Binding of nuclear proteins to HTLV-II cis-acting repressive sequence (CRS) RNA correlates with CRS function.
Binding of nuclear proteins to HTLV-II cis-acting repressive sequence (CRS) RNA correlates with CRS function.
复制标题
核蛋白与 HTLV-II 顺式作用抑制序列 (CRS) RNA 的结合与 CRS 功能相关。
DOI：
10.1006/viro.1994.1159
复制
发表时间：
1994
期刊：
影响因子：
3.7
通讯作者：
Rosenblatt,JD
中科院分区：
文献类型：
--
作者：
Black,AC;Ruland,CT;Luo,J;Bakker,A;Fraser,JK;Rosenblatt,JD
人类T细胞白血病病毒I型(HTLV-I)和II型(HTLV-II)从病毒调控基因表达到结构基因表达的转变是由Rex介导的。我们先前已经证明HTLV-II Rex通过5‘长末端重复序列(LTR)的R/U5中的一个元件，Rex反应元件(RxRE)起作用，并且Rex蛋白与RxRE中包含的特定RNA序列，Rex结合元件(RBE)结合(Blacket al.，J.Viroll.65,6645-6653,1991b)。通过RBE(NT 405-520)的REX作用克服了连续的LTR RNA调节元件对表达的抑制，该元件包含不与Rex蛋白结合的顺式作用抑制序列(CRS；NT 520-630)(Blacket al，病毒学，181,433-144,1991a)。我们现在通过电泳迁移率改变分析(EMSA)表明，HeLa核提取物中的细胞蛋白与含有HTLV-II CRS的RNA转录本特异结合。利用凝胶延迟带的紫外线(UV)交联，我们鉴定了一种约60 kDa的主要蛋白质物种p60CRS，它与CRS RNA结合，但与RBE RNA的亲和力较弱。此外，一种独特的40 kDa蛋白质p40CRS与CRS下游的U5 RNA(nt645-750)结合。CRS RNA中的特定缺失可以减少或取消与这个60 kDa蛋白质的结合。EMSA和UV交联实验也表明p60CRS和p40CRS都与CRS RNA相互作用。在5‘LTR连锁的基因表达分析中，CRS的功能与p60CRS和p40CRS在体外与5’LTRRNA相互作用的能力有关。
The shift from viral regulatory to structural gene expression in human T-cell leukemia virus types I (HTLV-I) and II (HTLV-II) is mediated by Rex. We have previously shown that HTLV-II Rex acts through an element in R/U5 of the 5′ long terminal repeat (LTR), the Rex-responsive element (RxRE), and that Rex protein binds to specific RNA sequences, the Rex binding element (RBE), contained within the RxRE (Blacket al., J. Virol.65, 6645-6653, 1991b). Rex action through the RBE (nt 405-520) overcomes the inhibition of expression conferred by a contiguous LTR RNA regulatory element, which contains cis-acting repressive sequences (CRS; nt 520-630) that are not bound by Rex protein (Blacket al., Virology,181, 433-144, 1991a). We now show by electrophoretic mobility shift assay (EMSA) that cellular proteins in a HeLa nuclear extract bind specifically to RNA transcripts containing the HTLV-II CRS. Using ultraviolet (uv) crosslinking of gel-retarded bands, we identified a major protein species of approximately 60 kDa, p60CRS, that binds to CRS RNA and, with weaker affinity, to RBE RNA. In addition, a distinct 40-kDa protein, p40CRS, binds to U5 RNA (nt 645-750) downstream from the CRS. Specific deletions within CRS RNA can reduce or abrogate binding to this 60-kDa protein. EMSA and uv crosslinking assays also suggest that both p60CRS and p40CRS interact with CRS RNA. CRS function in a 5′ LTR-linked gene expression assay correlates with the ability of both p60CRS and p40CRS to interact with 5′ LTR RNAin vitro.