Theresa A. Gurney
The Vaccine Revolution
Dr. Robert Siegel
20 March 1997
A PUZZLING PARADOX: HOW DOES TAT PRODUCTION INITIATE?
An Exploration of Current Research on
Tat/TAR Dependent Transcriptional Regulation
and its Implications in Developing a Model for Tat Origination
Much of the current research on the replication of HIV-1 focuses on the interactions between the virally encoded regulatory protein, the transactivator protein (Tat), and its RNA target, the trans-acting responsive element (TAR). It has been generally found that the presence of Tat greatly increases transcriptional initiation and elongation from the HIV-1 long terminal repeat (LTR) region. The activation of transcription conferred by Tat is dependent upon Tat's binding to TAR in most cell types. However, admist the diligent search for the intricacies of the Tat/TAR interaction, present research does not question the puzzling paradox that this model presents: if Tat is needed for transcriptional initiation and elongation, how is Tat produced initially?
This review will examine possible models for Tat's initial production. To begin this discussion, it is important to review current literature on the role of Tat in transcriptional regulation and the mechanism of this regulation via Tat's interaction with TAR. From this background, we can then better explore the possible models for the genesis of Tat; (1) virion packaged Tat, (2) a cellular homologue capable of inducing full-length transcription, (3) basal read through, and (4) a Tat-independent second initiator site.
TAT CONFERS TRANSCRIPTIONAL INITIATION AND ELONGATION
Tat is a virally encoded protein that is produced early in the life cycle of HIV-1. It is a 86 amino-acid made from a singly-spiced viral mRNA transcript (Fig. 1). Residues 48-57 are highly basic and have been found to be important for both TAR binding and nuclear localization (Fig. 2) (Kingsman, 1996). Tat also contains a highly conserved hydrophobic core region and a cysteine-rich region.
Research has demonstrated that Tat acts as a potent trans-activator of transcription from the HIV-1 LTR. In multiple in vitro experiments, Tat has been found to dramatically increase transcription. Marciniak and colleagues showed that purified Tat increased transcription from the HIV LTR greater than ten-fold (Marciniak, 1990). In Tat-deficient cell lines constiuatively producing low-levels of virus, Tat increased viral production by greater than 30,000 fold (Feinberg, 1991). In vivo experiments have also found that Tat trans-activates transcription. Primary peripheral blood mononuclear cells were found to be transcriptionally upregulated in the presence of Tat while a specific Tat inhibitor decreased the trans-activation (Thomas, 1994).
Tat's ability to increase transcription is primarily due to its stimulation of transcriptional elongation (Feinburg, 1991, Zhou, 1995, Laspia, 1989). In the absence of Tat, transcripts have been found to end prematurely at 55-59 nucleotides downstream of the initiator (Laspia, 1989). In the presence of Tat, transcripts are able to elongate efficiently (Laspia, 1989, Feinburg, 1991, Zhou, 1995, Rittner, 1995 and Keen, 1996). In addition to elongation, Laspia and colleagues have found that Tat can also stimulate transcriptional initiation (Laspia, 1989).
However, there are some instances found in which Tat function did not appear to increase transcription. Research by Cannon and coworkers found that viral production dramatically increased with the addition of purified Tat in chronically HIV-1 infected human monocyte cell lines, U1, but no increase in viral production was found with the addition of Tat to similarly chronically infected human T-lymphocyte cell lines, ACH-2 (Cannon, 1994). Similar results were found by Duan and colleagues (Duan, 1994). However the experiments by Cannon and colleagues used a reporter construct to measure the increase of Tat activity above endogenous levels and does not directly measure Tat's effect on transcription and elongation. Consequently some other block or rate-limiting factor may exist in this system by which viral production did not increase. The direct effects of transcriptional initiation and elongation of Tat were not assayed and it is possible that endogenous levels of Tat were sufficient to activate full-length transcription.
TAR: AN RNA SPECIFIC TARGET FOR TAT FUNCTION
TAR is a stem-loop structure formed on the 5' leader end of all RNA transcripts initiating from the HIV LTR (Fig. 3). TAR has been found to be essential for Tat dependent trans-activation (Laspia, 1989 and Marciniak, 1990). Of the TAR nucleotide span from +1 to +59, the region from +19 to +42 has been found to be both necessary and sufficient for Tat activation (Jones, 1994). Mutations that disrupted either the apical loop or the stem bulge knocked out Tat trans-activation (Laspia, 1989, Marciniak, 1990, Churcher, 1995, Murphy, 1993 and Garcia, 1989). Both the RNA sequence and structure are essential for a fully functional TAR (Selby, 1989). The position of TAR also influences trans-activation; placement significantly upstream or downstream obliterates Tat-dependent transactivation (Selby, 1989). Wright and colleagues found placement of TAR greater than 353 nucleotides knocked out Tat-dependent transcriptional activation (Wright, 1996). Biochemical assays have shown that Tat and TAR interacts directly in vitro (Marciniak, 1990, Slice, 1992 and Keen, 1996).
INITIATION AND ELONGATION: THE DUAL PROMOTER THEORY
Tat's ability to confer both initiation and elongation has led to the development of a dual promoter theory. Cullen proposes that the HIV-1 promoter may actually consist of two distinct overlapping promoters (Cullen, 1993). The first promoter is thought to be responsible for generating the short, non-processive transcripts in the absence of Tat. The second promoter is thought to be the Tat-responsive promoter, capable of generating full-length, processive transcripts (Fig. 4). A proposed mechanism for this model would be that the short non-processive promoter would provide a basal level of TAR RNA structures. Upon host Tat production, Tat could then bind to the TAR elements and instigate the switch over to the second processive promoter, thereby stimulating both initiation and elongation. In support of the dual promoter hypothesis, Pendergrast and colleagues have found that in the presence of Tat, the production of short transcripts is repressed and that of full-length transcripts is stimulated (Pendergrast, 1997).
Although this theory appears consistent with the majority of research regarding Tat and TAR interactions in transcriptional regulation, it fails to address how Tat is generated initially if it is indeed needed for full-length transcription. This question is conspicuously absent from most discussions regarding TAR-dependent Tat trans-activation. And its absence presents both a gap in our understanding of Tat function, as well as an opportunity to further explore the mechanisms of HIV-1 transcription regulation. Having established a background for Tat and TAR's role in regulation of full length transcription, we can now consider possible models for the original production of Tat.
MODEL 1: VIRION PACKAGED TAT
The introduction of Tat in a virally infected host cell, could possibly originate with the virion itself. A small number of Tat proteins could be packaged in each virus and delivered to the host upon infection. Regulation of Tat activity could then occur via cytoplasmic localization, post-transcriptional modification, or interaction with cellular cofactors. Upon activation, Tat could then bind with TAR to trans-activate processive transcription. This would provide the needed transcripts for host production of Tat and further upregulate genomic transcription in a positive feedback loop.
Current research does not show that Tat is packaged in HIV-1 virions. Reviews of HIV-1 virus structure cite that Tat is not found in the virion (Levy, 1993). However the sensitivity of these assays are not noted, leaving open the possibility that a small number of Tat proteins could be packaged into each virion. To assay for the presence of Tat in virions, viral lysates could be run out on polyacrylimide gels and incubated with Tat specific antibodies. Calibration with viral titers or known amounts of viral protein could be used to quantify the amount of Tat, if found. If Tat is found to be packaged within the virion itself, research should then focus on exploring the mechanisms by which Tat activity is regulated within the host cell.
MODEL 2: FUNCTIONALLY CELLULAR HOMOLOGUE OF TAT
A second possible origin of Tat, is the presence of a cellular protein which can stimulate sufficient full-length transcription for host Tat production. In this model, a host cellular protein would initiate full-length transcription, enabling cellular Tat production, and instigating a switch to Tat-dependent trans-activation. It may seem unnecessary to have Tat-dependent trans-activation if a cellular protein is capable of conferring processive transcription. However the production of Tat could be essential for a positive feedback loop and conferring continuous full-length transcription.
Bryan Cullen has proposed NF-kB as a candidate for a cellular homologue of Tat. There is sufficient evidence for NF-kB's involvement in transcription regulation of the HIV-1 LTR. First, NF-kB consensus regions are located upstream of the TATA box in the HIV-1 LTR (Fig. 5). Second, research has found that NF-kB homodimers are capable of binding this region in vitro and increase TFII-I binding protein (Montano, 1996). Finally, Tat responsive activation has been shown to require functional NF-kB elements both in vitro and in vivo (Berkhout, 1992, Mallardo, 1996 and Alcami, 1995). However these experiments do not demonstrate NF-kB's ability to act solely as a transcriptional initiator of full-length transcripts, only that NF-kB elements are necessary for Tat-dependent trans-activation. Future research is needed to determine if NF-kB, or a currently unidentified cellular protein, is capable of producing sufficient processive transcripts for Tat-production.
MODEL 3: BASAL READ THROUGH
In the absence of Tat, it is possible that a low-level of full-length transcriptional read through occurs, allowing the production of sufficient amounts of processive transcripts to produce Tat. The activity of Tat could then be controlled through a variety of mechanisms, such as exon splicing, post-translation modifications, or the activity of required cellular transcriptional enhancers and cofactors. Upon appropriate stimulation conditions, Tat could act to initiate processive transcripts and positively upregulate full-length read through.
Low level basal read through is generally assumed to be the origin for Tat production in the host cell. However it remains to be tested if low level basal read through is sufficient to produce biologically functional levels of Tat. If low levels of basal read through are sufficient to produce Tat, it then remains to be explored how Tat activity is regulated. It is possible that the activity of Tat produced from basal read through is regulated via the availability and activity of cellular cofactors, such as Sp1, NF-kB, TFII-I, TFII-D, etc.
MODEL 4: SECOND INITIATOR SITE HYPOTHESIS
Although not discussed in the current literature, it is also possible that the overlapping promoters influence two different initiation sites. Current research focuses on transcripts originating from the +1 initiation site, however evidence exists that there may also be a second initiator site between +30-50. As transcription initiating from this region does not allow for TAR formation, this would be a Tat-independent method of trans-activation. In the absense of Tat, short non-processive transcripts are generated from +1. However upon a cellular stimulation event, transcription could be switched to the second initiator site downstream to produce full-length transcripts to encode for Tat production. After sufficient full-length transcripts were generated, Tat could stimulate TAR-dependent processive transcription thorugh a positive feedback loop.
Research by Montano found evidence of transcripts originating from +32. 293 cells containing a lac Z reporter driven by the HIV-1 LTR were stimulated with phorbal myristyrate acetate (PMA) and incubated either with or without HIV-1 (Montano, 1994). Primer extension analysis found that in the absense of HIV-1 infection, transcripts originated from +32 while in the presence of HIV-1 infection, transcripts were found to originate from the +1 initiation site.
In a second system, Montano found additional supporting evidence of a second initiator site. U1 and ACH-2 cells, chronically HIV-1 infected human monocyte and T-lymphocyte cell lines were found to originate transcription at +1 in RNAse protection assays in the absense of TNF-µ. However upon TNF-µ stimulation, transcriptional initiation was found at both +32 and +1 in U1 cells, while transcriptional initiation remained at +1 only in ACH-2 cells.
Transcription start sites downstream of the +1 have also been found in published literature. Kao and colleagues using COS7 cells transfected with an HIV-1 LTR reporter construct and a Tat expression vector showed in a RNAse protection assay and primer extension assays bands that corresponded to the +1 start site, as well as another start site 30-50 nucleotides downstream (Kao, 1987). Kashanchi and coworkers also found transcripts that could correspond to a second initiation site 30-50 nucleotides downstream of the +1 start site with in vitro transcription assays using nuclear extracts depleted of TFIID (Kashanchi, 1994). Whether these bands correspond to legitimate start sites or are artifacts of RNAse activity remains to be demonstrated.
CONCLUSIONS AND DIRECTIONS FOR FUTURE RESEARCH
In this literature review of the current work on Tat's trans-activation of the HIV-1 LTR, no concrete answers have surfaced to our question, "if Tat is needed for full-length transcription, how is Tat originally produced?" However within this body of research lies "clues" from which a variety of explanatory models have been presented. Analyzing the strengths and weaknesses of these models can direct future research efforts.
The first model of virion packaging of Tat is the most unlikely model for Tat production. If Tat did indeed come from the infecting virion, it would necessarily be in low levels since it has not been detected in previous analyses of viral components. It is therefore unlikely that a low-level of Tat would be capable of functionally in biologically significant manner, although this should still be tested as explained previously.
The second model, that of a cellular homologue which would be capable of stimulating full-length transcription, is more plausible. The requirement of cellular cofactors has been demonstrated in numerous instances, such as Sp1, NF-kB and other endogenous transcription factors. However, the strict requirement for Tat to work in concert with these transcription factors undermines the feasibility that a cellular cofactor alone could confer sufficient full-length transcription in order to produce Tat. It is more likely that the current research examining these cellular transcription factors as cofactors is a more accurate way to view their role in transcriptional regulation. In other words, they play important supporting roles in transcriptional regulation of the HIV-1 LTR but are not capable of substituting for the lead actor, Tat.
Basal low-level read through is a more likely candidate than the previous two models. Although short non-processive transcripts are predominant, it is feasible that a low-level of processive transcripts are also produced. Subsequent regulation of Tat could occur through interactions with host transcription factors. Considering biological systems, this model presents an appealing mode of regulation--it would seem easier to fine tune transcription if starting at a baseline level rather than with an all or nothing event. An analogy of adjusting the water temperature of a shower aptly describes this model; it is easier to adjust to a desired temperature if the water is already on, than if it is off and must be both turned on and adjusted (Siegel, 1997). Relating to the model of HIV-1 transcription regulation, it may be more efficient to fine tune full-length transcription if starting from a baseline of low-level read through than starting with no full-length transcripts. Research to explore this model will require the use of RNAse protection and nuclear run-on assays to monitor the presence and amount of full-length transcripts in the absense of Tat. Analysis should employin vivo systems that contain full-length HIV-1 provirus to test this model.
In addition to basal read through, the second initiator site model is also a possible model for the original host production of Tat necessitating further exploration. The most compelling evidence for this model is that transcripts starting downstream of the +1 initiation site were found in a variety of systems assaying transcription and that transcripts starting at +30-+50 would necessarily be TAR independent since transcription initiating here would not permit TAR formation. To test this model experimentally, RNAse protection assays and nuclear run-on assays should be performed on stimulated and unstimulated HIV-1 infected cells of various types. To distinguish between a band corresponding to a true initiation site and that of a gel artifact, assays for the capping of the 5' end of the transcript should be performed for all bands found.
Exploration of the mechanism of the original production of Tat is essential for both developing a fuller understanding of the regulation of the expression of the HIV-1 genome and to provide insight into mechanisms of transcriptional regulation in other systems. On the level of viral infection, a better understanding of transcriptional regulation can aid in targeting therapies for HIV-1 disease. And on a broader scope, much of what we learn regarding virus transcriptional regulation can potentially expand our knowledge of the regulation of normal cellular gene expression.