Previously described dominant negative Tat proteins containing the Tat AD alone have shown relatively modest levels of inhibition (6
). The extraordinarily potent proteins described here represent a new mechanistic class of transcriptional inhibitor, and a splicing factor (U2AF65 or SF1) or short RS domain acts as a targeting/localization moiety for the tethered Tat AD. The localization function provided by this targeting moiety allows the inhibitor to function at stoichiometric levels, without the need for substantial overexpression typically required by dominant negatives that operate by squelching or other simple competition mechanisms (13
). Ptashne and Gann proposed the concept of “regulated localization,” activity and specificity being imposed by simple binding interactions between a locator (transcription factor), the transcriptional machinery, and the DNA (39
). Such localization can include subcellular compartmentalization, in which molecular crowding can enhance the assembly of large macromolecular complexes (30
), in our case, favoring assembly into holo-Pol II complexes. We further suggest that combining multiple targeting functions within a single polypeptide provides an entropic benefit, allowing T-RS to load efficiently into early transcription complexes through CTD and CycT1 interactions, thereby blocking the entry of wild-type Tat.
It is especially interesting that RNA binding is not required for the delivery of T-RS to the HIV-1 promoter, raising a number of questions about the mechanisms and timing of T-RS and Tat recruitment into HIV-1 transcription complexes and also whether other dominant negative transcription factors can be recruited efficiently to their promoters by a similar cotranscriptional tethering strategy. Preliminary experiments with HSF1, CIITA, and GAL4-VP16 activators show little or no effect of an appended RS domain (data not shown), suggesting that Tat assembly may represent a special case, perhaps related to its role in elongation or dependence on RNA binding. The Tat AD itself associates directly with holo-Pol II complexes (9
; this work), perhaps explaining why the U2AF65 RS moiety, known to associate with preinitiation or early transcription complexes (42
), enhances T-RS assembly. The proposed targeting function of the RS domain to the CTD is consistent with the observations that U2AF65 is found in early transcription complexes (42
) and is recruited to the HIV-1 promoter (5
). Our experiments demonstrate a direct interaction between the U2AF65 RS domain and CTD in vitro and a CTD-dependent interaction in vivo (Fig. ), agreeing with the observation that splicing factors copurify with nonphosphorylated RNAP II (10
). Mutagenesis of T-RS indicates that the serines of the RS dipeptides are important for CTD binding and dominant negative activity, whereas the positively charged arginines contribute to nonspecific RNA binding, as previously proposed (50
), but have little effect on inhibition (Fig. ).
Still, the CTD-tethering hypothesis is insufficient to explain the specificity of recruitment to the HIV-1 promoter, given that T-RS, which requires interactions with CycT1 in addition to the CTD, would be expected to bind to and inhibit activation of other P-TEFb-dependent promoters, yet this is not the case (Fig. and ). Perhaps the transient nature or precise timing of splicing factor-CTD interactions (29
), interactions of the Tat AD or P-TEFb with other HIV-1 promoter-specific factors, or the assembly of TBP-associated-factor-less TBP complexes (40
) differ among promoters and determine whether stable “ChIPable” complexes can form. Irrespective of the details of assembly, it is clear that mechanisms used to cotranscriptionally load RNA-processing factors at promoters (2
) can be co-opted to deliver the dominant negative Tat AD to the HIV-1 promoter and thereby generate an extraordinarily potent inhibitor.
Given the high inhibitor potency, viral replication is substantially inhibited by low-level expression of the dominant negative in stable cell lines, even without optimizing and selecting for lines with high activity. It is interesting that these cells establish a chronic infection without cytopathic effects, reminiscent of other cellular environments that may resemble latent stages of HIV-1 infection (27
). The amount of Tat clearly affects viral replication rates (51
) and also can drive phenotypic diversity (52
), and here, we show that expression of the dominant negative provides another means to alter Tat function. It will be interesting to examine mechanisms by which resistance to the Tat dominant negative might arise and to evaluate its therapeutic potential.