Properties of human foamy virus relevant to its development as a vector for gene therapy

Claire L. Hill1, Paul D. Bieniaszb,1 and Myra O. McClure1

Department of GU Medicine and Communicable Diseases, Jefferiss Research Trust Laboratories, Imperial College School of Medicine at St. Mary's, Praed Street, London W2 1NY, UK1

Author for correspondence: Myra McClure.Fax +44 20 7886 6648. e-mail m.mcclure{at}ic.ac.uk


   Abstract
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Abstract
Introduction
Methods
Results
Discussion
References
 
The Spumaviridae (foamy viruses) are increasingly being considered as potential vectors for gene therapy, yet little has been documented of their basic cell biology. This study demonstrates that human foamy virus (HFV) has a broad tropism and that the receptor for HFV is expressed not only on many mammalian, but on avian and reptilian cells. Receptor interference assays using an envelope-expressing cell line and a vesicular stomatitis virus/HFV pseudotype virus demonstrate that the cellular receptor is common to all primate members of the genus. The majority of foamy virus particles assemble and remain sequestered intracellularly. A rapid and quantitative method of assaying foamy virus infectivity by reverse transcriptase activity facilitates the use of classical protocols to increase infectious virus titres in vitro to >=106 TCID/ml.


   Introduction
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
Foamy viruses are so called because of the characteristic `foamy' appearance of the cytopathic effect (CPE) induced in cells following in vitro culture of the virus. They are apathogenic in accidentally infected humans (Schweizer et al., 1997 ) and naturally or experimentally infected animals (Schmidt et al., 1997 ), and early reports of natural infection in various parts of the world (Achong & Epstein, 1978 ) have not been confirmed using a range of modern techniques on much larger sample sizes (Ali et al., 1996 ). It is, therefore, not surprising that foamy viruses have remained, until recently, the least studied of the Retroviridae. However, they are emerging from obscurity to be considered as potential retrovirus vectors.

The human foamy virus (HFV) belongs to a group of complex retroviruses which encode three structural genes (gag, pol and env) in addition to three accessory open reading frames (bel-1, -2, -3) located in the 3' end of the genome. Of these only bel-1 (now called tas), which encodes the transcriptional transactivator (Tas), is essential for virus replication (Baunach et al., 1993 ). The Gag, Pol and Env proteins are synthesized independently (Enssle et al., 1996 ; Lochelt & Flügel, 1996 ; Yu et al., 1996b ) and, therefore, in terms of vector consideration, can be provided in trans on three different plasmids to create stable packaging cell lines, reducing the possibility of generating replication-competent helper virus (Bieniasz et al., 1997 ). First studies in which foamy viruses have been developed as gene delivery vehicles (Schmidt & Rethwilm, 1995 ; Russell & Miller, 1996 ; Bieniasz et al., 1997 ) have involved replacing some or all of the bel genes of pHSRV, an infectious molecular clone of HFV (Rethwilm et al., 1990 ), with a reporter gene to assess transduction. Replication-competent vectors induced the characteristic CPE when transfected into BHK cells (Schmidt & Rethwilm, 1995 ), while replication-incompetent vectors transduced a variety of cell lines (Russell & Miller, 1996 ) at maximum titres of 105 transducing units/ml (Schmidt & Rethwilm, 1995 ; Bieniasz et al., 1997 ).

This paper reports some basic aspects of foamy virus infection relevant to future vector development. The aims of this study were to find a simple assay of infectivity with which to optimize infection conditions and increase virus titre, to consider foamy virus tropism and, by means of an HFV envelope-expressing cell line, exploit the phenomenon of receptor interference to determine whether different foamy viruses share a common cellular receptor. This is based on the observation that chronically infected cell lines are resistant to superinfection by homologous viruses utilizing the same receptor. The identification of cellular receptors which mediate virus attachment and entry is a fundamental step in the investigation of the molecular events involved during retrovirus infection. Although several retrovirus receptors have been identified (Weiss & Tailor, 1995 ), nothing is known of the cell surface molecules which enable foamy viruses to enter cells. The BHK Env-expressing cell line and the vesicular stomatitis virus (VSV) pseudotypes expressing foamy virus envelopes described in this paper constitute useful tools with which to investigate further the mechanism of HFV attachment to cells.


   Methods
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Abstract
Introduction
Methods
Results
Discussion
References
 
{blacksquare} Cell lines.
Most cell lines used in the study (Table 1) were cultured in DMEM plus 10% foetal calf serum (FCS), 25000 units/ml penicillin and 25 mg/ml streptomycin. The exceptions were the cell lines MPK and QT6, which were grown in medium containing 20% FCS and 10% FCS+2% chicken serum, respectively.


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Table 1. Sensitivity of cell lines to HFV infection

 
{blacksquare} BHK-Env cells.
A eukaryotic expression plasmid, pEnv3.1, in which HFV envelope expression is driven by a simian virus 40 early promoter was constructed using the expression plasmid pSG5 (Stratagene). BHK-21 cells were co-transfected with 20 µg pEnv3.1 and 1 µg pBabepuro (Morgenstern & Land, 1990 ) using calcium phosphate co-precipitation. Single cell clones were selected in medium containing 2·5 µg/ml puromycin. Foamy virus-immune chimpanzee serum was used to identify envelope-expressing cells (Moebes et al., 1997 ). Functional expression of the HFV envelope was confirmed by co-cultivation of BHK-Env or BHK-21 cells with primary human embryonic lung fibroblasts for 24 h followed by staining with 0·5% methylene blue and 0·16% basic fuschin in methanol to detect cell fusion (data not shown).

{blacksquare} Wild-type viruses.
Virus isolates used were as follows: HFV (Achong et al., 1971 ), simian foamy viruses SFV-1 and SFV-2 different serotypes from rhesus macaques (Stiles, 1968 ), SFV-3 from an African green monkey (Stiles, 1968 ; Swack et al., 1970 ) and the chimpanzee isolates, SFV-6 and SFV-7 (Hooks et al., 1973 ).

{blacksquare} Production of VSV (HFV) pseudotype virus.
VSV (HFV) pseudotypes were generated as previously described (Schnitzer, 1982 ). BHK-21 cells or BHK-Env cells were acutely infected with VSV (Indiana serotype) at approximately 1 p.f.u. per cell. After 1 h the inoculum was removed and replaced with 4 ml fresh medium. The following day, culture supernatant was harvested, passed through a 0·45 µm filter and stored in 0·5 ml aliquots at -70 °C. For VSV pseudotype titration, 50 µl pseudotype stock was incubated with 50 µl of 1:4 diluted sheep anti-VSV serum for 1 h at 37 °C. Non-neutralized controls were incubated with DMEM/10% FCS. BHK-21 or BHK-Env cells, seeded at 8x104 cells per well in 24-well plates, were inoculated with 10-fold serial dilutions of virus or virus serum mixture. After 1 h incubation, the inoculum was removed and the cells overlaid with 250 µl agarose medium. After 5 min at room temperature, to permit solidification of the agarose, cells were cultured for 48 h. Two hundred and fifty µl of 0·1% neutral red in PBS was added to each well, and after 1 h plaques were observed microscopically.

{blacksquare} Focal immunoassay (FIA).
HFV infection in diverse cell lines was assayed by FIA (Bieniasz et al., 1995 ). Briefly, virus was added in 10-fold serial dilutions to cells seeded at 5x104 cells per well in 24-well plates. Forty-eight hours post-infection, cells were washed with PBS and fixed for 10 min with 500 µl of a 1:1 acetone:methanol solution at -20 °C. On removal of the fixative, cells were air-dried for 5 min, washed in PBS/1% FCS and incubated for 45 min at room temperature with 200 µl anti-HFV antibody (HFV-positive serum from an accidentally infected laboratory worker; Schweizer et al., 1997 ) diluted 1:200 in PBS/1% FCS. Unbound antibody was removed by washing three times in PBS/1% FCS and the cells were incubated for 45 min at room temperature with 200 µl of 1:100 anti-human IgG F(ab)2 fragments conjugated to horseradish peroxidase in PBS/1% FCS. Cells were washed twice in PBS before addition of 300 µl of 0·3 mg/ml 3'3'diaminobenzadine, 0·1% H2O2 in PBS for 15 min, and examined microscopically for stained foci of infection.

{blacksquare} Reverse transcriptase (RT) assay.
RT activity was assayed by the C-type-RT [manganese (Mn2+)-dependent] and Lenti-RT [magnesium (Mg2+)-dependent] activity assays from Cavidi Tech (Ekstrand et al., 1996 ; Malmsten et al., 1998 ). Briefly, polyadenylic acid [poly(rA)] was covalently coupled to the wells of a 96-well microtitre plate and used as a template for the incorporation of the nucleotide analogue 5-bromo-deoxyuridine 5'triphosphate (BrdUTP), with oligo(dT)22 as a primer. Incorporated BrdUMP product was quantitatively detected immunologically using alkaline phosphatase-conjugated anti-BrdU monoclonal antibody and para-nitrophenyl phosphate as substrate for colorimetric detection at 405 nm after 1 h incubation.

{blacksquare} Enhancement of HFV titre.
BHK-21 cells were seeded at 1x105 cells/ml in 25 cm2 flasks (6 ml per flask), infected with HFV at an m.o.i. of between 0 and 0·1 in 1 ml medium and incubated at 37 °C in a 5% CO2 environment. When using Lipofectin (Gibco BRL) to enhance adsorption to cells, virions were complexed with 20 µl lipid for 1 h at room temperature prior to maintenance under normal conditions. Cells were observed for CPE each day and passaged 1 in 10 at confluence. Virus was harvested from cells and cell supernatant using one or a combination of the following methods. (i) Infected cells were harvested in a small volume (2 ml per 25 cm2 flask) of fresh medium and subjected to three cycles of freezing on dry ice and thawing at 37 °C. (ii) Cellular debris from either supernatant or freeze–thawed infected cells was removed by centrifugation at 500 g for 5 min or by filtration (0·45 µm, Millipore). (iii) Virus supernatant was harvested and concentrated 200-fold by centrifugal ultrafiltration (Centricon, Amicon).

{blacksquare} HFV infection of cell lines.
Cells were seeded in either 6-well plates or in 25 cm2 flasks. When 30–50% confluent (approx. 1–5x105 cells per well or 3x105–2x106 cells per flask) cultures were inoculated with 3x105 focus-forming units (f.f.u.) virus per 25 cm2 flask or 1x105 f.f.u. per well in 6-well plates. Forty-eight hours post-infection cells were assayed for virus antigen by FIA.

{blacksquare} Receptor interference.
BHK-21 cells or BHK-Env cells were seeded in 24-well plates (2x104 cells in 500 µl medium) and the following day inoculated with 100 µl of 10-fold serial dilutions of SFV-1, SFV-2, SFV-3, SFV-6, SFV-7 and HFV. After 48 h, infectious titres were determined by FIA.


   Results
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Abstract
Introduction
Methods
Results
Discussion
References
 
RT assay and enhancement of virus titre
A C-type-RT assay (Cavidi Tech) which contains the divalent cation Mn2+ was compared with an identical assay for human immunodeficiency virus (HIV), the Lenti-RT, which uses Mg2+ cations. The Mn2+-based assay detected higher enzyme activity, down to 10 infectious virions/ml, as determined by FIA (data not shown). This assay provided a quick and reliable means of determining the time post-infection that virions were optimally released from infected cells. Cell-free supernatants (300 µl) were collected each day post-infection from BHK cells infected at an m.o.i. of 0·005 and RT activity assayed by the C-type-RT assay. The highest RT activity was produced on day 7 (Fig. 1 A), while syncytium formation was observed from day 4 onwards and by day 9 there was little cell growth and extensive CPE in the cultures.



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Fig. 1. Optimization of HFV infection in BHK-21 cells. (A) BHK-21 cells were infected at an m.o.i. of 0·005 and supernatant was collected each day post-infection and analysed for RT activity by the C-type RT assay. Cells were passaged on days 3 and 6. (B), (C) Infection of BHK-21 cells with HFV at a range of m.o.i. (0–0·1). At 7 days post-infection supernatant was collected and (B) RT activity determined by the C-type RT assay. (C) Number of infectious units produced per cell calculated by FIA and total cell count.

 
Cationic liposomes were used to maximize the virus–cell interaction. Thus, BHK-21 cells were infected at an m.o.i. of 0·005 in the presence and absence of Lipofectin. At 4–7 days post-infection there was no difference in CPE between the two cultures and virus titres were similar.

To determine the effect of m.o.i. on virus yields, BHK-21 cells were infected at an m.o.i. of between 0 and 0·1 virions per cell. Supernatants were harvested on day 7 post-infection (at optimal RT activity). Total cell number, RT activity in cell-free supernatant and extracellular virus titre (by FIA) were determined (Fig. 1B, C). Between an m.o.i. of 0 and 0·05, the higher the m.o.i., the greater the RT activity and virus titre. There was a sixfold increase in RT activity at an m.o.i. of 0·05 compared with infection at an m.o.i. of 0·001 (Fig. 1B). Cells infected at an m.o.i. of 0·1 produced a marked CPE on day 4, and by day 7 there were few viable cells remaining to support virus replication and although RT activity was detected, no infectious virus was observed by FIA (Fig. 1C). Fig. 1 shows that the optimal m.o.i. for infection was between 0·01 and 0·05.

To facilitate virus release, cultures showing extensive CPE were subjected to lysis by freeze–thawing. Following removal of the cellular debris the resulting virus titre was 104–105 compared to 102–103 for virus-containing supernatant harvested from infected cells. Using a Centricon filter (Amicon/Millipore) with a pore size of 100 kDa it was possible to concentrate 200 ml (titre 5x102) HFV virus-containing supernatant to 1 ml with a titre of 1x105, with no loss of infectivity to the filtrate. A combination of all the above-mentioned procedures provided HFV stocks with a titre of >106.

HFV infection of cell lines
The results of infecting diverse cell lines with HFV are shown in Table 1. Inoculation of a variety of mammalian, two avian and one reptilian cell lines resulted in immunoperoxidase staining of virus antigen by FIA, while mock-inoculated control cells remained unstained. These data demonstrate that the HFV receptor is not only widely distributed among mammalian cell lines, but it is also present on cells of avian and reptilian origin.

Receptor interference assays
To investigate whether SFVs use the same cell surface receptor as HFV, the infectivity of isolates either closely related (SFV-6, SFV-7) or more distantly related (SFV-1, SFV-2, SFV-3) to HFV were determined using FIA, with BHK-21 and BHK-Env cells as target cells (Figs 2 and 3). None of the sera used stained BHK-Env cells in the absence of virus infection. Immunoperoxidase staining of BHK-21 and BHK-Env cells (Fig. 2), each inoculated with SFV-6 (Fig. 2A, B), SFV-7 (Fig. 2C, D), HFV (Fig. 2E, F), SFV-1 (Fig. 2I, J), SFV-2 (Fig. 2K, L) or SFV-3 (Fig. 2M, N) showed fewer infected BHK-Env cells compared with BHK-21 cells. In fact, end-point titres, determined using BHK-Env cells (used within 1 month of cloning), were 500- to 1500-fold lower than on BHK-21 cells (Fig. 3), suggesting that these viruses use the same cell surface receptor as HFV. To confirm that this phenotype was determined at the level of entry, a VSV pseudotype bearing the HFV envelope was generated by passage of VSV in BHK-Env cells. After neutralization with anti-VSV serum, a pseudotype titre of 2x103 p.f.u./ml was observed on BHK-21 cells, whereas no plaques were observed when BHK-Env cells were inoculated with 20 µl VSV (HFV). Thus, VSV (HFV) was at least 40-fold less infectious on BHK-Env cells compared with BHK-21 cells (Fig. 3). In contrast, when non-pseudotyped VSV was titred on BHK-21 and BHK-Env target cells, an equal number of plaques was observed, and no infectivity was observed after neutralization with anti-VSV serum. These results suggest that HFV and distinct SFV neutralization serotypes share a common saturable receptor on BHK-21 cells.



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Fig. 2. Receptor interference assays. HFV-infected BHK-21 cells and BHK-Env cells were detected by immunoperoxidase staining using HFV-positive human antiserum (A–H) or pooled foamy virus-positive macaque sera (I–P): SFV-6-infected BHK-21 cells (A), SFV-6-infected BHK-Env cells (B), SFV-7-infected BHK-21 cells (C), SFV-7-infected BHK-Env cells (D), HFV-infected BHK-21 cells (E), HFV-infected BHK-Env cells (F), uninfected BHK-21 cells (G), uninfected BHK-Env cells (H), SFV-1-infected BHK-21 cells (I), SFV-1-infected BHK-Env cells (J), SFV-2-infected BHK-21 cells (K), SFV-2-infected BHK-Env cells (L), SFV-3-infected BHK-21 cells (M), SFV-3-infected BHK-Env cells (N), uninfected BHK-21 cells (O) and uninfected BHK-Env cells (P).

 


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Fig. 3. Titration of foamy viruses and VSV (foamy virus) pseudotypes using BHK-21 and BHK-Env target cells. End-point titres of foamy virus isolates and VSV pseudotypes were determined on BHK-21 cells (solid shaded bars) or BHK-Env cells (line shaded bars) by immunoperoxidase staining. VSV and VSV (HFV) titres were determined by plaque assay.

 

   Discussion
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Abstract
Introduction
Methods
Results
Discussion
References
 
Foamy viruses bud from intracellular membranes and remain sequestered in the cell, resulting in low extracellular titres and constituting a practical disadvantage to their investigation and exploitation as vectors. Traditional methods for virus titre enhancement were tested and the effects monitored using a commercially available RT assay designed for C-type retroviruses, the RTs of which are Mn2+-dependent. Cation dependence of the foamy virus RT is not exclusive, but the Mg2+ assay for HIV-1 proved less sensitive, in agreement with previous published data (Kögel et al., 1995 ). The greatest RT activity, an indication of virus particles, in cell-free supernatant of infected cells occurred 7 days post-infection. Thus, maximal release of virions from infected cells occurs approximately 72 h after the first syncytia were observed. The optimal m.o.i. for infection was found to be between 0·01 and 0·05. A lower m.o.i. did not provide a productive infection, and a higher m.o.i., although producing CPE on day 4, caused extensive cell death by day 7 and although the RT activity remained high the end-point titre, determined by serial dilution of the supernatant, was greatly reduced. This suggests that RT production is not a limiting factor in the production of replication-competent virions from infected cells or alternatively that RT is more stable than the virus.

In comparison with previous reports of HIV and ecotropic murine leukaemia virus infections which were enhanced by surface charge reduction (Faller & Baltimore, 1984 ; Innes et al., 1990 ; Konopka et al., 1990 ), the cationic liposome formulation of Lipofectin was utilized to attempt to enhance the HFV infection rate by reducing surface charge. Lipofectin had no effect on HFV production, demonstrating that virion surface charge is not a limiting factor during HFV infection.

During foamy virus infection in vitro, the majority of virus particles remain within host cells, where they bud from intracellular membranes (Bodem et al., 1997 ). Release of virions from infected cells by disruption of the cell membrane by freeze–thawing, removal of cell debris by filtration or low speed centrifugation and virion concentration by ultrafiltration increased HFV titre from 103 to 106 virions/ml. It has been reported that HFV titres of approx. 106 can also be obtained by transfection of BHK-21 cells with human spumaretrovirus and placing the CPE-producing lysates onto low passage HEL-299 cells (Yu & Linial, 1993 ). Further increases may require a better understanding of the role of the HFV endoplasmic reticulum retrieval signal present in the envelope glycoprotein (Goepfert et al., 1997 ).

In this paper, analysis of a variety of cell lines from diverse species confirmed that the receptor for HFV is widely expressed in mammals and is also present in birds and reptiles. In addition to the cell lines studied here, HFV has also been reported to productively infect cell lines derived from rabbits, cows, dogs, cats, sheep and chickens (Hooks & Gibbs, 1975 ; Russell & Miller, 1996 ) as well as many cell types of human origin including cell lines of epithelial, neuronal, myeloid and fibroblastoid origin (Hooks & Gibbs, 1975 ; Hooks & Dentrick-Hooks, 1981 ; Neumann-Haefelin et al., 1983 ; Yu et al., 1996a ). The ubiquitous nature of the HFV receptor suggests that it may perform an essential conserved cellular function in eukaryotes. The apparent absence of a cell line refractory to foamy virus infection has hampered attempts to clone the cellular receptor by genetic means. However, the ubiquitous presence of the receptor in cell lines of diverse lineage and derived from both mammalian and invertebrate species might indicate that the receptor is not protein in nature. Nevertheless using a BHK-21-derived cell line constructed to express HFV envelope, we demonstrate from receptor interference assays that the receptor is saturable and is shared by several different primate foamy virus isolates. Overall, these results indicate that (i) foamy viruses can be produced in reasonable quantities, (ii) foamy viruses are concentratable without loss of infectivity and (iii) HFV-based vectors should be useful for gene transfer in a wide variety of cell types.


   Acknowledgments
 
We are grateful to the Wellcome Trust and the EC for their support of this study.


   Footnotes
 
b Present address: Howard Hughes Medical Institute, Duke University Medical Center, Durham, North Carolina 27710, USA


   References
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
Achong, B. G. & Epstein, M. A. (1978). Preliminary seroepidemiological studies on the human syncytial virus. Journal of General Virology 40, 175-181.[Abstract]

Achong, B. G., Mansell, P. W. A., Epstein, M. A. & Clifford, P. (1971). An unusual virus in cultures from a human nasopharyngeal carcinoma patient. Journal of the National Cancer Institute 46, 299-307.[Medline]

Ali, M., Rethwilm, A., Cheingsong-Popov, R., Taylor, J. N., Bradley, J. & McClure, M. O. (1996). Seroepidemiology of human foamy virus infection. AIDS & Human Retroviruses 12, 1473-1483.

Baunach, G., Maurer, B., Hahn, H., Kranz, M. & Rethwilm, A. (1993). Functional analysis of human foamy virus accessory reading frames. Journal of Virology 67, 5411-5418.[Abstract]

Bieniasz, P. D., Weiss, R. A. & McClure, M. O. (1995). Cell cycle dependence of foamy retrovirus infection. Journal of Virology 69, 7295-7299.[Abstract]

Bieniasz, P. D., Erlwien, O., Aguzzi, A., Rethwilm, A. & McClure, M. O. (1997). Gene transfer using replication-defective human foamy virus vectors. Virology 235, 65-72.[Medline]

Bodem, J., Löchely, M., Yang, P. & Flügel, R. M. (1997). Regulation of gene expression by human foamy virus and potentials of foamy viral vectors. Hematopoietic Stem Cells 15, 141-147.

Ekstrand, D. H. L., Awad, R. J. K., Kallander, C. F. R. & Gronowitz, J. S. (1996). A sensitive assay for the quantification of reverse transcriptase activity based on the use of carrier-bound template and non-radioactive-product detection, with special reference to human immunodeficiency virus isolation. Biotechnology and Applied Biochemistry 23, 95-105.[Medline]

Enssle, J., Jordan, I., Mauer, B. & Rethwilm, A. (1996). Foamy virus reverse transcriptase is expressed independently from the Gag protein. Proceedings of the National Academy of Sciences, USA 93, 4137-4141.[Abstract/Free Full Text]

Faller, D. V. & Baltimore, D. (1984). Liposome encapsulation of retrovirus allows efficient superinfection of resistant cell lines. Journal of Virology 49, 269-272.[Medline]

Goepfert, P. A., Shaw, K. L., Ritter, G. D.Jr & Mulligan, M. J. (1997). A sorting motif localises the foamy virus glycoprotein to the endoplasmic reticulum. Journal of Virology 71, 778-784.[Abstract]

Hooks, J. J. & Dentrick-Hooks, B. (1981). Spumavirinae foamy virus group infections: comparative aspects and diagnosis. In Comparative Diagnosis of Viral Disease, pp. 559-618. Edited by E. Kurstak & C. Kurstak. New York: Academic Press.

Hooks, J. J. & Gibbs, C. J.Jr. (1975). The foamy viruses. Bacteriology Review 39, 169-185.

Hooks, J. J., Gibbs, C. J.Jr., Chou, S., Howk, R., Lewi, M. & Gajdusek, D. C. (1973). Isolation of a new simian foamy virus from a spider monkey brain culture. Infection and Immunity 8, 804-813.[Medline]

Innes, C. L., Smith, P. B., Langenbach, R., Tindall, K. R. & Boone, L. R. (1990). Cationic liposomes (Lipofectin) mediate retroviral infection in the absence of specific receptors. Journal of Virology 64, 957-961.[Medline]

Kögel, D., Mordechai, A. & Flügel, R. M. (1995). Molecular biological characterization of the human foamy virus reverse transcriptase and ribonuclease H domains. Virology 213, 97-108.[Medline]

Konopka, K., Davis, B. R., Larsen, C. E., Alford, D. R., Debs, R. J. & Düzgüne, N. (1990). Liposomes modulate human immunodeficiency virus infectivity. Journal of General Virology 71, 2899-2907.[Abstract]

Lochelt, M. & Flügel, R. M. (1996). Human foamy virus pol gene is expressed as a pro-pol polyprotein and not as a gag–pol fusion protein. Journal of Virology 62, 1120-1124.

Malmsten, A., Ekstrand, D. H. L., Akerbolm, L., Gronowitz, J. S., Kallander, C. F. R., Bendinelli, M. & Matteucci, D. (1998). A colorimetric reverse transcriptase (RT) assay optimized for Moloney murine leukemia virus, and its use for characterization of RTs of unknown identity. Journal of Virological Methods 75, 9-20.[Medline]

Moebes, A., Enssle, J., Bieniasz, P. D., Heinkelein, M., Lindemann, D., Bock, M., McClure, M. O. & Rethwilm, A. (1997). Human foamy virus reverse transcription that occurs late in the viral replication cycle. Journal of Virology 71, 7305-7311.[Abstract]

Morgenstern, J. P. & Land, H. (1990). Advanced mammalian gene transfer: high titre retroviral vectors and a complementary helper free packaging cell line. Nucleic Acids Research 18, 3587-3596.[Abstract]

Neumann-Haefelin, D., Rethwilm, A., Bauer, G., Gudat, F. & zur Hausen, H. H. (1983). Characterization of a foamy virus isolated from Ceropithecus aethiops lymphoblastoid cells. Medical Microbiology and Immunology 172, 75-86.[Medline]

Rethwilm, A., Baunach, G., Netzer, K., Maurer, B., Borisch, B. & Meulen, V. (1990). Infectious DNA of the human spumaretrovirus. Nucleic Acids Research 18, 733-738.[Abstract]

Russell, D. W. & Miller, A. D. (1996). Foamy virus vectors. Journal of Virology 70, 217-222.[Abstract]

Schmidt, M. & Rethwilm, A. (1995). Replicating foamy virus based vectors directing high level expression of foreign genes. Virology 210, 167-178.[Medline]

Schmidt, M., Niewiesk, S., Heeny, J., Aguzzi, A. & Rethwilm, A. (1997). Mouse model to study the replication of primate foamy viruses. Journal of General Virology 78, 1929-1933.[Abstract]

Schnitzer, T. J. (1982). Simian foamy virus pseudotypes of vesicular stomatitis virus: production and use in sero-epidemiological investigations. Journal of General Virology 59, 203-206.[Abstract]

Schweizer, M., Falcone, V., Gange, J., Turek, R. & Neumann-Haefelin, D. (1997). Simian foamy virus isolated from an accidentally infected human individual. Journal of Virology 71, 217-222.

Stiles, G. E. (1968). Serological screening of rhesus and grivet monkeys for SV40 and the foamy viruses. Proceedings of the Society for Experimental Biology and Medicine 127, 225-230.

Swack, N. S., Schoentag, R. A. & Hsiung, G. D. (1970). Foamy virus infection of rhesus and green monkeys in captivity. American Journal of Epidemiology 92, 79-83.[Medline]

Weiss, R. A. & Tailor, C. S. (1995). Retrovirus receptors. Cell 82, 531-533.[Medline]

Yu, S. F. & Linial, M. L. (1993). Analysis of the role of the bel and bet open reading frames of human foamy virus by using a new quantitative assay. Journal of Virology 67, 6618-6624.[Abstract]

Yu, S. F., Stone, J. & Linial, M. L. (1996a). Productive persistent infection of haemopoietic cells by human foamy virus. Journal of Virology 70, 1250-1254.[Abstract]

Yu, S. F., Baldwin, D. N., Gwynn, S. R., Yendapalli, S. & Linial, M. L. (1996b). Human foamy virus replication: a pathway distinct from that of retroviruses and hepadnaviruses. Science 271, 1579-1582.[Abstract]

Received 15 March 1999; accepted 26 April 1999.