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The activation domain of a hormone inducible HTLV-1 Rex protein determines colocalization with the nuclear pore
S Rehberger1, F Gounari, M DucDodon
1Department of Tumour Immunology, German Cancer Research Centre, Heidelberg.
Abstract:
Human T-cell leukemia/lymphoma virus type 1 (HTLV-1) Rex is an essential regulatory protein that acts at the posttranscriptional level to promote expression of unspliced and singly spliced genes of the virus. Rex functions have been attributed to at least three separate domains of the protein determining nuclear/nucleolar accumulation and RNA binding (overlapping), multimerization, and nuclear export of Rex-responsive RNA. The steady-state intracellular localization of functional Rex molecules is mainly nucleolar. Fusions of wild-type Rex and the ligand binding domain of human estrogen receptor (ER) produced conditional molecules (ERRex and ERalaRex), which remained cytoplasmic in the absence of hormone and in response to hormone colocalized with the nuclear pore complex (NPC). These molecules induced in a hormone-dependent manner the expression of a Rex reporter plasmid and of the HTLV-1 Env protein and fusion of Env expressing cells. In contrast, activation domain mutants (ERRex delta and ERRexGly) translocated from the cytoplasm and acquired a diffuse nuclear localization. These mutants did not associate with the NPC and failed to show any of the expected Rex functions. Rex functions were perturbed by inactivating the RNA binding domain (mutant ERM2) or the oligomerization domain (mutant ERM7). However, these two mutant fusion proteins exhibited a hormone-dependent NPC colocalization. These observations provide in vivo evidence that intranuclear translocation of intact Rex to the NPC is dependent exclusively on a functional activation domain and is not influenced by binding to the target RNA.
Insights
The Human T-cell leukemia/lymphoma virus type 1 (HTLV-1) Rex protein
Area of Science:
- Virology
- Molecular Biology
- Cell Biology
Background:
- Human T-cell leukemia/lymphoma virus type 1 (HTLV-1) Rex is crucial for viral gene expression.
- Rex protein regulates viral gene expression post-transcriptionally.
- Rex protein has distinct domains for nuclear localization, RNA binding, multimerization, and nuclear export.
Purpose of the Study:
- To investigate the in vivo role of the HTLV-1 Rex protein's activation domain in its function and localization.
- To determine the relationship between Rex protein's activation domain, RNA binding, and nuclear pore complex (NPC) association.
- To elucidate the mechanism of Rex-mediated viral gene expression.
Main Methods:
- Created conditional Rex fusion proteins with the human estrogen receptor (ER) ligand-binding domain.
- Utilized hormone-dependent translocation assays to track Rex localization and function.
- Assessed the impact of mutations in the activation, RNA-binding, and oligomerization domains on Rex function and NPC association.
Main Results:
- Conditional Rex fusion proteins translocated to the nuclear pore complex (NPC) and induced viral gene expression upon hormone addition.
- Mutants lacking a functional activation domain translocated diffusely within the nucleus but failed to associate with the NPC or exhibit Rex functions.
- Mutations in RNA binding or oligomerization domains did not prevent NPC colocalization, despite impairing Rex function.
Conclusions:
- A functional activation domain is essential for the intranuclear translocation of intact Rex to the NPC.
- Rex's association with the NPC is independent of its RNA-binding or oligomerization capabilities.
- These findings clarify the distinct roles of Rex domains in viral gene regulation and intracellular trafficking.