Alternate usage of two dimerization initiation sites in HIV-2 viral RNA in vitro.
Alternate usage of two dimerization initiation sites in HIV-2 viral RNA in vitro.
复制标题
DOI：
10.1016/s0022-2836(02)00369-8
复制
发表时间：
2002-06
影响因子：
5.6
通讯作者：
J. Lanchy;J. Lodmell
中科院分区：
文献类型：
--
作者：
J. Lanchy;J. Lodmell
所有逆转录病毒复制周期中的一个重要步骤是在出芽和出芽之前或期间基因组RNA的二聚化。在HIV-1中，被称为二聚化起始位点（DIS）的基因组RNA中的茎环结构已经得到了很好的表征。然而，鉴定HIV-2基因组RNA二聚化所需的结构并不那么简单，这反映在最近相互矛盾的报道中。在这里，使用各种突变体和野生型RNA构建体和实验条件的系统分析，我们证明了在不同的实验条件下，HIV-2 RNA中的两个二聚化位点是清晰可辨的。与引物结合位点重叠的短序列充当几种长度的野生型病毒RNA转录物的默认二聚化位点，条件是二聚化孵育条件不包括高温步骤（>50°C），并且电泳在不耗尽RNA的镁的温和条件下进行。然而，在某些实验条件下，一些RNA构建体能够通过茎环1（SL 1）二聚化，所述茎环1是与HIV-1 DIS同源的结构。有趣的是，缺省PBS二聚化位点的缺失或突变导致SL 1二聚化位点的有效利用。本研究定义了每个位点可用于二聚化的条件，并证明了两个位点可以相互替代的设施。这提示可能在病毒复制周期中使用的转换机制。
An essential step in the replication cycle of all retroviruses is the dimerization of genomic RNA prior to or during encapsidation and budding. In HIV-1, a stem-loop structure in the genomic RNA called the dimerization initiation site, or DIS, has been well characterized. However, the identification of the structure(s) necessary for dimerization of HIV-2 genomic RNA has been less straightforward, as reflected by recent conflicting reports. Here, using a variety of mutant and wild-type RNA constructs and a systematic analysis of experimental conditions, we demonstrate that two dimerization sites in HIV-2 RNA are clearly discernible under different experimental conditions. A short sequence overlapping the primer binding site acts as the default dimerization site for wild-type viral RNA transcripts of several lengths provided that dimerization incubation conditions do not include a high heat step (>50°C), and electrophoresis is carried out under mild conditions that do not deplete the RNA of magnesium. However, some RNA constructs are able to dimerize through stem-loop 1 (SL1), which is the structure homologous to the HIV-1 DIS, under certain experimental conditions. Interestingly, deletion or mutation of the default PBS dimerization site leads to efficient usage of the SL1 dimerization site. This study defines conditions under which each site may be used for dimerization and demonstrates, furthermore, the facility with which the two sites can substitute for each other. This is suggestive of a switching mechanism that may be used in the viral replication cycle.