One of the key controlling events in HTLV-1 biology is transcription from the viral LTR-mediated by the HTLV-1 Tax protein. The trans-acting function of Tax, in addition to another HTLV-1 protein Rex, is essential for efficient replication of HTLV-1 (5
). Tax by itself does not bind to the viral LTR and does not posses an intrinsic enzymatic activity and it is thought that the viral transactivator functions via interaction with cellular transcription factors. It is also clear that in addition to the recruitment of basal transcription factors, Tax exerts influence upon viral transcription through interaction with cellular factors that are themselves either positive or negative regulators of transcription. Therefore, the ability of Tax to interact with cellular factors is a critical component of transcriptional regulation and thus HTLV-1 biology.
Transactivation by Tax via interaction with positive transcription factors is a complex process mediated via factor recruitment at one or more of the three 21-bp Tax response elements (TxREs) present in the U3 region of the HTLV-1 LTR (4
). The TxREs are composed of a central imperfect cyclic AMP response element (CRE). Similar to other CREs, TxRE binding transcription factors are globally members of the CREB/ATF family of proteins with a conserved C-terminal bZip domain which has been shown to be essential for interaction with Tax, and binding to TxRE. A significant number of studies have suggested that, in general, Tax-mediated transactivation of the LTR involves three mechanistic steps; Tax promotes homodimerization and recruitment of CREB/ATF proteins; Tax mediates stabilization of the LTR bound complexes; TxRE associated Tax recruits transcriptional coactivators like CBP/p300 (15, 22, 26-28, 48).
Negative regulation of the viral LTR by cellular proteins is likely an important feature of the integrated virus that facilitates repression of virus replication and is believed to be necessary to escape the cytotoxic-T-lymphocyte response, allowing the virus to survive inside the host (23
). The b-ZIP protein CCAAT/enhancer binding protein β (C/EBPβ), forms a heterodimeric complex with CREB2, which binds to the CRE site in TxREs and competes with the ability of Tax to recruit active complexes to the viral promoter (23
). Further, the inducible cAMP early repressor (ICER), another member of CREB/ATF/CREM family, has been shown to repress Tax-mediated activation of the HTLV-1 LTR (29
). Interestingly, the affinity for binding of ICER to the TxRE is increased in presence of Tax. Thus, Tax-mediated regulation of viral transcription is governed by multiple factors. Specifically, in addition to possessing an activation domain and recruiting basal transcription machinery, Tax-mediated transcription is influenced by interaction with transcriptional activating factors, and with transcriptional repressing factors.
We have identified ATFx, a member of the CREB/ATF factor family, as a novel repressor of HTLV-1 LTR transcription. ATFx is identical to human ATF5 and has 87% amino acid sequence similarity to mouse ATF5. Mouse ATF5 has also been named as ATF-7 (38
). We observed specific repression of the HTLV-1 LTR that was not seen in other heterologous promoter and the repression was apparent in the presence of Tax. Therefore, the Tax-activated promoter was repressed by exogenously added ATFx. This suggested that the repression of ATFx is competitive with Tax-activation since Tax is able to reverse the repression of ATFx. The binding of ATFx to the viral LTR is mediated by the bZIP domain within ATFx and this same region is required for interaction with Tax. Thus, the interaction of ATFx with Tax would prevent ATFx repression of the HTLV-1-LTR.
The observation that the same domain within ATFx mediates a functionally competitive bridge is suggestive of several mechanistic models. One intriguing model is that the presence of ATFx on the viral promoter is involved in the recruitment of Tax to the promoter and that reversal of ATFx repression contributes to overall Tax activation. Thus, ATFx would be part of the molecular topology of the HTLV-1 promoter to which Tax binds. In light of the competitive relationship we observed between ATFx repression and CREB/CREB2 activation, it is reasonable to conclude that these positive and negative regulatory mechanisms interact to achieve promoter function. This interaction could also extend to the cellular promoter targets of ATFx with which Tax may exert activation.
Unlike other members of the ATF family, ATFx is not known to affect cell cycle or proliferation and the known downstream targets for ATFx seem to be restricted to those genes that are involved in the regulation of apoptosis (37
). Specifically, ATFx is an anti-apoptotic factor and the expression of ATFx is down-regulated in a variety of cell lines undergoing apoptosis following growth factor deprivation. The action of ATFx is believed to be conditionally related to apoptosis in that ATFx levels must be reduced to allow apoptosis to proceed. It has been established that the transcriptional activity of ATFx is required for cell survival, since a dominant negative mutant of ATFx (retaining the bZIP DNA-binding domain) suppresses the anti-apoptotic effects of ATFx.
Unique among the ATF family members, ATFx expression is cell cycle regulated and peaks in G1
/S and is undetectable in G2
/M. It is noteworthy that repression of ATFx is required in order for apoptotic signals to be effective and that presumably the requirement for ATFx changes with cell cycle. Specifically, ATFx is expressed highest during G1
/S, the most critical cell cycle period for initiation of apoptosis (37
). Conversely, in G2
/M the levels of ATFx are very low perhaps indicating the cells are less susceptible to apoptosis. The consequence of ATFx repression of viral transcription to HTLV-1 infection is that transcription activity would increase during G2
/M and become suppressed at G1
It is interesting to speculate that HTLV-I may have evolved this mechanism of “monitoring” the levels of ATFx in order to regulate viral transcription around apoptotic signaling. We further note that we and others have shown that de novo Tax expression leads to G2/M accumulation during which ATFx and suppression of the viral LTR would be lowest. One of the potential outcomes of Tax-induced G2/M accumulation would be to insure viral transcription during a cell cycle period when the cell is least susceptible to apoptosis. Thus, the interplay between Tax and ATFx could provide a mechanism by which viral transcription is tied to cell homeostasis. Understanding the relationship between ATFx and Tax may help decipher the underlying mechanism that allows HTLV-1 infection to survive “dormancy” for so many years.