Structural investigations of the HTLV Tax protein – SinTax

Tax protein and its cellular partners are co- expressed in a prokaryotic system to purify the native complexes. Complexes will be used in parallel for structural studies by X-ray crystallography and electron microscopy. Screening of crystallogenesis conditions will be performed with the use of a nanopipetter Mosquito and crystal farms at 19°C and 4°C (Formulatrix, Explora Nova). Optimization from the lead conditions will be performed manually. Diffraction of the obtained crystals will be performed at synchrotron facilities at Grenoble, France (ESRF facility) and Villigen, Switzerland (PSI facility) for which the consortium has regular access.
Electron Microscopy (EM) will be used to determine the structure of the different complexes. Indeed, it is now possible to obtain 3D structure of small complexes by cryo-EM thanks to the use of a direct electron detector on a Polara EM (team 3). If cryo-EM does not succeed, we will use negative staining EM to determine the envelope of the complexes. Fitting of the high resolution structures of the partners into the EM densities will be performed. These EM results will be decisive if crystals of the complexes appear difficult to obtain. The structure of the Tax/CREB complex will also be studied in the presence of its 21bp-DNA target, the TxRE (Tax responsive element) of the viral promoter, to get insights in the functional ternary complex, when compared with the published CREB/DNA structure in the absence of Tax.
The importance of the residues present at the different interfaces that we will identify on the 3D structures of Tax in complex with cellular partners will be assessed by structure-based site-directed mutagenesis through molecular modeling. These mutants will be tested in functional assays to characterize precisely the residues and interfaces that are mandatory for the functions of Tax.

We have first expressed Tax alone in a prokaryotic system. After purification, electron microscopy analysis suggested that Tax was in complex with the bacterial chaperone GroEL. X-ray crystallography experiments on these samples led to the obtaining of diffraction data at ~4Å resolution. These data are currently being analyzed to assess if the Tax protein is present in the crystal and, in that case, to perform the first structural studies of the protein.
We have co-expressed the Tax protein with the p65 subunit of NF-kB. Conditions allowing the co-expression of these two proteins have been explored, but the purification proved to be difficult due to the tags that were used. Experiments are ongoing for the co-expression of p65 and Tax with other purification tags.
We have also performed the co-expression of Tax with the transcription factor CREB. In these conditions, purification of Tax allows the co-purification of CREB, demonstrating the presence of Tax/CREB complexes and its partial purification. Moreover, this complex seems functional, as electrophoretic mobility shift assays show the interaction of the complex with the target viral DNA. However, these complexes are not pure enough for structural studies. Therefore, new attempts of purification of this complex are under way, including purification of the Tax/CREB complex in the presence of the target viral DNA TxRE, which has been described to increase the affinity of the Tax/CREB complex.
Finally, the project has been extended to the structural study of the Tax protein in complex with UPF-1, a cellular helicase of known structure. This complex can be purified in its active form in small amounts. A scale-up has been initiated to obtain enough Tax/UPF-1 complexes for structural studies.

We are confident that we will obtain the complexes of Tax with various cellular partners within the time limits of the financial support of this project. Therefore, the different approaches that we have developed should allow us to reach our objective, which is to obtain the first structural data of the Tax protein of HTLV.
Moreover, the structural study of the Tax/UPF1 complex, which has been added recently to the project, will increase the odds of getting a complex allowing the elucidation of the structure of the Tax protein.
Finally, the very recent obtaining of structural data of Tax/GroEL complexes could allow us to get the first structural information of the Tax protein and give insights for the first directed mutagenesis of Tax, in order to characterize the structures/functions relationships of this protein.

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Human T-cell Leukemia Viruses (HTLV) infect at least 20 million people worldwide and comprise four types (HTLV-1 to -4), the most frequent types being HTLV-1 and -2. HTLV-1 causes Adult T-cell Leukemia/Lymphoma (ATL) or Tropical Spastic Paraparesis/HTLV-1 Associated Myelopathy (TSP/HAM), a neurodegenerative disease, while HTLV-2 promotes only a persistent lymphocytosis. The protein Tax of HTLV is a viral transactivator that is required for activating transcription from the viral long-terminal repeat (LTR) promoter. Tax also interacts with numerous cellular proteins, leading to an alteration of cellular gene transcription that is linked to pathogenesis. HTLV-1 Tax (Tax-1) and HTLV-2 Tax (Tax-2) share the ability to bind to the CREB transcription factor for activating viral transcription, while they do not interact with the same NF-kB members. This might explain the differences in gene dysregulation and outcomes of infection. As Tax is expressed in vivo, it represents a valuable target for the development of anti-HTLV molecules, but its structure remains unknown.
This project is aimed at obtaining the first experimental structures of Tax-1 and Tax-2 proteins, alone and in complex with cellular transcription factors interacting with Tax-1 and/or Tax-2, with or without viral DNA. For this purpose, the atomic structures of the Tax-1 and Tax-2 proteins (full-length or domains) will be sought by X-ray crystallography. Cellular transcription factors interacting with Tax-1 and/or Tax-2 will also be produced in two different expression systems (E. coli and mammalian cells) for functional and structural studies (X-ray crystallography, electron microscopy (EM)). The factors that have been selected for this study are CREB, p65/RelA, and NF-kB2/p100, which are involved in three different functions of Tax i.e. viral LTR transcription, interaction with the canonical NF-kB pathway, and with the non-canonical NF-kB pathway, respectively. Atomic structures are already available for the functional domains of these three cellular partners of Tax.
Following expression (or co-expression) of the individual proteins, complexes between Tax-1 or Tax-2 and the cellular factors will be characterized using standard biophysical and biochemical methods. Macromolecular complexes will then be studied by cryo-electron microscopy to obtain medium resolution envelopes in which we will fit the high-resolution structures of the different components of the complexes, including Tax structures that we expect to obtain. Complexes between Tax proteins and CREB domain will also be studied in the presence of the target viral DNA (Tax Responsive Element present in the viral LTR) to get structural insights in the functional complex that initiates activation of HTLV transcription by recruiting Pol II on the viral promoter.
Medium and high resolution structures of the macromolecular complexes, combined with their biochemical and biophysical characterization, will be used to solve the protein:protein and protein:DNA interfaces involved in Tax functions. The functional relevance of these interfaces will be assessed by structure-based mutagenesis of Tax proteins. To validate our observations, the impact of these mutations will be determined in in vitro functional assays. This will allow the identification of amino acid residues and interfaces involved in Tax functions. They would represent interesting targets for the development of anti-HTLV molecules targeting Tax.
Ultimately, this study will allow us to decipher the precise molecular mechanisms of Tax function both at the level of viral transcription (Tax/CREB complexes) and at the level of Tax-induced cellular transformation (Tax/NF-kB complexes). Understanding these mechanisms will provide a rationale to develop anti-HTLV therapeutic molecules targeting Tax. More generally, this study will provide new insights on how functional adaptability of viral proteins can arise despite the structural constraints they undergo.