. Experiments in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 28. Muranyi, W., and R. M. Flu ¨gel. 1991. Analysis of splicing patterns of human spumaretrovirus by polymerase chain reaction reveals complex RNA structures. J. Virol. 65:727–735. 29. Nara, P., W. Robey, L. Arthur, M. Gonda, D. Asher, R. Yanagihara, C. Gibbs, D. Gajdusek, and P. Fischinger. 1987. Simultaneous isolation of simian foamy virus and HTLV-III/LAV from chimpanzee lymphocytes following HTLV-III or LAV inoculation. Arch. Virol. 92:183–186. 30. Neumann-Haefelin, D., U. Fleps, R. Renne, and M. Schweizer. 1993. Foamy viruses. Intervirology 35:196–207. 31. O’Brien, T., P. Albrecht, H. Schumacher, and N. Tauraso. 1971. Isolation of foamy virus type 1 and 2 from primary rhesus monkey brain cultures. Proc. Soc. Exp. Biol. Med. 137:1318–1323. 32. Rethwilm, A. 1995. Regulation of foamy virus expression. Curr. Top. Microbiol. Immunol. 193:1–24. 33. Rethwilm, A., G. Baunach, K.-O. Netzer, B. Maurer, B. Borisch, and V. terMeulen. 1990. Infectious DNA of the human spumaretrovirus. Nucleic Acids Res. 18:733–738. 34. Rethwilm, A., G. Darai, A. Rosen, B. Maurer, and R. M. Flugel. 1987. Molecular cloning of the genome of the human spumaretrovirus. Gene 59:19–28. 35. Rethwilm, A., O. Erlwein, G. Baunach, B. Maurer, and V. terMeulen. 1991. The transcriptional transactivator of human foamy virus maps to the bel 1 genomic region. Proc. Natl. Acad. Sci. USA 88:941–945. 36. Rogers, N. C., M. Basnight, C. J. Gibbs, and D. C. Gajdusek. 1967. Latent virus in chimpanzees with experimental kuru. Nature (London) 216:446–449. 37. Ruscetti, F. W., J. A. Mikovits, V. S. Kalyanaraman, R. Overton, H. Stevenson, K. Stromberg, R. B. Herberman, W. L. Farrar, and J. R. Ortaldo. 1986. Analysis of effector mechanisms against HTLV-I- and HTLV-III/LAV-infected lymphoid cells. J. Immunol. 136:3619–3624. 38. Saib, A., J. Peries, and H. de The. 1995. Recent insights into the biology of human foamy virus. Trends Microbiol. 3:173–178. 39. Schliephake, A. W., and A. Rethwilm. 1994. Nuclear localization of foamy virus Gag precursor protein. J. Virol. 68:4946–4954. 40. Schmidt, M., and A. Rethwilm. 1995. Replicating foamy virus-based vectors directing high level expression of foreign genes. Virology 210:167–178. 41. Schweizer, M., U. Fleps, A. Jackle, R. Renne, R. Turek, and D. NeumannHaefelin. 1993. Simian foamy virus type 3 (SFV-3) in latently infected vero cells: reactivation by demethylation of proviral DNA. Virology 192:663–666. 42. Schweizer, M., R. Turek, H. Hahn, A. Schliephake, K. Netzer, G. Eder, M. Reinhardt, A. Rethwilm, and D. Neumann-Haefelin. 1995. Markers of foamy virus infection in monkeys, apes and accidently infected humans: appropriate testing fails to confirm suspected foamy virus prevalence in humans. AIDS Res. Hum. Retroviruses 11:161–170. 43. Schweizer, M., R. Turek, M. Reinhardt, and D. Neumann-Haefelin. 1994. Absence of foamy virus DNA in Graves’ disease. AIDS Res. Hum. Retroviruses 10:601–605. 44. Stevenson, H., P. Miller, Y. Akiyama, T. Favilla, J. Beman, R. Herberman, H. Stull, G. Thurman, A. Maluish, and R. Oldham. 1983. A system for obtaining large numbers of human monocytes purified by leukopheresis and counter-current elutriation. J. Immunol. Methods 62:353–364. 45. Watkins, B., H. Dorn, W. Kelly, R. Armstrong, B. Potts, F. Michaels, C. Kufta, and M. Dubois-Dalcq. 1990. Specific tropism of HIV-1 for microglial cells in primary brain cultures. Science 249:549–553. 46. Weinberg, J., T. Mattews, B. Cullen, and M. Malim. 1991. Productive human immunodeficiency virus type 1 infection of nonproliferating human monocytes. J. Exp. Med. 174:1477–1482. 47. Weiss, R., N. Teich, H. Varmus, and J. Coffin (ed.). 1985. RNA tumor viruses, 2nd ed., vol. 1, p. 157–163. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 48. Weiss, R. A. 1988. A virus in search of a disease. Nature (London) 333:497– 498. 49. Yu, S. F., and M. L. Linial. 1993. Analysis of the role of the bel and bet open reading frames of human foamy virus by using a new quantitative assay. J. Virol. 67:6618–6624. 50. Yu, S. F., J. Stone, and M. L. Linial. 1996. Productive persistent infection of hematopoietic cells by human foamy virus. J. Virol. 70:1250–1254.