Bel1 Transactivator Research Articles

Identification and Functional Characterization of an Intragenic DNA Binding Site for the Spumaretroviral trans-Activator in the Human p57Kip2 Gene

Expression of the human cyclin-dependent protein kinase inhibitor p57(Kip2) gene was previously shown to be specifically and strongly activated by the retroviral trans-activator Bel1 of human foamy virus by means of expression profiling, Northern, and Western blot analysis. Here we report that Bel1-mediated trans-activation was conferred by a Bel1 response element (BRE) located in the second exon of p57(Kip2). The intragenic Kip2-BRE was capable of trans-activating the luciferase reporter gene upon cotransfection with Bel1. In electrophoretic mobility shift assays using 293T nuclear extracts or a purified glutathione S-transferase (GST) small middle dotBel1 fusion protein, we identified the 55-nucleotide-long Kip2-BRE site that mainly consists of three direct repeats of 14-mers partially homologous to a functionally active BRE in the viral internal promoter. The specificity of the transactivator-DNA binding was shown by using mutated and shortened Kip2-BRE oligodeoxynucleotides in competition experiments with the authentic viral internal promoter and by Bel1-specific antibody that led to a supershift of the nuclear protein small middle dotKip2-BRE and GST small middle dotBel1 small middle dotKip2-BRE complex. The data indicate that Bel1 can directly bind to BRE sites. The cellular Kip2-BRE can be used to predict those human genes that are directly or indirectly activated by the Bel1 trans-activator.

Induction of cellular genes is mediated by the Bel1 transactivator in foamy virus-infected human cells.

To gain insight into human foamy virus (HFV; also called spumaretrovirus)-induced alterations of cellular genes, the expression profiles of defined genes in HFV-infected primary human cells were analyzed by cDNA array assays. Several distinct cellular genes activated by HFV infection were identified; the identities of the cellular genes were confirmed by RNA blot analyses. Compared with mock-infected controls, the concentrations of cellular Kip2, Egr-1, COUP-TF1, insulin-like growth factor II (IGF-II), and EphB3 mRNAs were significantly increased in HFV-infected cells and showed a gene-specific and time-dependent induction. Immunoblot analyses with antibodies against some of the cellular gene products revealed increased levels of the corresponding proteins. To investigate mechanisms of HFV-induced alterations in cellular gene expression, the capacity of known HFV genes to increase expression of defined cellular genes was analyzed by transient expression experiments. Plasmids that encode the HFV Bel1 transcriptional transactivator were necessary and sufficient to strongly increase expression of p57Kip2, IGF-II, and EphB3 genes in 293T cells. Potential mechanisms and consequences of activation of cellular genes during HFV infection and Bel1 transactivation of the Kip2 gene are discussed.

Induction of Cellular Genes Is Mediated by the Bel1 Transactivator in Foamy Virus-Infected Human Cells

Induction of Cellular Genes Is Mediated by the Bel1 Transactivator in Foamy Virus-Infected Human Cells

Helper-free foamy virus vectors.

Retroviral vectors based on human foamy virus (HFV) have been developed and show promise as gene therapy vehicles. Here we describe a method for the production of HFV vector stocks free of detectable helper virus. The helper and vector plasmid constructs used both lack the HFV bel genes, so recombination between these constructs cannot create a wild-type virus. A fusion promoter that combines portions of the cytomegalovirus (CMV) immediate-early and HFV long terminal repeat (LTR) promoters was used to drive expression of both the helper and vector constructs. The CMV-LTR fusion promoter allows for HFV vector production in the absence of the Bel-1 trans-activator protein, which would otherwise be necessary for efficient transcription from the HFV LTR. Vector stocks containing either neomycin phosphotransferase or alkaline phosphatase reporter genes were produced by transient transfection at titers greater than 10(5) transducing units/ml. G418-resistant BHK-21 cells obtained by transduction with neo vectors contained randomly integrated HFV vector proviruses without detectable deletions or rearrangements. The vector stocks generated were free of replication-competent retrovirus (RCR), as determined by assays for LTR trans-activation and a marker rescue assay developed here for the detection of Bel-independent RCR.

Gene Transfer Using Replication-Defective Human Foamy Virus Vectors

Replication-defective vectors based on an infectious molecular clone of human foamy virus (HFV) were constructed by deletion and replacement of the accessory genes with expression cassettes for puromycin-resistance and β-glucouronidase. Cell lines which produced in excess of 105helper virus-free transducing units/ml were generated bytrans-complementation of the replication defect using a BHK-21-derived cell line expressing the Bel-1 transactivator. Vectors based on the HFV genome may provide useful alternatives to existing retroviral vectors.

Human Foamy Virus DNA Forms and Expression in Persistently Infected Dami Megakaryocytic Cells

We have characterized human foamy virus (HFV) proviral DNA and determined HFV expression in a persistent infection model, the Dami megakaryocytic cell line. Molecular studies were performed on parental persistently infected cells (Dami-P), as well as on derived clones (Dami-Cl). We report that in these nonlytic and non-HFV producer cells, viral DNA was found to be integrated into the cellular genome and that the few free proviral forms detected in Dami-P cells were deleted in their 5' LTR. Our molecular analysis indicates the presence of undeleted 5' LTR forms in the integrated provirus within a proviral population mainly composed of deleted forms. In addition, the deletion in the bel1 trans-activator gene, previously described by Saïb et al., was found to be highly predominant. However, in 5-iodo-2'-deoxyuridine treated Dami-Cl cultures, virus production occurred, providing evidence for the presence of complete viral genome. Analysis of HFV expression in Dami-Cl cells, by Northern blot and immunoprecipitation, shows that the most striking difference between cytolytic and persistent HFV infection was the lack of expression of structural viral proteins, in contrast with Bet protein expression, which is maintained. Our data suggest that the Bet protein could be involved in the maintenance of viral persistency and that the persistently infected Dami system provides a suitable model for clarifying its function.

Human foamy virus Bel1 transactivator contains a bipartite nuclear localization determinant which is sensitive to protein context and triple multimerization domains.

The Bel1 protein of human foamy virus is a 300-amino-acid nuclear regulatory protein which transactivates the gene expression directed by the homologous long terminal repeat and the human immunodeficiency virus type 1 long terminal repeat. While previous reports suggested that the single basic domain of Bel1 from residues 211 to 222 and/or 209 to 226 is necessary and sufficient for efficient nuclear localization (L. K. Venkatesh, C. Yang, P. A. Theodorakis, and G. Chinnandurai, J. Virol. 67:161-169, 1993; F. He, J. D. Sun, E. D. Garrett, and B. R. Cullen, J. Virol. 67:1896-1904, 1993), our recent data showed that another basic domain, from amino acid residues 199 to 200, is also required for nuclear localization of Bel1 (C. W. Lee, C. Jun, K. J. Lee, and Y. C. Sung, J. Virol. 68:2708-2719, 1994). To clarify this discrepancy, we constructed various bel1-lacZ chimeric constructs and several linker insertion mutants and determined their subcellular localization. When the region of Bel1 containing basic domains was placed at an internal site of the lacZ gene, the nuclear localization signal (NLS) of Bel1 consisted of two discontinuous basic regions separated by an intervening sequence. Moreover, insertion of specific amino acids between two basic regions disrupted the activity of the Bel1 NLS. On the other hand, Bel1 residues 199 and 200 were not required to direct the Bel1-beta-galactosidase chimeric protein to the nucleus when the Bel1 NLS was appended to the amino terminus of beta-galactosidase. These results indicate that the function of the Bel1 NLS is sensitive to the protein context within which the sequence is present. In addition, we demonstrated that the Bel1 protein forms a multimeric complex in the nuclei of mammalian cells by using a sensitive in vivo protein-protein interaction assay. Mutational analyses revealed that the regions which mediate multimer formation map to three domains of Bel1, i.e., residues 1 to 31, 42 to 82, and 82 to 111. Furthermore, our results show that the region of Bel1 from residues 202 to 226 prevents Bel1 from forming a multimeric complex.

The Bel1 protein of human foamy virus contains one positive and two negative control regions which regulate a distinct activation domain of 30 amino acids.

The Bel1 transactivator is essential for the replication of human foamy virus (HFV). To define the functional domains of HFV Bel1, we generated random missense mutations throughout the entire coding sequence of Bel1. Functional analyses of 24 missense mutations have revealed the presence of at least two functional domains in Bel1. One domain corresponds to a basic amino acid-rich motif which acts as a bipartite nuclear targeting sequence. A second, central domain corresponds to a presumed effector region which, when mutated, leads to dominant-negative mutants and/or lacks transactivating ability. In addition, deletion analyses and domain-swapping experiments further showed that Bel1 protein contains a strong carboxy-terminal activation domain. The activating region is also capable of functioning as a transcription-activating domain in yeast cells, although it does not bear any significant sequence homology to the well-characterized acidic activation domain which is known to function only in yeast and mammalian cells. We also demonstrated that the regions of Bel1 from residues 1 to 76 and from residues 153 to 225 repressed transcriptional activation exerted by the Bel1 activation domain. In contrast, the region from residues 82 to 150 appears to overcome an inhibitory effect. These results indicate that Bel1 contains one positive and two negative regulatory domains that modulate a distinct activation domain of Bel1. These regulatory domains of Bel1 cannot affect the function of the VP16 activation domain, suggesting that these domains specifically regulate the activation domain of Bel1. Furthermore, in vivo competition experiments showed that the positive regulatory domain acts in trans. Thus, our results demonstrate that Bel1-mediated transactivation appears to undergo a complex regulatory pathway which provides a novel mode of regulation for a transcriptional activation domain.

A defective human foamy provirus generated by pregenome splicing.

Foamy viruses are a group of retroviruses of complex structure which were thought to be non-pathogenic. The recent demonstration of neurological diseases in mice transgenic for human foamy virus (HFV) and the high prevalence of HFV sequences in Graves' disease question this idea. By PCR, we have detected HFV sequences with a non-random deletion in the bel1 transactivator gene in other autoimmune conditions. Sequence analysis revealed that this deleted area corresponds to the excision of a known intron in bet, one of HFV's regulatory genes. The same phenomenon was observed in both acute and chronic infections, in vitro or in vivo, although the deleted forms were distinctly more abundant in chronic states. The viral DNA containing the bel1 deletion is apparently part of an otherwise complete genome, strongly suggesting that this provirus derives from the reverse transcription of a spliced pregenomic RNA. Bel1-spliced provirus was shown to be defective when transfected into permissive cells. However, co-expression with the Bel1 transactivator led to functional trans-complementation and formation of viral particles. Splicing of the genome may be an important factor in HFV biology: genomes with the deletion may either interfere with wild-type virus expression or alter host cell functions through background expression of viral regulatory proteins.

Expression of the human foamy virus bel-1 transactivator in insect cells

The human foamy virus (HFV) bel-1 transactivator protein was expressed in insect cells by a recombinant baculovirus. For the generation of the recombinant baculovirus, Acbel-1, the bel-1 gene of an HFV mutant was used, that bears truncations in the bel-1 overlapping bel-2 open reading frame. Acbel-1 infected Sf9 cells produced high amounts of recombinant protein of the same electrophoretic mobility (36 kD) as bel-1 expressed in mammalian cells. The baculovirus expressed bel-1 protein was readily identified by a polyclonal rabbit serum directed against bel-1 in immunoblot assay. As in mammalian cells, bel-1 was predominantly localized to the nucleus of Acbel-1 infected insect cells. The baculovirus expressed bel-1 protein will be of use to determine the action of this novel viral transactivator more precisely.

BEL-1 Transactivator Responsive Sequences in the Long Terminal Repeat of Human Foamy Virus

Cis-regulatory elements in the long terminal repeat (LTR) of human foamy virus (HFV) were identified by using LTR mutants to transiently express the chloramphenicol acetyl-transferase gene after co-transfection with an expression plasmid for the virus bel-1 (transactivator) gene. The R-U5 region and an element in the 5′ U3 region were found to negatively influence HFV gene expression. The complete BEL-1 responsive region was mapped to extend from nucleotide position -471 to position -93 relative to the start of transcription. Within this region, three elements were identified that in the homologous or a heterologous (SV40) promoter context can, independently and irrespective of their orientation, act as targets for BEL-1. These elements are located between nucleotide positions -413/-378, -361/-291, and 124/-93. The target elements do not share obvious sequence homologies. The mechanism of HFV transactivation appears to be novel among the complex retroviruses and is likely to involve, as yet, undiscovered cellular DNA binding factors.

Characterization of the transcriptional trans activator of human foamy retrovirus

The human foamy viruses, or spumaviruses, a distinct subfamily of complex human retroviruses, remain poorly understood both in terms of their pathogenic potential and in terms of the regulatory mechanisms that govern their replication. Here, we demonstrate that the human spumaretrovirus shares with other complex human retroviruses the property of encoding a transcriptional trans activator of the homologous viral long terminal repeat. This regulatory protein is encoded by the viral Bel-1 open reading frame and is localized to the nucleus of expressing cells. The Bel-1 trans activator is shown to function effectively in cell lines derived from human, simian, murine, and avian sources. The viral target sequence for Bel-1 has been mapped 5' to the start of viral transcription and is therefore likely to be recognized as a DNA sequence. Our results further suggest that the mechanism of action of the Bel-1 protein may be distinct from those reported for the transcriptional trans activators encoded by members of the other human retroviral subfamilies.