Bordier B, Tarrago-Litvak L, Sallafranque-Andreola M L, Robert D, Tharaud D, Fournier M, Barr P J, Litvak S, Sarih-Cottin L
Institut de Biochimie Cellulaire et Neurochimie du CNRS, Bordeaux, France.
Nucleic Acids Res. 1990 Feb 11;18(3):429-36. doi: 10.1093/nar/18.3.429.
Human immunodeficiency virus (HIV) reverse transcriptase (RT) uses host tRNA(Lys) partially annealed to the primer binding site (PBS) as primer for the initiation of cDNA synthesis. When assaying cDNA synthesis with a template-primer complex formed by an RNA fragment carrying the PBS site and bovine tRNA(Lys) we noticed that an excess of primer tRNA inhibited strongly the DNA polymerase activity of a recombinant HIV RT (p66-p51 heterodimeric form) produced in transformed yeast cells. The same inhibitory effect was observed with animal DNA polymerase alpha, while avian retrovirus RT was neither affected by tRNA(Lys) nor by its specific primer tRNA(Trp). Although the strongest inhibition was observed with tRNA(Lys), other tRNas like tRNA(Phe) and tRNA(Trp) inhibited also the HIV RT, whereas tRNAs specific for valine, proline and glycine had no effect on enzyme activity. Digestion of tRNA(Lys) with pancreatic RNase abolished the inhibition; on the other hand T1 RNase digestion had no effect on the inhibition suggesting a role of the anticodon region in this effect. The 12- and 14-mers corresponding to the anticodon regions of the three bovine tRNA(Lys) isoacceptors inhibited RT activity, indicating that at least an important part of the inhibitory effect could be ascribed to this tRNA region. A strong stimulation of DNA polymerase activity was observed when the effect of tRNA(Lys) was assayed on a recombinant HIV reverse transcriptase produced in a protease deficient yeast strain, which leads to the production of an active p66 enzyme. The same tRNAs that inhibited strongly the heterodimeric form stimulated the p66 form of HIV reverse transcriptase. The results suggest that although both enzymatic forms are able to interact with tRNA(Lys) the topography, as well as the functional implications of the interaction between the precursor and the mature form of HIV reverse transcriptase with the tRNA(Lys) primer, are different.
人类免疫缺陷病毒（HIV）逆转录酶（RT）利用部分退火至引物结合位点（PBS）的宿主tRNA（Lys）作为引物起始cDNA合成。在用携带PBS位点的RNA片段与牛tRNA（Lys）形成的模板 - 引物复合物检测cDNA合成时，我们注意到过量的引物tRNA强烈抑制转化酵母细胞中产生的重组HIV RT（p66 - p51异二聚体形式）的DNA聚合酶活性。动物DNA聚合酶α也观察到相同的抑制作用，而禽逆转录病毒RT既不受tRNA（Lys）影响，也不受其特异性引物tRNA（Trp）影响。尽管tRNA（Lys）的抑制作用最强，但其他tRNA如tRNA（Phe）和tRNA（Trp）也抑制HIV RT，而缬氨酸、脯氨酸和甘氨酸特异性的tRNA对酶活性没有影响。用胰核糖核酸酶消化tRNA（Lys）可消除抑制作用；另一方面，T1核糖核酸酶消化对抑制作用没有影响，这表明反密码子区域在这种作用中发挥作用。对应于三种牛tRNA（Lys）同工受体反密码子区域的12聚体和14聚体抑制RT活性，表明至少抑制作用的一个重要部分可归因于该tRNA区域。在用蛋白酶缺陷酵母菌株产生的重组HIV逆转录酶上检测tRNA（Lys）的作用时，观察到DNA聚合酶活性受到强烈刺激，该酵母菌株可产生有活性的p66酶。强烈抑制异二聚体形式的相同tRNA刺激HIV逆转录酶的p66形式。结果表明，尽管两种酶形式都能够与tRNA（Lys）相互作用，但HIV逆转录酶前体和成熟形式与tRNA（Lys）引物之间相互作用的拓扑结构以及功能意义是不同的。
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