Simian Foamy Virus Type Research Articles

Simian Immunodeficiency Virus seroreactivity in inhabitants from rural Cameroon frequently in contact with non-human primates

Central African tropical forests are home to several species of non-human primates (NHPs), infected by Simian Immunodeficiency Virus (SIV). It is well-known that HIV-1 epidemic is due to cross-transmission and adaptation of SIV to humans. The main goal of this work was to investigate if a NHP bite is a risk factor for SIV acquisition. A cross-sectional study was performed in rural Cameroon on 246 bitten individuals (mostly by adult NHPs), matched, according to sex, age, and ethnicity (Bantus and Pygmies), with an equal number of not-bitten subjects. Following a serological assay for a wide range of SIVs, we observed a high level of indeterminate seroreactivity (25.8%) in the total population, whereas 68.9% were sero-negative and 5.3% HIV-1 positive. Bites do not appear to be a risk factor for SIV seroreactivity, in contrast to Simian Foamy Virus and Simian T-Lymphotropic Virus type 1 in the same studied population.

Preparation of Simian Foamy Virus Type-1 Vectors

Foamy viruses (FVs) are nonpathogenic retroviruses that offer opportunities for efficient and safe gene transfer in various cell types from different species. These viruses have unique replication mechanisms that are distinct from other retroviruses, which may give an advantage to FV-mediated gene transfer. This protocol describes a method for simian foamy virus type-1 (SFV-1) vector preparation and concentration. A transient transfection of vector and packaging constructs allows generation of the SFV-1 vector with titers of 10(7)/mL. The vectors can be further concentrated by 100-200-fold without significant loss of vector titer.

Inhibition of simian immunodeficiency virus by foamy virus vectors expressing siRNAs

Viral vectors available for gene therapy are either inefficient or suffer from safety concerns for human applications. Foamy viruses are non-pathogenic retroviruses that offer several unique opportunities for gene transfer in various cell types from different species. In this report, we describe the use of simian foamy virus type 1 (SFV-1) vector to examine the efficacy of therapeutic genes. Hairpin short-interfering RNA (siRNA) that targets the simian immunodeficiency virus (SIV) rev/env was placed under the control of the PolIII U6 snRNA promoter for expression and screened for silencing target genes using cognate target–reporter fusions. We have identified an effective siRNA (designated R2) which reduces the rev and env gene expression by 89% and 95%, respectively. Using the simian foamy virus type 1 (SFV-1) based vector, we delivered the PolIII expressed R2 siRNA into cultured cells and challenged with SIV. The results show that the R2 siRNA is a potent inhibitor of SIV replication as determined by p27 expression and reverse transcriptase assays. Vectors based on a non-pathogenic SFV-1 vector may provide a safe and efficient alternative to currently available vectors, and the SIV model will help devise protocols for effective anti-HIV gene therapy.

Transduction of Umbilical Cord Blood CD34+ NOD/SCID-Repopulating Cells by Simian Foamy Virus Type 1 (SFV-1) Vector

Foamy viruses are nonpathogenic retroviruses that offer unique opportunities for gene transfer into various cell types including hematopoietic stem cells. We used a simian foamy virus type 1 vector (SFV-1) containing a LacZ reporter gene with a titer of 1–5 × 10 6 viral particles/ml that was free of replication-competent retrovirus to transduce human umbilical cord blood CD34+ cells. Transduced CD34+ cord blood cells were transplanted into NOD/SCID mice and plated in serum-free methylcellulose culture to determine the transduction efficiency of human hematopoietic progenitor cells. A transduction efficiency of about 20% was obtained. At 6–10 weeks posttransplantation, human hematopoietic cell engraftment and marking were determined. Marrow from transplanted mice demonstrated human cell engraftment by the presence of human (CD45+) cells containing both CD19+ lymphoid and CD33+ myeloid cells. Serial sampling of NOD/SCID bone marrow revealed the presence of 6.7–14.0% CD45+ cells at 6 weeks posttransplant as compared to 3.6–27.2% CD45+ cells at 9–10 weeks posttransplant. Human progenitors examined from NOD/SCID bone marrow cells 9 weeks posttransplant revealed from 7.4 to 25.9% of the colonies exhibiting X-gal staining. Our study demonstrates the ability of a simian foamy virus vector to transduce the SCID-repopulating cell and offers a promising new gene delivery system for use in hematopoietic stem cell gene therapy.

A Minimal Genome Simian Foamy Virus Type 1 Vector System with Efficient Gene Transfer

Foamy viruses have several inherent features for the opportunity to develop efficient and versatile vectors for gene therapy. We have constructed a series of vectors and helper plasmids based on simian foamy virus type 1 (SFV-1) to establish the minimum vector genome required for efficient gene transduction. To characterize the efficiency of gene transduction by these vectors, the green fluorescent protein (GFP) coding sequence is linked to the human cytomegalovirus immediate gene promoter. Several deletion analyses of SFV-1 vectors revealed that the minimum genome with efficient GFP transduction contained the 5′ untranslated region extending to the first 637 nucleotides of the gag gene, a 596 nucleotides of pol sequence from position 3137–3733, the 3′ pol region at position 5200–5693, the 3′ end polypurine tract, and the 3′ LTR. An additional 1131 nucleotides can be removed from the 3′ end LTR without affecting the efficiency of vector transduction. SFV-1 vector can therefore accommodate a minimum 8930 base-size heterologous DNA fragment. Furthermore, the efficiency of SFV-1 vector transduction was analyzed using different packaging plasmids. GFP transduction with packaging plasmid that contained the 5′ R-U5 region of the LTR was compared with helper plasmids that had deletions in this region except for 22 nucleotides (positions 21–41), the first 61, 77, or 140 nucleotides of the R of the LTR. Transduction efficiencies were significantly reduced with the deletion mutations implicating that for optimum SFV-1 vector productions a packaging construct that includes the 5′ R-U5 is required.

Mutational Analysis of the 5′ Leader Region of Simian Foamy Virus Type 1

The sequence within the 5′ untranslated region of the retroviral genome contains important cis elements for many steps in viral replication. There is limited information available on the role of this region in foamy virus replication. Similar to other retroviruses, the 5′ untranslated region of foamy viruses predicts extensive RNA secondary structure. Serial mutations that could change parts of the predicted secondary structure were introduced in the 5′ leader sequence including the R-U5 region of simian foamy virus type 1 (SFV-1) to investigate their role in virus genome packaging and virus replication. Point mutations in the R-U5 regions at nucleotide positions 7–12 (I), 241–243 (B), and 256–257 (D) had no effect on virus replication. Base substitution mutation at positions 193–195 (C), however, severely impaired virus replication. Deletion of sequences in the leader region, between the primer-binding site and the gag gene, at positions 364–399 (d1), 397–435 (d2), or 364–435 (d3), which included sequences for RNA genome dimerization, also blocked SFV-1 replication. Interestingly, none of these mutations affected genome packaging or the synthesis of viral transcripts, suggesting that a step(s) of virus replication following packaging is affected. The region between the primer-binding site and the gag gene, therefore, is not essential for foamy virus genome packaging. Furthermore, the cis-acting elements for genome dimerization and packaging appear to be localized in separate regions for foamy viruses.

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.

Derivation and functional characterization of a consensus DNA binding sequence for the tas transcriptional activator of simian foamy virus type 1.

Although DNA binding sites specific for the Bel-1 and Tas transcriptional activators, encoded, respectively, by the human and simian foamy viruses, have been mutationally defined, they show little evident sequence identity. As a result, the sequence determinants for DNA binding by both Bel-1 and Tas have remained unclear. Here, we report the use of a novel in vivo randomization and selection strategy to identify a Tas DNA binding site consensus. This approach takes advantage of the fact that Tas can effectively activate gene expression in yeast cells via a Tas DNA binding site derived from the simian foamy virus type 1 (SFV-1) internal promoter. The defined Tas DNA binding site consensus extends over approximately 25 bp and contains a critical core sequence of approximately 5 bp. Positions adjacent to this core sequence, while clearly also subject to selection, show a significantly higher level of sequence variation. Surprisingly, the wild-type SFV-1 internal promoter Tas DNA binding site fails to conform to the consensus at several positions. Further analysis demonstrated that the consensus sequence bound Tas more effectively than did the wild-type sequence in vitro and could mediate an enhanced Tas response in vivo when substituted into the SFV-1 internal promoter context. These findings explain the limited sequence identity observed for mutationally defined Tas or Bel-1 response elements and should facilitate the identification of Tas DNA target sites located elsewhere in the SFV-1 genome.

Cis-Acting sequences required for simian foamy virus type 1 vectors.

We have constructed a series of vectors based on simian foamy virus type 1 (SFV-1) to define the minimum cis-acting elements required for gene transfer. To characterize these vectors, we inserted the coding sequence of the bacterial lacZ gene linked to the cytomegalovirus immediate-early gene promoter. Introduction of a deletion mutation in the leader region between the 5' long terminal repeat and the start of the gag gene at position 1659 to 1694 completely abrogated gene transfer by the SFV-1 vector. Deletion of 39 nucleotides from position 1692 to 1731 in the leader region resulted in a significant reduction in the transducing-particle titer. Furthermore, we have identified a second cis-acting element located at the 3' end of the pol gene between position 6486 and 6975 to be critical for SFV-1 vector transduction. These results identify the two important cis-acting elements required for SFV-1 vector construction, and the finding of a cis-acting element in the pol gene is unique among retroviruses.

Characterization of provirus clones of simian foamy virus type 1.

We have cloned proviral DNA of simian foamy virus type 1 (SFV-1) from linear unintegrated DNA (pSFV-1). Transfection of pSFV-1 induces cytopathology in several cell lines with supernatants from the transfected cell culture containing infectious viral particles. Electron microscopy of the transfected cells revealed foamy virus particles. Deletion analysis of pSFV-1 indicated that the transcriptional transactivator (tas) gene located between env and the long terminal repeat is critical for virus replication, whereas the second open reading frame (ORF-2) in this region is dispensable. Although the tas and ORF-2 regions of foamy viruses have significantly diverged, the results presented here suggested that the gene products have similar functions. Recombinant pSFV-1 containing the cat gene was able to transduce the heterologous gene, indicating the utility of SFV-1 as a vector. An infectious clone of SFV-1 which is distantly related to the human foamy virus will provide a means to understand the biology of this unique group of viruses.

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.

Transactivation of the Two Promoters of SFV-3 by Different Mechanisms

Simian foamy virus type 3 (SFV-3), a member of the spumavirus genus of retroviruses, has a complex genome organization and encodes two open reading frames (ORFs), in addition to the structural genesgag, pol,andenv.ORF-1 encodes a viral transcriptional transactivator designated Taf (transactivator of foamy viruses) which augments transcription from the viral long terminal repeat (LTR). It was recently shown that human foamy virus, as well as the simian viruses SFV-1 and SFV-3, contains a second internal transcriptional promoter in the transmembrane domain of theenvgene; this promoter also is transactivated by Taf. Here we report the characterization of the internal promoter of SFV-3. The transcriptional start site of this promoter has been mapped in two different SFV-3-infected cell lines to nt position 9761 in the proviral genome of SFV-3. Allcis-regulatory elements required for transactivation by Taf are located between −202 and −32 (+1 representing the transcription initiation site in the internal promoter). Analysis of hybrid promoter constructs and deletion mutants in transient expression assays revealed that this region contains two elements which are independently responsive to Taf. In addition, we employed anin vivoDNA competition assay to determine whether the transactivation mechanisms of both SFV-3 promoters are similar or different. The differences observed utilizing this competition assay suggest that Taf transactivates the internal promoter and the LTR through different cellular transcription factors.

The transcriptional transactivator of simian foamy virus 1 binds to a DNA target element in the viral internal promoter.

The transcriptional transactivator (Tas) of simian foamy virus type 1 strongly augments gene expression directed by both the promoter in the viral long terminal repeat and the newly discovered internal promoter located within the env gene. A region of 121 bp, located immediately 5' to the TATA box in the internal promoter, is required for transactivation by Tas. The present study aimed to identify the precise Tas-responsive target(s) in this region and to determine the role of Tas in transcriptional regulation. By analysis of both clustered-site mutations and hybrid promoters in transient expression assays in murine and simian cells, two separate sequence elements within this 121-bp region were shown to be Tas-dependent transcriptional enhancers. These targets, each < 30 bp in length and displaying no apparent sequence homology one to the other, are designated the promoter-proximal and promoter-distal elements. By means of the gel electrophoresis mobility-shift assays, using purified glutathione S-transferase-Tas fusion protein expressed in Escherichia coli, the target proximal to the TATA box exhibited strong binding to glutathione S-transferase-Tas, whereas the distal element appears not to bind. In addition, footprint analysis revealed that 26 bp in the promoter proximal element was protected by glutathione S-transferase-Tas from DNase I. We propose a model for transactivation of the simian foamy virus type 1 internal promoter in which Tas interacts directly with the proximal target element positioned immediately 5' to the TATA box. In this model, Tas attached to this element is presumed to interact with a component(s) of the cellular RNA polymerase II initiation complex and thereby enhance transcription directed by the viral internal promoter.

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.

Simian Foamy Virus Type 1 Contains a Second Promoter Located at the 3′ End of the env Gene

Simian foamy virus type 1 (SFV-1) is a member of a distinct subfamily of retroviruses that possess a complex genome organization as well as complex means of regulating gene expression. This virus encodes a transcriptional transactivator ( taf) that dramatically increases gene expression directed by the viral long terminal repeat. In this report, the identification of a second promoter which is significantly transactivated by SFV-1 Taf is described. The second promoter is located at the 3′ end of SFV-1 env gene downstream from position 8158 (1 represents the first nucleotide sequence of the vital genome). Deletion analysis indicated that there are at least two targets for Taf in the internal promoter. A time-course analysis of SFV-1 gene expression showed that the messages for the regulatory genes were generated prior to the transcription of the structural proteins. Therefore, SFV-1 replication may be regulated by mechanisms that involve a temporal pattern of gene expression. The potential role of the internal promoter in SFV-1 gene expression and replication is discussed.

Transcriptional trans activators of human and simian foamy viruses contain a small, highly conserved activation domain.

The Bel-1 protein of human foamy virus is a potent transcriptional trans activator of its homologous long terminal repeat promoter element. Here, we demonstrate that Bel-1 can also efficiently activate gene expression when targeted to a heterologous promoter by fusion to the DNA-binding motif of the yeast GAL4 protein. Analysis of a series of deletion mutants of Bel-1 generated in this hybrid protein context suggests the presence of a single transcription activation domain that is fully contained within a discrete, approximately 30-amino-acid segment located proximal to the Bel-1 carboxy terminus. Although this short motif can be shown to function effectively in eukaryotic cells of mammalian, avian, and fungal origin, it does not bear any evident sequence homology to the known classes of eukaryotic activation domain. However, this Bel-1 activation domain was found to be fully conserved, in terms of both biological activity and location, in the distantly related Taf trans activator of simian foamy virus type 1.

Simian Foamy Virus Type 3 (SFV-3) in Latently Infected Vero Cells: Reactivation by Demethylation of Proviral DNA

Cell cultures latently infected with simian foamy virus type 3 (SFV-3) were established by suppressing lytic infection in Vero cells with 3′-azido-3′-deoxythymidine (AZT) and homologous antibodies (African green monkey serum immune to SFV-3). The resulting cell line, designated Vero-L, was shown to contain at least one copy per cell of SFV-3 DNA stably integrated at a defined site of the host cell genome. Sequencing of 669 bp at the integration site did not identify a coding region and revealed a 4-bp imperfect repeat in host cell DNA due to SFV-3 integration. Over 2 years of subcultivation, no spontaneous expression of proviral genes could be detected. However, the demethylating agent 5′-azacytidine reactivated lytic infection, proving conservation of the complete viral genome. Comparison of proviral DNA from latently and lyrically infected cells supports the notion that methylation is instrumental in keeping SFV-3 infection in latency.

Regulatory Elements in the Long Terminal Repeat (LTR) of Simian Foamy Virus Type 3 (SFV-3)

Simian foamy virus type 3 (SFV-3) is a retrovirus that has a complex genome organization and encodes two open reading frames (ORF-1 and ORF-2) in addition to the genes coding for gag, pol, and env. In this report, we demonstrate that ORF-1 of SFV-3 encodes a transcriptional transactivator designated taf (transactivator of foamy virus) which augments gene expression directed by the viral long terminal repeat (LTR). The taf responsive elements have been mapped to the U3 region of the LTR, between positions —637 and —180 (+1 represents the transcription initiation site). Two regions between —637 and —180 in the LTR are targets for taf transactivation. These target sequences for taf confer responsiveness to a heterologous promoter independent of orientation; thus, they function like conditional enhancers. The R-U5 region of the viral LTR is shown to have an inhibitory effect on gene expression. SFV-1 is a related spumavirus and encodes a taf gene that augments expression directed by the SFV-3 LTR as well as the SFV-1 LTR; however, the taf gene of SFV-3 transactivates the SFV-3 LTR but not the SFV-1 LTR. These data on regulatory elements in the SFV-3 LTR show that the mechanism of foamy virus transactivation is significantly different from lentiviruses as well as from the HTLV group of viruses.

Genomic organization and expression of simian foamy virus type 3 (SFV-3)

The complete nucleotide sequence of simian foamy virus type 3 (SFV-3) strain LK-3, isolated from an African green monkey, was determined. In addition to translation frames representing the gag, pol, and env genes, two open reading frames are located in the region between the env gene and the 3′ long terminal repeat (LTR). Both SFV-3 and SFV-1 encode two open reading frames between env and the 3′ LTR, whereas HFV encodes three open reading frames in this region. Northern blot analysis of cell cultures infected with SFV-3 revealed subgenomic RNAs for these open reading frames. The protease of SFV-3 is encoded by the pol gene in contrast to HFV which encodes the protease in the gag gene. Notably, the pol gene of SFV-3 is in the +1 translational frame relative to the gag gene; this observation is in agreement with SFV-1, but differs for HFV and all other retrovirus genomes reported. Thus, gag-pol precursors of the SFVs appear to be expressed by a + 1 frameshift. Nucleotide and deduced amino acid alignments of SFV-3, SFV-1, and HFB revealed an unexpected homology pattern; highest homologies are observed in the pol and env genes but low homologies are noted in the gag genes and the additional open reading frames. Analysis of phylogenetic trees confirms the classification of foamy viruses as a subfamily of retroviruses, distinct from the lentiviruses and oncoviruses.

Cis-acting regulatory regions in the long terminal repeat of simian foamy virus type 1.

Simian foamy virus type 1 (SFV-1), a member of the Spumavirinae subfamily of retroviruses, encodes a transcriptional transactivator (taf) that strongly augments gene expression directed by the viral long terminal repeat (LTR) (A. Mergia, K. E. S. Shaw, E. Pratt-Lowe, P. A. Barry, and P. A. Luciw, J. Virol. 65:2903-2909, 1991). This report describes cis-acting regulatory elements in the LTR that control viral gene expression. A series of LTR mutants and hybrid promoter constructs have been analyzed in transient expression assays for responsiveness to Taf. The targets for transactivation have been mapped to two regions of the U3 domain of the LTR, between positions -1196 and -880 and between positions -403 and -125 (+1 represents the transcription initiation site). No significant nucleotide sequence homology between these two regions is noted; thus, the SFV-1 taf gene acts through at least two distinct sequence elements in the LTR. The target contained between positions -403 and -125 acts independently of orientation, in different cell types and species, and in the context of a heterologous promoter. Thus, the target element between positions -403 and -125 has properties of a transcriptional enhancer. The observation that two distinct elements in the SFV-1 LTR are targets for transcriptional transactivation is novel with respect to observations for other retroviral systems. The R-U5 region of the SFV-1 LTR down-regulates transactivation by severalfold. Computer analysis of the R-U5 region revealed a secondary structure with a free-energy level of -74 kcal (ca. -310,000 J); this structural feature may account for the inhibitory effect on gene expression directed by the LTR. Taf of SFV-1 had no effect on gene expression directed by the LTR of the related human foamy virus, whereas Taf transactivates gene expression directed by the LTRs of the human and simian immunodeficiency viruses. Comparative functional analysis of Taf on homologous and heterologous LTRs may facilitate elucidation of the mechanism of transactivation of foamy viruses.