Burton M, Upadhyaya C D, Maier B, Hope T J, Semmes O J
The Myles H. Thaler Center for AIDS and Human Retroviruses, Department of Microbiology, University of Virginia, Charlottesville, Virginia 23060, USA.
J Virol. 2000 Mar;74(5):2351-64. doi: 10.1128/jvi.74.5.2351-2364.2000.
Human T-cell leukemia virus type 1 (HTLV-1) Tax is a nuclear protein with striking pleiotropic functionality. We recently demonstrated that Tax localizes to a multicomponent nuclear speckled structure (Tax speckled structure [TSS]). Here, we examine these structures further and identify a partial overlap of TSS with transcription hot spots. We used a strategy of directed expression via fusion proteins to determine if these transcription sites are the subtargets within TSS required for Tax function. When fused to human immunodeficiency virus type 1 (HIV-1) Tat, the resulting Tat-Tax fusion protein displayed neither a Tat-like nor a Tax-like pattern but rather was targeted specifically to the transcription subsites. The Tat-Tax fusion was able to activate both the HIV-1 long terminal repeat (LTR) and the HTVL-1 LTR at the same level as the individual component; thus, targeting proteins to transcription hot spots was compatible with both Tax and Tat transcription function. In contrast, the fusion with HIV-1 Rev, Rev-Tax, resulted in a pattern of expression that was largely Rev-like (nucleolar and cytoplasmic). The reduced localization of Rev-Tax to transcription sites was reflected in a 10-fold drop in activation of the HTLV-1 LTR. However, there was no loss in the ability of Tax to activate via NF-kappaB. Thus, NF-kappaB-dependent Tax function does not require targeting of Tax to these transcription sites and suggests that activation via NF-kappaB is a cytoplasmic function. Selective mutation of the nuclear localization signal site in the Rev portion resulted in retargeting of Rev-Tax to TSS and subsequent restoration of transcription function, demonstrating that inappropriate localization preceded loss of function. Mutation of the nuclear export signal site in the Rev portion had no effect on transcription, although the relative amount of Rev-Tax in the cytoplasm was reduced. Finally, in explaining how Tax can occupy multiple subcellular sites, we show that Tax shuttles from the nucleus to the cytoplasm in a heterokaryon fusion assay. Thus, pleiotropic functionality by Tax is regulatable via shuttling between discrete subcellular compartments.
人类T细胞白血病病毒1型（HTLV-1）Tax是一种具有显著多效性功能的核蛋白。我们最近证明Tax定位于一种多组分核斑点结构（Tax斑点结构 [TSS]）。在此，我们进一步研究这些结构，并确定TSS与转录热点存在部分重叠。我们采用通过融合蛋白进行定向表达的策略，以确定这些转录位点是否是Tax功能所需的TSS内的亚靶点。当与1型人类免疫缺陷病毒（HIV-1）Tat融合时，产生的Tat-Tax融合蛋白既不显示类似Tat的模式，也不显示类似Tax的模式，而是特异性地靶向转录亚位点。Tat-Tax融合蛋白能够以与单个组分相同的水平激活HIV-1长末端重复序列（LTR）和HTVL-1 LTR；因此，将蛋白质靶向转录热点与Tax和Tat的转录功能均兼容。相比之下，与HIV-1 Rev融合，即Rev-Tax，导致的表达模式在很大程度上类似Rev（核仁及细胞质）。Rev-Tax在转录位点的定位减少反映在HTLV-1 LTR激活下降10倍。然而，Tax通过核因子κB（NF-κB）激活的能力并未丧失。因此，依赖NF-κB的Tax功能并不需要将Tax靶向这些转录位点，这表明通过NF-κB激活是一种细胞质功能。Rev部分中核定位信号位点的选择性突变导致Rev-Tax重新靶向TSS，并随后恢复转录功能，表明功能丧失之前存在定位不当。Rev部分中核输出信号位点的突变对转录没有影响，尽管Rev-Tax在细胞质中的相对量减少。最后，在解释Tax如何能占据多个亚细胞位点时，我们在异核体融合试验中表明Tax在细胞核与细胞质之间穿梭。因此，Tax的多效性功能可通过在离散亚细胞区室之间穿梭来调节。
