Chen Z, Li Y, Krug R M
Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, New Jersey 08855, USA.
Virology. 1998 Feb 15;241(2):234-50. doi: 10.1006/viro.1997.8949.
Nuclear RNA export mediated by the HIV-1 Rev protein is inhibited by chimeric proteins in which the wild-type Rev protein is covalently linked to amino acid sequences of the NS1 protein of influenza A virus (NS1A protein), a protein that inhibits nuclear RNA export. These chimeric molecules function not only in cis but also in trans: they inhibit nuclear RNA export mediated by Rev protein molecules that are not covalently linked to the NS1A protein sequence. Here we show that inhibition occurs with a NS1-Rev chimera in which the 78 amino-terminal amino acids of the NS1A protein comprising its entire RNA-binding domain is deleted, thereby establishing that this carboxyl portion of the NS1A protein can function as an independent effector domain. The mechanism by which this NS1-Rev chimera inhibits Rev function in trans was determined. The Rev sequence in this chimera oligomerizes with Rev molecules in trans, and the resulting mixed oligomers are retained in the nucleus because the nuclear retention activity of the NS1 effector domain is dominant over the nuclear transport activity of the Rev effector domain. Binding of the FG-containing nucleoporin-like Rab protein to this NS1-Rev chimera, as measured in yeast two-hybrid assays, is much stronger than that to the Rev protein itself, yet nuclear export does not occur in the presence of the chimera. Unexpectedly, the introduction of specific mutations into the NS1A portion of this NS1-Rev chimera not only restores Rev-mediated unclear export of RNA but also eliminates detectable Rab binding, indicating that this nuclear export can occur without detectable Rab binding. A different NS1-Rev chimera, one in which the NS1A protein is full-length but contains a mutated RNA-binding domain, effectively inhibits Rev-mediated nuclear export of RNA without blocking the nuclear export of the Rev protein, indicating that nuclear export of the carrier Rev protein can be uncoupled from nuclear export of its passenger RNA.
由HIV-1 Rev蛋白介导的核RNA输出受到嵌合蛋白的抑制，在这些嵌合蛋白中，野生型Rev蛋白与甲型流感病毒NS1蛋白（NS1A蛋白）的氨基酸序列共价连接，NS1A蛋白是一种抑制核RNA输出的蛋白。这些嵌合分子不仅顺式起作用，也反式起作用：它们抑制由未与NS1A蛋白序列共价连接的Rev蛋白分子介导的核RNA输出。在此我们表明，一种NS1-Rev嵌合体可产生抑制作用，其中NS1A蛋白包含其整个RNA结合结构域的78个氨基末端氨基酸被缺失，从而确定NS1A蛋白的该羧基部分可作为一个独立的效应结构域发挥作用。确定了这种NS1-Rev嵌合体反式抑制Rev功能的机制。该嵌合体中的Rev序列与反式Rev分子寡聚化，并且产生的混合寡聚体保留在细胞核中，因为NS1效应结构域的核保留活性强于Rev效应结构域的核转运活性。在酵母双杂交试验中检测到，含FG的核孔蛋白样Rab蛋白与这种NS1-Rev嵌合体的结合比与Rev蛋白本身的结合要强得多，但在存在该嵌合体的情况下不会发生核输出。出乎意料的是，在这种NS1-Rev嵌合体的NS1A部分引入特定突变不仅恢复了Rev介导的RNA的核输出，而且消除了可检测到的Rab结合，表明这种核输出可在没有可检测到的Rab结合的情况下发生。另一种NS1-Rev嵌合体，其中NS1A蛋白是全长的但含有一个突变的RNA结合结构域，可有效抑制Rev介导的RNA的核输出，而不阻断Rev蛋白的核输出，表明载体Rev蛋白的核输出可与其携带的RNA的核输出解偶联。
