Foamy virus vectors.

Gene therapy is a promising novel treatment for a variety of human diseases. Successful application of gene therapy requires the availability of vehicles with the ability to efficiently deliver and express genes. Viral vectors are efficient means of transferring a gene of interest into target cells. Current available vehicles for gene transfer are either inefficient or potentially unsafe for human gene therapy applications. Foamy viruses offer a fresh alternative vector system for gene transfer with the potential to overcome the concerns of the current vectors. Foamy viruses are nonpathogenic and have a broad host range with the ability to infect various types of cells from different species. Foamy virus replication is distinct and may provide an edge for foamy virus vector usage over other retroviral vectors. These features offer the foamy vectors unique opportunities to deliver several genes into a number of different cell types in vivo safely and efficiently. The principal problems for the design of foamy virus vectors have been solved, and several foamy virus vectors that efficiently transduce a variety of cell types are available. This chapter reviews specific features of foamy virus vector systems and recent advances in the development and use of these vectors.

Partial characterization of retroviruses from boid snakes with inclusion body disease.

OBJECTIVE: To characterize retroviruses isolated from boid snakes with inclusion body disease (IBD). ANIMALS: 2 boa constrictors with IBD and 1 boa exposed to an affected snake. PROCEDURE: Snakes were euthanatized, and tissue specimens and blood samples were submitted for virus isolation. Tissue specimens were cultured with or without commercially available viper heart cells and examined by use of transmission electron microscopy (TEM) for evidence of viral replication. Reverse transcriptase activ ty was determined in sucrose gradient-purified virus. Western blotting was performed, using polyclonal antibodies against 1 of the isolated viruses. Specificity of the rabbit anti-virus antibody was evaluated, using an immunogold-labeling TEM technique. RESULTS: 3 viruses (RV-1, RV-2, and RV-3) were isolated. The isolates were morphologically comparable to members of the Retroviridae family. Reverse transcriptase activity was high in sucrose gradient fractions that were rich in virus. Polyclonal antibody against RV-1 reacted with proteins of similar relative mobility in RV-1 and RV-2. By use of immunogold labeling, this antibody also recognized virions of both RV-1 and RV-2. CONCLUSIONS AND CLINICAL RELEVANCE: A retrovirus was isolated from boid snakes with IBD or exposed to IBD. Western blot analysis of viral proteins indicated that viruses isolated from the different snakes were similar. Whether this virus represents the causative agent of IBD is yet to be determined. The isolation of retroviruses from boid snakes with IBD is an important step n the process of identifying the causative agent of this disease.

The efficiency of simian foamy virus vector type-1 (SFV-1) in nondividing cells and in human PBLs.

Current retroviral vectors based on murine leukemia virus (MuLV) are unable to efficiently transduce nondividing cells. Lentiviruses, such as the human immunodeficiency virus 1 (HIV-1) are efficient at transducing nondividing, growth-arrested, and post-mitotic cells, but due to complex safety considerations, they may have limited potential for human clinical gene transfer. For this reason, alternatives to MuLV and HIV-1 vectors need to be explored. In this paper, we have found that simian foamy virus vector (SFV-1) containing a CMV-LacZ expression cassette is able to efficiently transduce multiple cell types of various species that include epithelial, lymphoid, and hematopoietic-derived human cell lines and fibroblast cell lines of several species. Previously it was reported that foamy virus replication is cell cycle dependent (P. D. Bieniasz, R. A. Weiss, and M. O. McClure, 1995. J. Virol. 69, 7295-7299). However, others studies demonstrated nuclear import of viral DNA in arrested cells (A. Saibi, F. Puvion-Dutilleul, M. Schmid, J. Peries, and H. d. The 1997. J. Virol. 71, 1155-1161). Here, we show efficient LacZ transduction by SFV-1 vectors in several chemically arrested cell lines and terminally differentiated human neurons. SFV-1 vector can transduce cell lines arrested in G1/S phase of the cell cycle by aphidicolin treatment with similar efficiencies to that of dividing cells. The terminally differentiated human neural cell line, NT2N, was transduced with 30-50% efficiency, corroborating our data obtained with the arrested cell lines. To further examine whether the SFV-1 vector can efficiently deliver a gene into clinically important cells for gene therapy, we transduced primary human peripheral blood cells (PBLs) in the presence and absence of phytohemagglutanin (PHA) stimulation. We observed 81% transduction efficiency in non-stimulated PBLs and 87% in PHA-stimulated PBLs with vector infection carried out twice in 8 hours intervals at a multiplicity of infection of 1. Together, these data indicate that SFV-1 based retroviral vectors may provide a safe, efficient alternative to current onco- and lentiviral vectors for gene transfer in cells from a broad spectrum of lineages across species boundaries.

Packaging cell lines for simian foamy virus type 1 vectors.

Foamy viruses are nonpathogenic retroviruses that offer several unique opportunities for gene transfer in various cell types from different species. We have previously demonstrated the utility of simian foamy virus type 1 (SFV-1) as a vector system by transient expression assay (M. Wu et al., J. Virol. 72:3451-3454, 1998). In this report, we describe the first stable packaging cell lines for foamy virus vectors based on SFV-1. We developed two packaging cell lines in which the helper DNA is placed under the control of either a constitutive cytomegalovirus (CMV) immediate-early gene or inducible tetracycline promoter for expression. Although the constitutive packaging expressing cell line had a higher copy number of packaging DNA, the inducible packaging cell line produced four times more vector particles. This result suggested that the structural gene products in the constitutively expressing packaging cell line were expressed at a level that is not toxic to the cells, and thus vector production was reduced. The SFV-1 vector in the presence of vesicular stomatitis virus envelope protein G (VSV-G) produced an insignificant level of transduction, indicating that foamy viruses could not be pseudotyped with VSV-G to generate high-titer vectors. The availability of stable packaging cell lines represents a step toward the use of an SFV-1 vector delivery system that will allow scaled-up production of vector stocks for gene therapy.

Inhibition of FIV replication by a ribozyme that targets the Rev response element.

The feline immunodeficiency virus (FIV) Rev protein and its cognate sequence the Rev response element (RRE) are essential for virus replication. Thus, the inhibition of either Rev or RRE function can significantly inhibit FIV replication. In the present study, we constructed a ribozyme that targets the RRE sequence and determined its ability to inhibit FIV replication. The RRE ribozyme cleaved the target molecule both in vitro and in FIV-infected cells. Furthermore, FIV replication was reduced significantly in the presence of the RRE ribozyme. FIV provides a good animal model system with which to develop novel antiviral strategies for the human immunodeficiency virus.

Simian foamy virus type 1 (SFV-1) induces apoptosis.

Foamy virus infection causes cytopathology in several cell types from different species. The mechanism of cell killing by foamy viruses is not known. In this report, the mechanism of cell death induced by simian foamy virus type 1 (SFV-1) infection was investigated in fibroblast and lymphoid derived cells lines. Infected L-929 (fibroblast) and Raji (B cell) cells showed chromatin condensation, chromatin cleavage into nucleosome oligomers, and ultrastructural changes consistent with apoptosis. These data suggest that SFV-1 induced apoptotic cell death in different cell lines from different species. The degree of apoptotic cell death in both L-929 and Raji cell lines correlated with increased virus replication. Apoptosis, therefore, is one mechanism by which SFV-1 causes cell death.

Cell tropism of the simian foamy virus type 1 (SFV-1).

Several cell lines representing different species and cell types were tested for simian foamy virus type 1 (SFV-1) infection. SFV-1 infections were monitored by polymerase chain reaction, reverse transcriptase, cytopathology, and immunofluorescent assays. All cells tested were permissive for SFV-1, demonstrating that SFV-1 has a broad host range with respect to species and cell types. Infected fibroblasts, epithelial cells, and neural cells all showed extensive cytopathology that is characteristic of foamy virus infection. No cytopathology was induced in T cell-derived Jurkats and Hut-78 cell lines. The cytopathic effects in B and macrophage originated cells were delayed by several days. Cytopathology in these cell lines was not as dramatic as seen in the infected fibroblast and epithelial cells. The reverse transcriptase values in the SFV-1 infected lymphoid and macrophage cell lines were severalfold lower than that of the fibroblasts and epithelial cells. Therefore, SFV-1 appears to establish a low level persistent infection in lymphoid and macrophage cell lines.

Reactivity of primate sera to foamy virus Gag and Bet proteins.

In order to establish criteria for the serodiagnosis of foamy virus infections we investigated the extent to which sera from infected individuals of human and primate origin react with structural and non-structural virus proteins in immunoblot assays. Using lysates from infected cells as the source of virus antigen, antibodies were preferentially detected against the Gag proteins and the non-structural Bet protein. Both the Gag precursor molecules of 70 and 74K apparent M(r) and the cytoplasmic 60K M(r) Bet protein were found to be phosphorylated, the latter being synthesized in large amounts in infected cells. Rabbit antiserum raised against recombinant human foamy virus (HFV) Gag major capsid protein cross-reacted with foamy viruses of chimpanzee, gorilla, orang-utan, rhesus monkey and African green monkey origin. This was reflected by a broad cross-reactivity of the respective monkey sera to the Gag proteins of the various foamy virus isolates. Cross-reactivity of antisera against the Bet protein was restricted to viruses from man and the great apes. Recombinant Gag and Bet proteins expressed in prokaryotes or in insect cells were readily recognized by foamy virus-positive primate sera. Screening serum samples from chimpanzees with HFV Gag and Bet proteins expressed by recombinant baculoviruses revealed that 18 out of 35 (52%) were positive for Gag antibodies. Of these, 13 (72%) showed antibodies against the Bet protein, indicating that Bet antigen is of value in serological screening for foamy virus infections.

Glutathione-S-transferase activates novel alkylating agents.

Alkylating agents which are activated by glutathion-S-transferases (GSTs) have been designed and synthesized. The model compound gamma-glutamyl-alpha-amino-beta-[(2-ethyl N,N,N',N'-tetraethylphosphorodiamidate) sulfonyl]propionylglycine (1) and the nitrogen mustards gamma-glutamyl-alpha- amino-beta-[[2-ethyl N,N,N',N'-tetrakis (2-chloroethyl)phosphorodiamidate] sulfonyl]propionylglycine (2) and gamma-glutamyl-alpha-amino-beta-[[2-ethyl-N,N,N',N'-tetrakis(2- chloroethyl)phosphorodiamidate]sulfonyl]-propionyl-(R)-(-)-phenylg lycine (3) were prepared via multistep chemical synthesis. The compounds were tested with recombinant human A1-1, M1a-1a and P1-1 GSTs. HPLC studies showed that the compounds were differentially and catalytically cleaved by biologically relevant concentrations of the GSTs. Mass spectral studies of the cleavage mixture of 2 showed that M1a-1a GST liberated the cytotoxic phosphate moiety needed for efficacy as an alkylating agent. Cell culture studies with MCF-7 breast cancer cells showed that 1 was not toxic at 200 microM, while 2 and 3 showed IC50S of 40.6 and 37.5 microM, respectively, for the same cell line. MCF-7 cells transfected to overexpress P1-1 GST showed enhanced sensitivity with 2 and 3, with IC50S of 20.9 and 9.5 microM, respectively. This result correlates well with the rates of cleavage of 2 and 3 by P1-1 GST observed in vitro and demonstrates that higher levels of cellular P1-1 GST will give increased sensitivity to these drugs.

Identification of the simian foamy virus transcriptional transactivator gene (taf).

Simian foamy virus type 1 (SFV-1), a member of spumavirus subfamily of retroviruses, encodes a transcriptional transactivator that functions to strongly augment gene expression directed by the viral long terminal repeat (LTR). The objective of this study was to identify the viral gene responsible for transactivation. Nucleotide sequences between the env gene and the LTR of SFV-1 were determined. The predicted amino acid sequence revealed two large open reading frames (ORFs), designated ORF-1 (311 amino acids) and ORF-2 (422 amino acids). In the corresponding region of the human foamy virus, three ORFs (bel-1, bel-2, and bel-3) have been identified (R. M. Flugel, A. Rethwilm, B. Maurer, and G. Darai, EMBO J. 6:2077-2084, 1987). Pairwise comparisons of the ORF-1 and ORF-2 with bel-1 and bel-2 show small clusters of homology; less than 39% overall homology of conserved amino acids is observed. A counterpart for human foamy virus bel-3 is not present in the SFV-1 sequence. Three species of viral RNA have been identified in cells infected with SFV-1; an 11.5-kb RNA representing full-length transcripts, a 6.5-kb RNA representing the env message, and a 2.8-kb RNA from the ORF region. Analysis of a cDNA clone encoding the ORF region of SFV-1 reveals that the 2.8-kb message is generated by complex splicing events involving the 3' end of the env gene. In transient expression assays in cell lines representing several species. ORF-1 was shown to be necessary and sufficient for transactivating viral gene expression directed by the SFV-1 LTR. The target for transactivation is located in the U3 domain of the LTR, upstream from position - 125 (+ 1 represents the transcription initiation site). We propose that OFF-1 of SFV-1 be designated the transcriptional transactivator of foamy virus (taf).

Simian foamy virus type 1 is a retrovirus which encodes a transcriptional transactivator.

Simian foamy viruses, members of the spumavirus subfamily of retroviruses, are found in a variety of nonhuman primates and, as yet, remain to be characterized with respect to genetic structure and regulation of viral gene expression. The genome of simian foamy virus type 1 (SFV-1), an isolate from rhesus macaques, has been molecularly cloned, and the role of the viral long terminal repeat (LTR) in transcriptional control has been investigated. The SFV-1 LTR is 1,621 base pairs long, and sequence comparisons with human foamy virus revealed a pattern of clustered homology. A cap site in the LTR was identified by analysis of SFV-1 transcripts in infected cells. Transient expression assays in cell lines representing several species and different cell types showed that the SFV-1 LTR has low basal activity in uninfected cells, whereas LTR-directed expression is greatly increased in cells infected with SFV-1. This transactivation is mediated by a mechanism involving increases in steady-state levels of viral transcripts. Thus, the SFV-1 genome encodes a transactivator that functions on the LTR at the transcriptional level.

Relationship of the env genes and the endonuclease domain of the pol genes of simian foamy virus type 1 and human foamy virus.

We have molecularly cloned and sequenced a portion of the simian foamy virus type 1 (SFV-1); open reading frames representing the endonuclease domain of the polymerase (pol) and the envelope (env) genes were identified by comparison with the human foamy virus (HFV). Unlike the HFV genomic organization, the SFV-1 pol gene overlaps the env gene; thus, the open reading frames reported for HFV between pol and env is not present in SFV-1. Comparisons of predicted amino acid sequences of HFV and SFV-1 reveal that the endonuclease domains of the pol genes are about 84% related. The region predicted to encode the SFV-1 extracellular env domain is 569 codons; SFV-1 and HFV have 64% amino acid similarity in this env domain. The predicted hydrophobic transmembrane env proteins of both HFV and SFV-1 show about 73% similarity. A total of 16 potential glycosylation sites are found in SFV-1 env, and 15 are found in HFV; 11 are shared. SFV-1 has 25 cysteine residues, and HFV has 23 residues; all 23 cysteine residues of HFV are conserved in SFV-1. This sequence analysis reveals that the human and simian foamy viruses are highly related.

Basic fibroblast growth factor in human rhabdomyosarcoma cells: implications for the proliferation and neovascularization of myoblast-derived tumors.

Cultured human embryonal rhabdomyosarcoma cells express the basic fibroblast growth factor (bFGF) gene and they produce bFGF, which is apparently composed of two microheterogenous forms with Mrs of 16,500 and 17,200, respectively. bFGF derived from the rhabdomyosarcoma cells stimulates their own proliferation and that of human or bovine vascular endothelial cells. It is conceivable that the rhabdomyosarcoma-derived bFGF stimulates the growth and neovascularization of human rhabdomyosarcomas and that it may thereby contribute to the development of these tumors.

Human basic fibroblast growth factor: nucleotide sequence and genomic organization.

Clones encoding the angiogenic endothelial cell mitogen, basic fibroblast growth factor (FGF), have been isolated from human cDNA libraries made from kidney, fetal heart, fetal liver, term placenta, and a breast carcinoma. Basic FGF cDNA clones are present in these libraries at very low levels when compared to the quantity of the growth factor in the tissues. This observation, combined with the fact that several of the clones represent unspliced transcripts, suggests that cytoplasmic basic FGF mRNA is unstable and that the protein is stored in tissues. The amino acid sequence of human basic FGF, deduced from the sequence of these cDNAs and from genomic clones, is 99% homologous to that of bovine basic FGF, implying a strong selection pressure for maintenance of function and structure. As with the bovine factor, human basic FGF does not appear to have a signal peptide sequence. Southern blot analysis of human genomic DNA and mapping of the cloned gene shows that there is only one basic FGF gene. All of the basic, heparin-binding endothelial cell mitogens of similar amino acid composition that have been described must therefore be products of this single gene.

Nucleotide sequence of a bovine clone encoding the angiogenic protein, basic fibroblast growth factor.

Basic and acidic fibroblast growth factors (FGF's) are potent mitogens for capillary endothelial cells in vitro, stimulate angiogenesis in vivo, and may participate in tissue repair. An oligonucleotide probe for bovine basic FGF was designed from the nucleotide sequence of the amino-terminal exon of bovine acidic FGF, taking into account the 55 percent amino acid sequence homology between the two factors. With this oligonucleotide probe, a full length complementary DNA for basic FGF was isolated from bovine pituitary. Basic FGF in bovine hypothalamus was shown to be encoded by a single 5.0-kilobase messenger RNA; in a human hepatoma cell line, both 4.6- and 2.2-kilobase basic FGF messenger RNA's were present. Both growth factors seem to be synthesized with short amino-terminal extensions that are not found on the isolated forms for which the amino acid sequences have been determined. Neither basic nor acidic FGF has a classic signal peptide.