Tas is the transcriptional trans-activator of foamy retroviruses. It activates gene expression from both the viral long terminal repeat (LTR) promoter and an internal promoter that drives the expression of Tas itself. Understanding the mechanism of transcriptional activation and temporal control of retroviral gene expression by Tas is the key to understand the mechanism of foamy viruses replication. In this study I intend to investigate the following aspects of transcriptional activation by Tas:

1. Accurate mapping of binding sites for Tas in foamy virus genomes, particularly in the LTR and in the internal promoter.
2. DNA sequence specificity of human and simian foamy virus transcriptional activator Tas.
3. Identification of the cellular cofactor for Tas transcriptional activation.

B. Introduction

Foamy viruses, including human foamy virus (HFV) and the simian foamy viruses (SFVs), are unusual among retroviruses in that they contain two distinct promoter elements (Rethwilm, 1995, see the attached figure). As in all retroviruses, a promoter element located in the viral long terminal repeat (LTR) directs the synthesis of a genome-length transcript that gives rise to the viral structural proteins Gag, Pol, and Env. The second foamy virus promoter, termed the internal promoter (IP), is located at the 3' end of the envelope gene (Campbell et al., 1996; Lochelt et al., 1993, Mergia, 1994). The IP is primarily responsible for the expression of two open reading frames located between env and the 3' LTR, one of which encodes a transcriptional transactivator termed Tas (also known as Bel-1 in HFV). Importantly, both of these promoter elements are strongly activated upon expression of the cognate Tas/Bel-1 regulatory protein (Rethwilm, 1995). The internal promoter element is thought to activate expression of these auxiliary proteins early in viral life cycle and is clearly critical for effective virus replication in culture. (Lochelt et al., 1994)
Analysis of the mechanism of action of Bel-1 in HFV and of Tas in SFV-1 has demonstrated that these virally encoded transcription factors are DNA binding proteins, a property which distinguishes Tas/Bel-1 from the functionally equivalent human immunodeficiency virus type 1 Tat and human T-cell leukemia virus type 1 Tax proteins (He et al., 1996; Zou et a;. 1996; Cullen, 1992). Domain organization analysis of the HFV Bel-1 protein has identified an acidic transcription activation domain located within the carboxy-terminal ~40 amino acids (aa) of this 300-aa viral regulatory protein and has also defined a DNA targeting domain occupying ~120 aa in the core of Bel-1 (Blair et al., 1994; Garrett et al., 1993; He et al., 1993,Venkatesh et al., 1993). While the domain organization of the related SFV type 1 (SFV-1) Tas protein appears to be very similar to that observed in Bel-1 (Mergia et al., 1993), Tas and Bel-1 both fail to activate transcription directed by promoters containing functional DNA target sites specific for the other protein (Garrett et al., 1993).
Although several DNA target sites for Bel-1 have been mutationally defined, these have little evident sequence homology (Erlwein et al., 1993; Keller et al., 1992, Keller et al., 1991; Lee et al., 1993, Lochelt et al., 1993, Venkatesh et al., 1991). Nevertheless, it has been demonstrated that Bel-1 can directly and specifically bind to the major, cap-proximal Bel-1 response element (BRE) located in the viral LTR promoter and also to sequences present in the HFV internal promoter element (He et al., 1996). Similarly, specific Tas binding to the SFV-1 internal promoter, and to a proposed Tas-dependent enhancer element located in the SFV-1 gag gene, has also been reported (Campbell et al., 1996, Zou et al., 1996). Surprisingly, for both Tas and Bel-1, DNA sequences that are sufficient for DNA binding in vitro were found to be necessary but not sufficient for optimal Tas or Bel-1 function in vivo (He et al., 1996, Zou, et al., 1996). This observation raises the possibility that other, cellular DNA binding proteins may play a critical role in mediating Bel 1 and Tas function in vivo. There has been speculations that Tas/Bel-1 plays an important role in the temporal control of foamy virus early and late gene expression (Lochelt et al., 1993). However, the mechanism by which Tas exerts this control has remained unclear.

C. Experimental Design and Methods

Aim 1. Although target sequences for the Bel-1 protein have been loosely defined both based on functional criteria and by in vitro DNA binding (Erlwein et al., 1993; Keller et al., 1992, Keller et al., 1991; Lee et al., 1993, Lochelt et al., 1993, Venlatesh et al., 1991), the actual DNA target specificity of Bel-1 remains far from clear. I had demonstrated that Bel-1 binds to the BRE present in the internal promoter with significantly higher affinity than to the major LTR promoter BRE in both in vivo and in vitro assay systems. This difference in affinity may, at least in part, explain the observation that the HFV IP is activated significantly earlier than the LTR promoter during the foamy virus life cycle (Lochelt et al., 1994). This difference in affinity, if functionally important, could also explain the limited homology between the HFV LTR and IP Bel-1 binding site. Using modification interference, I had identified individual bases within both the internal and LTR promoters that are critical for Bel-1 DNA binding in vitro. Subsequently a minimal, 25-bp DNA sequence derived from Bel-1 bind site at IP was shown to be sufficient to mediate Bel-1 binding both in vitro and in vivo. This work has been published in the Journal of Virology (Kang et al., 1998a).
There are at least three BREs in the HFV LTR and preliminary data demonstrate that there are at least two more Bel-1 binding sites in the LTR in addition to the site in the cap-proximal BRE. I will combine in vitro footprinting assay and in vivo DNA binding assay in yeast and mammalian cells to precisely map these additional Bel-1 binding sites in the LTR. All these Bel-1 binding site will then be individually and combinatorially mutated or changed to higher affinity binding sequence for Bel-1, and the effects of such changes on the response of LTR promoter to Bel-1 will be examined. Results obtained from these experiments will facilitate understanding of the temporal regulation of viral gene expression by Bel-1.

Aim 2. There is very little homology between the HFV LTR and IP Bel-1 binding sites (He et al., 1996). Similarly, the SFV-1 IP and gag gene Tas binding sites also display only limited homology to each other and to Tas-responsive DNA sequences present in the SFV-1 LTR (Campbell et al., 1996; Mergia et al., 1992; Zou et al., 1996). These findings, combined with the observation that Bel-1 and Tas are specific only for target sequences present in their cognate viral genome (Campbell et al., 1994; Keller et al., 1991), have meant that no consensus DNA binding site for either Bel-1 or Tas has been defined. The sequence specificity of Tas/Bel-1 therefore is poorly understood.
The initial attempt to use PCR-based in vitro randomization/selection (Wright et al., 1991) to derive a consensus binding sequence for Tas/Bel-1 was not successful. Therefore I designed a novel in vivo randomization/selection procedure to define a consensus DNA binding site for the SFV-1 Tas protein. A minimal 25 bp SFV-1 IP that binds strongly to Tas was divided into five 5-bp segments and individually randomized and cloned into the promoter region of the yeast lacZ reporter. Tas responsive sequences were selected by virtue of their ability to drive the LacZ expression in response to Tas. Compilation of favored nucleotide sequence at each position of the minimal SFV IP results in a consensus Tas binding site. I demonstrate that the most-favored DNA sequence binds Tas with a higher affinity than does the wild-type IP Tas binding site both in vivo and in vitro. In addition, I have constructed a highly variant Tas DNA binding site that, while identical to the minimal IP Tas binding site at only 8 of 25 positions, can nevertheless binds to Tas effectively both in vivo and in vitro. The flexibility of the DNA binding specificity of Tas appears to explain the marked variability in the sequence of Tas responsive sequences previously identified in the SFV-1 genome. This work has also been published in the Journal of Virology (Kang et al., 1998b).
I plan to apply the same technique to derive a consensus binding sequence for HFV Bel-1. Comparison of the consensus binding sequences of SFV-1 Tas and HFV Bel-1 will answer the question of why these two viral proteins can not cross-transactivate each other's cognate target.

Aim 3. Several lines of evidence suggests that cellular factors play a role in the transcriptional regulation by Bel-1/Tas. Mutation of sequences outside the Bel-1 binding site in the cap proximal BRE in HFV LTR severely reduce the transactivation response to Bel-1(He et al., 1996); the distal element in SFV-1 IP is able to recruit Tas to the promoter independent of the actual Tas binding site in the SFV-1 IP (Zou et al., 1996). Presumably these functionally important non-Bel 1/Tas binding sites are recognized by cellular transcription factors which might also interact with Tas/Bel-1 to regulate the viral promoter. The yeast one-hybrid system will be utilized to search for cellular proteins that interact with these DNA sequences. Once candidate proteins are identified further in vitro and in vivo protein-protein and protein-DNA interaction assays will be applied to address the functional importance of these cellular cofactor in the regulation of foamy virus gene expression.

Reference

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Blair, W. S., H. Bogerd, and B. R. Cullen. 1994. Genetic analysis indicates that the human foamy virus Bel-1 protein contains a transcription activation domain of the acidic class. J. Virol. 68:3803-3808.
Campbell, M., C. Eng, and P. A. Luciw. 1996. The simian foamy virus type 1 transcriptional transactivator (Tas) binds and activates an enhancer element in the gag gene. J. Virol. 70:6847 6855.
Campbell, M., L. Renshaw-Gegg, R. Renne, and P. A. Luciw. 1994. Characterization of the internal promoter of simian foamy viruses. J. Virol. 68:4811-4820.
Cullen, B. R. 1992. Mechanism of action of regulatory proteins encoded by complex retroviruses. Microbiol. Rev. 56:375-394.
Erlwein, O., and A. Rethwilm. 1993. Bel-1 transactivator responsive sequences in the long terminal repeat of human foamy virus. Virology 196:256-268.
Flügel, R. M. 1991. Spumaviruses: a group of complex retroviruses. J. Acquired Immune Defic. Syndr. 4:739-750.
Garrett, E. D., F. He, H. P. Bogerd, and B. R. Cullen. 1993. Transcriptional trans activators of human and simian foamy viruses contain a small, highly conserved activation domain. J. Virol. 67:6824-6827.
He, F., W. S. Blair, J. Fukushima, and B. R. Cullen. 1996. The human foamy virus Bel-1 transcription factor is a sequence-specific DNA binding protein. J. Virol. 70:3902-3908.
He, F., J. D. Sun, E. D. Garrett, and B. R. Cullen. 1993. Functional organization of the Bel-1 trans activator of human foamy virus. J.Virol. 67:1896-1904.
Kang, Y., W. S. Blair, and B. R. Cullen. 1998a. Identification and functional characterization of a high-affinity Bel-1 DNA binding site located in the human foamy virus internal promoter. J. Virol. 72:504-511.
Kang, Y. and B. R. Cullen. 1998b. Derivation and Functional Characterization of the Consensus DNA Binding sequence for the Tas Transcriptional Activator of Simian Foamy Virus Type 1. J. Virol. 72: 5502-5509.
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Part II: Cellular cofactor for CTE-dependent nuclear export of type D retroviral RNA.

A. Specific Aims:

The constitutive transport element (CTE) of the type D retroviruses promotes nuclear export of unspliced viral RNAs by recruiting host factor(s) required for export of cellular messenger RNAs. Recently, the human Tap protein was shown to specifically bind to the CTE in vitro, and stimulate CTE-dependent mRNA export in Xenopus oocytes. Mex67p, the yeast homolog of Tap, is also involved in the nuclear export of yeast cellular mRNAs. Here, I propose to study the following aspects of Tap:

1. Functional dissection of human Tap protein.
2. Nuclear import/export receptor and functional partners of Tap.
3. The cellular role of Tap.

B. Introduction:

Most pre-mRNAs are completely spliced and polyadenylated prior to their export from the nucleus to the cytoplasm (reviewed by Nakielny and Dreyfuss, 1997) while unspliced pre mRNAs are retained in the nucleus. Nevertheless, replication of retroviruses requires export of unspliced viral RNAs, both as templates for the synthesis of structural proteins and as genomic RNA to be packaged in progeny virions. All retroviruses have therefore evolved some mechanism to bypass the requirement for splicing prior to export. Many complex retroviruses, like the human immunodeficiency virus type 1 (HIV-1), encode trans-acting factors that facilitate export of unspliced and incompletely spliced RNAs. The Rev protein of HIV-1 binds an intronic sequence in HIV-1 RNA, the Rev response element (RRE), and promotes export of the intron containing HIV-1 RNAs (Malim et al., 1989a, b). This export requires a leucine-rich nuclear export signal (NES) in the Rev protein (Meyer and Malim, 1994, Wen et al., 1995). Cellular nuclear export receptor CRM1 then recognizes the leucine-rich NES and promotes export of the RNP complex. As simpler retroviruses do not encode trans-acting regulatory proteins, export of their unspliced RNA must therefore rely on the interaction of cis-acting RNA elements with cellular factors.. A cis-acting RNA element located near the 3' end of one such retrovirus, Mason Pfizer monkey virus (MPMV), has been shown to be important for export of the intron containing viral RNA (Bray et al., 1994; Ernst et al., 1997a). Both the sequence and function of this element, termed the constitutive transport element (CTE), are conserved in related type D retroviruses (Zolotukhin et al., 1994; Tabernero et al., 1996; Ernst et al., 1997b). Because the CTE can substitute for RRE/Rev in promoting export of intron-containing RNAs (Bray et al., 1994; Tabernero et al., 1996; Ernst et al., 1997a), it is likely that the CTE functions by interacting with cellular export factors. However, although Rev uses the same export pathway as snRNAs and 5S ribosomal RNA, the CTE export pathway shares cellular factors that are essential for cellular mRNA export. (Saavedra et al., 1997; Pasquinelli et al., 1997).
The CTE folds into an extended RNA stem-lop structure (see the attached figure), comprising two conserved internal loops, A and B, which are believed to represent the interaction sites for cellular CTE-binding proteins. Their 180 degree symmetric arrangement indicates duplication of the binding site. Recently, Gruter, et al, identified Tap (Yoon et al, 1997), the human homolog of yeast mRNA export factor Mex67p (Segref et al, 1997), as the CTE-binding protein. Tap binds specifically to wild-type but not mutant CTE RNA in vitro and stimulate CTE-dependent RNA export in Xenopus oocytes. In addition, Tap overcomes the mRNA export block caused by the presence of saturating amounts of CTE RNA. Therefore Tap is a strong candidate as the bona fide cellular cofactor for the CTE-dependent RNA export. However, the binding to Tap to CTE has not yet been demonstrated in vivo, and the domain organization and mechanism of Tap function are still poorly understood. Furthermore, the cellular role of Tap remains unknown. Several sets of experiments discussed below are designed to answer these questions.

C. Experimental Design and Methods

Aim 1. Yeast three-hybrid system and a Tat-based mammalian RNA binding assay system were applied to analyze CTE binding to Tap in vivo. Microinjection experiments using GST fusion proteins of full length and various truncated forms of Tap were performed to map the NLS and NES in Tap. Further in vitro and in vivo experiments will address the functional importance of Tap in CTE-dependent nuclear export of mRNA.
a. Tap binds specifically to CTE in vivo, and aa 20-312 is the minimal RNA binding domain. In the yeast-three hybrid assay, binding of Tap-VP16 fusion protein to MS2-CTE chimeric RNA recruits the VP16 activation domain to the promoter of a LacZ reporter gene and subsequently activates its transcription. Tap specifically binds to wild-type CTE, half CTE and the terminal loop mutant of CTE. Mutation in either internal loop A or B reduces the affinity of CTE for Tap, while a double mutation in loop A and B completely knocked out Tap binding. Deletion mutant 1-312 surprisingly has a much higher affinity for CTE than the full length protein. Tap (20-312) still processes the ability to bind specifically to CTE while mutants 42 312 and 1-298 do not. Therefore the RNA binding domain is mapped to aa 20-312 in Tap.
A similar study was carried out in the mammalian RNA binding assay system. Tat-Tap fusion proteins recognize the CTE RNA inserted in the place of TAR in HIV-LTR promoter, which drives the expression of the CAT reporter gene. The binding specificity data obtained from the mammalian assay system is consistent with the data collected in the yeast three-hybrid system. Eight alanine scanning mutants made in the region of 42-312 all knocked out the ability of Tat Tap(1-312) to bind to CTE, suggesting that this region indeed is critical for RNA binding. Importantly in contrast to published results that both loop A and B must be intact for CTE function in mammalian cells (Tabernero et al., 1996), Tap clearly is able to binds to either half of the CTE in mammalian cells in our assay system. Therefore, just as the case with Rev, multimerization of Tap on RNA may be necessary for its function in export even though it's not indispensable for RNA binding.
b. Mapping of NLS and NES in Tap.
Hela cell microinjection experiments indicate that there are two nuclear localization signals in Tap, one at each ends of the protein. However, neither of these has any obvious similarity to currently known nuclear localization signals. The N-terminal NLS is composed of the first 42 amino acids of Tap. Mutations in aa 9-11, aa 20-22 and aa 28-30 knocked out this NLS. The C terminal NLS of Tap (aa 480-559) also functions as a potent nuclear export signal. Surprisingly full length Tap does not seem to have NES activity in our assay. One possible explanation is binding of CTE to Tap changes its conformation and exposes the NES, which then enables the loaded Tap to export out of the nucleus. Further experiments such as co-microinjection of Tap and CTE RNA are needed to address this possibility.
c. In vivo randomization/selection of functional CTE variants.
To functionally correlate the binding of Tap to CTE and the enhancement of CTE-dependent RNA export, we design an in vivo randomization/selection strategy to select CTE RNA sequence variants that can still be recognized by Tap, and subsequently tested the ability of these variants to support CTE-dependent nuclear export of intron-containing mRNA. Seven nucleotides at internal loop B of a half CTE construct in the yeast three-hybrid system were randomized and selected for Tap binding by in situ b-gal assay of the transformed yeast colonies. I was able to select the wild-type CTE as well as four sequence variants from the library. All these variants have lower affinity for Tap compared to wild-type CTE sequences but nevertheless are able to at least partially substitute for wild-type CTE in the nuclear export of mRNA in the intron-CAT reporter assay. I am currently making Rev- RRE- HIV-1 construct and will test the ability of wild-type and the variant CTE sequences to support the expression of HIV-1 Gag proteins.
d. Functional analysis of Tap mediated export in mammalian cells. Even though enhancement of CTE-dependent RNA export by Tap has been demonstrated in Xenopus oocytes (Gruter et al., 1998), a similar experiment has not been done in mammalian cells. Initially I tried to enhance CTE -dependent nuclear export of intron containing mRNA by overexpressing Tap. However, the experiment did not succeed, possibly because Tap protein is already expressed at a saturating level in the cell. Overexpression of CTE RNA also failed to drive the cellular Tap to a limiting level. In addition, none of the deletion mutants of Tap were able to function as a strong dominant negative suppressor for CTE function even though Tap (20-312), which retained the RNA binding ability but lost all NLS and NES signals, seems to be able to weakly suppress CTE function when it's targeted into nucleus by fusing to Tat. In an alternative approach to directly demonstrate the involvement of Tap in the nuclear export of mRNA, I plan to co-tranfect sense or antisense Tap expression plasmid with cell surface marker (e.g. CD4, CD19) expression plasmids into human cells. Forty-eight hours after transfection, cells will be sorted for high level of surface marker expression (and thus high level of Tap protein or antisense Tap RNA expression). This population of cells will then be transfected with reporter construct for CTE function and the effect of overexpression of Tap protein or antisense Tap RNA will be observed. In the end, I intend to establish a dependable functional assay for Tap in CTE-dependent RNA export in mammalian cells.
Our preliminary data suggests that yeast Mex67p does not bind to CTE by yeast three hybrid assay. As many factors in import/export pathway are highly conserved between human and yeast, we may have a reasonably good chance that Tap will retain its ability to function properly in yeast. Therefore I will try to reconstitute an CTE-dependent RNA export system in yeast by expressing Tap in it. In a separate experiment, I will use randomization/selection to identify CTE variants that are able to be recognized by Mex67p and subsequently examine if Mex67p also can facilitate export of such an RNA target in yeast or mammalian cells.
e. Fine mapping of Tap-interaction site on CTE and derivation of a consensus Tap binding site.
I will use chemical interference and footprinting techniques to precisely map the Tap interaction sites on the CTE. Published data on the mutagenesis analysis of CTE suggest that the primary sequences of the internal loops as well as the portions of the stem proximal to the internal loops are important for CTE function. Mapping of the RNA-protein interaction sites in these functionally important sequences may provide another strong evidence that Tap is indeed the genuine cellular cofactor for CTE function. In vitro or in vivo randomization/selection technique will be applied to thoroughly study the sequence flexibility of a functional CTE. These results will enable us to derive a consensus Tap binding sequence, which will be useful in searching the DNA sequence database for cellular targets for Tap.

Aim 2. The Tap-CTE export pathway is still largely a black box so far. Identification of other cellular components of this pathway, especially the import/export receptors for Tap will be a major subject of future efforts.
a. Identification of nuclear import/export receptor of Tap. Initially I will test if Tap interacts with currently known nuclear import/export receptors by either two-hybrid assay or in vitro binding and uptake assays. Peptide competition experiments to address the specificity of such interaction will then be carried out if Tap does use any of these known import/export receptors. If Tap does not interact with any of the know import/export receptors I will use yeast two hybrid system in vivo and affinity column purification technique in vitro to try to identify novel import/export receptors for Tap. Further biochemical and genetic characterization will be applied to these receptors.
b. Identification of Tap interacting protein(s).
Even though Tap and Mex67p both are suggested to involve in the nuclear export of mRNA, no evidence of a direct interaction of these proteins with cellular mRNA has been presented. A RNA binding protein, Mip6, was identified through yeast two hybrid system that specifically interacts with the C-terminal part of yeast Mex67p (Segref et al., 1997). Even though Tap can directly and specifically binds to CTE, it's still possible that Tap might not directly interact with cellular mRNA. Instead, it might enhance the export of cellular mRNA by interacting with a cellular mRNA binding protein. In addition, the import/export pathways of Tap might be drastically different from known pathways and require novel cellular cofactors. Yeast two hybrid and in vivo affinity column purification techniques will be applied to identify cellular Tap interacting proteins.

Aim 3. Mex67, the yeast homolog of Tap, is involved in nuclear export of cellular mRNAs (Segref et al., 1997). The role of Tap in cellular mRNA export is unclear. I plan to test if Tap binds to cellular mRNA in vivo and subsequently try to identify specific RNA targets that is recognized by Tap.
a. UV-crossing linking of Tap to cellular RNA. As the initial step to investigate the cellular role of Tap, I will use UV cross-linking technique to test if Tap is in close contact with any cellular mRNA. Positive result will justify further effort to identify these RNA targets.
b. Identification of cellular RNA targets of Tap.
Search of DNA sequence database using CTE as a probe only identify a RNA sequence within an intracisternal-A particle element in mouse genome that has high homology to CTE sequence and also functions as a CTE (Tabernero et al., 1997). It's still possible though that there are other cellular RNA targets that have low sequence homology to CTE but nevertheless can be recognized by Tap and play a role in cellular RNA export. Definition of a consensus Tap binding site in specific aim 1 therefore will be useful for a more complete search of such sequences. An alternative strategy is to directly identify cellular RNA target for Tap by virtue of their ability to specifically bind to Tap. The yeast three hybrid system will be used to identify such cellular RNA target in vivo. Binding of cellular RNA to immobilized Tap and amplification of these RNA by differential display RT-PCR is an alternative in vitro approach. After such RNA targets are identified further experiment will be applied to address the functional role of such RNA elements.

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