Impact of the terminal bulges of HIV-1 cTAR DNA on its stability and the destabilizing activity of the nucleocapsid protein NCp7
Impact of the terminal bulges of HIV-1 cTAR DNA on its stability and the destabilizing activity of the nucleocapsid protein NCp7
复制标题
DOI：
10.1016/s0022-2836(03)00244-4
复制
发表时间：
2003-04-18
影响因子：
5.6
通讯作者：
Mély, Y
中科院分区：
文献类型：
--
作者：
Beltz, H;Azoulay, J;Mély, Y
逆转录酶（RT）将HIV-1基因组RNA逆转录为双链DNA是HIV-1复制的关键步骤。该过程依赖于两种病毒蛋白，RT酶和核衣壳蛋白NCp 7，其具有充分记录的核酸伴侣特性。在线性DNA合成开始时，通过反式激活反应元件（TAR）RNA和cTAR DNA序列之间的杂交反应，将新制备的负链强终止DNA（（-）ssDNA）转移到基因组RNA的Y末端。由于两个TAR序列都表现出稳定的发夹结构，因此NCp 7需要使TAR结构不稳定，以便陪伴它们的杂交。为了进一步表征TAR稳定性和NC介导的去稳定化之间的关系，研究了A（49）和G（52）凸起残基在cTAR DNA稳定性中的作用。检查了cTAR和突变体的稳定性，其中一个或两个末端凸起被碱基对取代，以及NCp 7介导的这些cTAR序列的不稳定。热力学数据表明，这两个凸起合作，通过减少碱基之间的堆积相互作用，使cTAR不稳定。这导致约6.4千卡/摩尔的自由能变化，似乎是至关重要的NC活动。双重标记的cTAR衍生物的时间分辨荧光数据表明，NC介导的cTAR末端的解链传播至C-10.A（44）错配或T-40凸起。还使用使用双光子激发的荧光相关光谱来监测NC引起的cTAR末端磨损。结果表明，NC引起cTAR末端磨损的非常显著的增加，可能仅限于cTAR突变体的情况下的末端碱基对。由于TAR RNA和cTAR DNA凸起或错配似乎在所有HIV-1毒株中都很保守，因此目前的数据支持TAR和NC活性之间存在共进化关系的概念。(C)2003爱思唯尔科技有限公司版权所有。
Reverse transcription of HIV-1 genomic RNA to double-stranded DNA by reverse transcriptase (RT) is a critical step in HIV-1 replication. This process relies on two viral proteins, the RT enzyme and nucleocapsid protein NCp7 that has well documented nucleic acid chaperone properties. At the beginning of the linear DNA synthesis, the newly made minus-strand strongstop DNA ((-)ssDNA) is transferred to the Yend of the genomic RNA by means of an hybridization reaction between transactivation response element (TAR) RNA and cTAR DNA sequences. Since both TAR sequences exhibit stable hairpin structures, NCp7 needs to destabilize the TAR structures in order to chaperone their hybridization. To further characterize the relationships between TAR stability and NC-mediated destabilization, the role of the A(49) and G(52) bulged residues in cTAR DNA stability was investigated. The stability of cTAR and mutants where one or the two terminal bulges were replaced by base-pairs as well as the NCp7-mediated destabilization of these cTAR sequences were examined. Thermodynamic data indicate that the two bulges cooperatively destabilize cTAR by reducing the stacking interactions between the bases. This causes a free energy change of about 6.4 kcal/mol and seems to be critical for NC activity. Time-resolved fluorescence data of doubly labelled cTAR derivatives suggest that NC-mediated melting of cTAR ends propagates up to the C-10.A(44) mismatch or T-40 bulge. Fluorescence correlation spectroscopy using two-photon excitation was also used to monitor cTAR ends fraying by NC. Results show that NC causes a very significant increase of cTAR ends fraying, probably limited to the terminal base-pair in the case of cTAR mutants. Since the TAR RNA and cTAR DNA bulges or mismatches appear well conserved among all HIV-1 strains, the present data support the notion of a co-evolutionary relationship between TAR and NC activity. (C) 2003 Elsevier Science Ltd. All rights reserved.