Department of Cell and Developmental Biology, Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel.
J Virol. 2012 Jun;86(11):6222-30. doi: 10.1128/JVI.00009-12. Epub 2012 Apr 4.
The Tf1 retrotransposon represents a group of long terminal repeat retroelements that use an RNA self-primer for initiating reverse transcription while synthesizing the minus-sense DNA strand. Tf1 reverse transcriptase (RT) was found earlier to generate the self-primer in vitro. Here, we show that this RT can remove from the synthesized cDNA the entire self-primer as well as the complete polypurine tract (PPT) sequence (serving as a second primer for cDNA synthesis). However, these primer removals, mediated by the RNase H activity of Tf1 RT, are quite inefficient. Interestingly, the integrase of Tf1 stimulated the specific Tf1 RT-directed cleavage of both the self-primer and PPT, although there was no general enhancement of the RT's RNase H activity (and the integrase by itself is devoid of any primer cleavage). The RTs of two prototype retroviruses, murine leukemia virus and human immunodeficiency virus, showed only a partial and nonspecific cleavage of both Tf1-associated primers with no stimulation by Tf1 integrase. Mutagenesis of Tf1 integrase revealed that the complete Tf1 integrase protein (excluding its chromodomain) is required for stimulating the Tf1 RT primer removal activity. Nonetheless, a double mutant integrase that has lost its integration functions can still stimulate the RT's activity, though heat-inactivated integrase cannot enhance primer removals. These findings suggest that the enzymatic activity of Tf1 integrase is not essential for stimulating the RT-mediated primer removal, while the proper folding of this protein is obligatory for this function. These results highlight possible new functions of Tf1 integrase in the retrotransposon's reverse transcription process.
Tf1 反转录转座子代表了一组长末端重复反转录元件，它们使用 RNA 自我引物启动逆转录，同时合成负链 DNA。Tf1 反转录酶（RT）较早被发现可在体外生成自我引物。在这里，我们表明，这种 RT 可以从合成的 cDNA 中去除整个自我引物以及完整的多嘧啶序列（作为 cDNA 合成的第二个引物）。然而，这些引物的去除是由 Tf1 RT 的 RNase H 活性介导的，但效率相当低。有趣的是，Tf1 整合酶刺激 Tf1 RT 定向切割自我引物和 PPT，尽管没有普遍增强 RT 的 RNase H 活性（并且整合酶本身没有任何引物切割活性）。两种原型逆转录病毒（鼠白血病病毒和人类免疫缺陷病毒）的 RT 仅对 Tf1 相关引物进行部分非特异性切割，Tf1 整合酶没有任何刺激作用。Tf1 整合酶的突变显示，完整的 Tf1 整合酶蛋白（不包括其染色质域）是刺激 Tf1 RT 引物去除活性所必需的。尽管如此，已经失去整合功能的双突变整合酶仍然可以刺激 RT 的活性，尽管失活的整合酶不能增强引物去除。这些发现表明，Tf1 整合酶的酶活性对于刺激 RT 介导的引物去除不是必需的，而该蛋白的正确折叠对于该功能是必需的。这些结果突出了 Tf1 整合酶在反转录转座子逆转录过程中可能具有新的功能。
