Dominant Role of the 5′ TAR Bulge in Dimerization of HIV-1 Genomic RNA, but No Evidence of TAR-TAR Kissing during in Vivo Virus Assembly
Dominant Role of the 5′ TAR Bulge in Dimerization of HIV-1 Genomic RNA, but No Evidence of TAR-TAR Kissing during in Vivo Virus Assembly
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DOI：
10.1021/bi300111p
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发表时间：
2012-05-08
期刊：
影响因子：
2.9
通讯作者：
Laughrea, Michael
中科院分区：
文献类型：
--
作者：
Jalalirad, Mohammad;Saadatmand, Jenan;Laughrea, Michael
HIV-1基因组RNA（gRNA）的5'非翻译区含有两个茎环结构，这两个茎环结构似乎对gRNA二聚化同样重要：位于5'末端的57个核苷酸的5' TAR和35个核苷酸的SL 1（核苷酸243-277）。众所周知，SL 1在其顶端环中含有二聚化起始位点（DIS）。DIS是一个六核苷酸回文。在这里，我们研究了TAR指导的gRNA二聚化的机制。我们发现，5' TAR的三核苷酸凸起（UCU 24）对HIV-1 RNA二聚体的形成和所形成的二聚体的成熟都具有主导影响。Δ UCU三核苷酸缺失强烈抑制第一个过程并阻断另一个过程，因此与整个5' TAR的缺失一样严重地损害gRNA二聚化，并且比DIS的缺失、病毒蛋白酶的失活或核衣壳蛋白中的最严重突变更严重。TAR的顶端环含有10个核苷酸的回文，其被假定通过类似于SL 1用于刺激二聚化的TAR-TAR接吻机制来刺激gRNA二聚化。使用突变，强烈破坏TAR回文双链体的形成，以及补偿突变，恢复双链体的形成野生型样的水平，我们没有发现任何证据的TAR-TAR接吻，即使突变无效的接吻潜力的TAR回文可能会损害二聚化的机制以外的阻碍SL 1。然而，消除TAR的接吻潜力的影响远不如Delta UCU严重。由于没有揭示TAR固有的二聚化机制，我们的数据表明TAR突变在TAR的3'端发挥作用，但不对SL 1发挥作用，因为TAR和SL 1突变对gRNA二聚化具有协同作用。
The 5' untranslated region of HIV-1 genomic RNA (gRNA) contains two stem-loop structures that appear to be equally important for gRNA dimerization: the 57-nucleotide 5' TAR, at the very 5' end, and the 35-nucleotide SL1 (nucleotides 243-277). SL1 is well-known for containing the dimerization initiation site (DIS) in its apical loop. The DIS is a six-nucleotide palindrome. Here, we investigated the mechanism of TAR-directed gRNA dimerization. We found that the trinucleotide bulge (UCU24) of the 5' TAR has dominant impacts on both formation of HIV-1 RNA dimers and maturation of the formed dimers. The Delta UCU trinucleotide deletion strongly inhibited the first process and blocked the other, thus impairing gRNA dimerization as severely as deletion of the entire 5' TAR, and more severely than deletion of the DIS, inactivation of the viral protease, or most severe mutations in the nucleocapsid protein. The apical loop of TAR contains a 10-nucleotide palindrome that has been postulated to stimulate gRNA dimerization by a TAR-TAR kissing mechanism analogous to the one used by SL1 to stimulate dimerization. Using mutations that strongly destabilize formation of the TAR palindrome duplex, as well as compensatory mutations that restore duplex formation to a wild-type-like level, we found no evidence of TAR-TAR kissing, even though mutations nullifying the kissing potential of the TAR palindrome could impair dimerization by a mechanism other than hindering of SL1. However, nullifying the kissing potential of TAR had much less severe effects than Delta UCU. By not uncovering a dimerization mechanism intrinsic to TAR, our data suggest that TAR mutations exert their effect 3' of TAR, yet not on SL1, because TAR and SL1 mutations have synergistic effects on gRNA dimerization.