Specific implications of the HIV-1 nucleocapsid zinc fingers in the annealing of the primer binding site complementary sequences during the obligatory plus strand transfer
Specific implications of the HIV-1 nucleocapsid zinc fingers in the annealing of the primer binding site complementary sequences during the obligatory plus strand transfer
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DOI：
10.1093/nar/gkr274
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发表时间：
2011-08-01
影响因子：
14.9
通讯作者：
Mely, Yves
中科院分区：
文献类型：
--
作者：
Godet, Julien;Ramalanjaona, Nick;Mely, Yves
通过逆转录酶合成 HIV-1 病毒 DNA 涉及两个必然的链转移反应。第二条链转移对应于由核衣壳蛋白 (NCp7) 陪伴的引物结合位点 (PBS) 序列的 (-) 和 (+) DNA 拷贝的退火。 NCp7 通过激活环-环接吻途径来修改 (+)/(-)PBS 退火机制，如果没有 NCp7，该途径可以忽略不计。为了深入表征 NCp7/PBS 核蛋白复合物中环的动力学，我们研究了在位置 6、8 或 10 处含有荧光核苷类似物 2-氨基嘌呤的 (-)PBS 衍生物的时间分辨荧光参数。 (+)/(-)PBS 退火向环途径的 NCp7 定向转换与局部 DNA 动力学的严格限制相关，表明 NCp7 可以“冻结”能够通过环退火的 PBS 构象。此外，控制退火反应的 PBS 环结构和动力学的修改严格依赖于锌指疏水平台的完整性。我们的数据表明，需要两个 NCp7 锌指来确保第二链转移的特异性和保真度，进一步强调了 NCp7 在控制病毒 HIV-1 DNA 的忠实合成中发挥的关键作用。
Synthesis of the HIV-1 viral DNA by reverse transcriptase involves two obligatory strand transfer reactions. The second strand transfer corresponds to the annealing of the (-) and (+) DNA copies of the primer binding site (PBS) sequence which is chaperoned by the nucleocapsid protein (NCp7). NCp7 modifies the (+)/(-)PBS annealing mechanism by activating a loop-loop kissing pathway that is negligible without NCp7. To characterize in depth the dynamics of the loop in the NCp7/PBS nucleoprotein complexes, we investigated the time-resolved fluorescence parameters of a (-)PBS derivative containing the fluorescent nucleoside analogue 2-aminopurine at positions 6, 8 or 10. The NCp7-directed switch of (+)/(-)PBS annealing towards the loop pathway was associated to a drastic restriction of the local DNA dynamics, indicating that NCp7 can 'freeze' PBS conformations competent for annealing via the loops. Moreover, the modifications of the PBS loop structure and dynamics that govern the annealing reaction were found strictly dependent on the integrity of the zinc finger hydrophobic platform. Our data suggest that the two NCp7 zinc fingers are required to ensure the specificity and fidelity of the second strand transfer, further underlining the pivotal role played by NCp7 to control the faithful synthesis of viral HIV-1 DNA.