Apolloni Ann, Meredith Luke W, Suhrbier Andreas, Kiernan Rosemary, Harrich David
Division of Immunology and Infectious Disease, Queensland Institute of Medical Research, Brisbane, Australia.
Curr HIV Res. 2007 Sep;5(5):473-83. doi: 10.2174/157016207781662443.
The role of Tat in HIV-1 reverse transcription has been controversial largely because different studies have observed disparate effects of the Tat protein on reverse transcription. Studies of HIV-1 lacking a functional tat gene demonstrated a decrease in reverse transcription efficiency following infection of T-cells however, in vitro recombinant Tat(1-86) has been shown to inhibit RT activity. Here we show that 20-200 nM of both N-terminally histidine-tagged recombinant Tat(1-72) and Tat(1-86) stimulated reverse transcription by HIV-1 reverse transcriptase (RT) in vitro by 2-3 fold. However, both Tat species were efficient inhibitors of RT activity at 400 nM. The lower concentrations of Tat increased reverse transcription efficiency by facilitating multiple rounds of DNA synthesis, and this increase was either not seen or reduced when Tat proteins with multiply-mutated cysteine or basic domains were used. Tat-enhanced reverse transcription occurred in a RNA-independent manner, and required formation of a Tat-RT complex. Pull-down and immunoprecipitation experiments confirmed that Tat could interact with the RT p51 subunit, and mammalian two-hybrid experiments showed interaction between Tat and both the p51 and p66 subunits. Together these results provide evidence that Tat can stimulate reverse transcription through an interaction with RT.
Tat在HIV-1逆转录过程中的作用一直存在争议，主要原因是不同研究观察到Tat蛋白对逆转录有不同的影响。对缺乏功能性tat基因的HIV-1进行的研究表明，感染T细胞后逆转录效率降低；然而，体外重组Tat(1-86)已被证明可抑制RT活性。在此我们表明，20 - 200 nM的N端带组氨酸标签的重组Tat(1-72)和Tat(1-86)在体外可使HIV-1逆转录酶（RT）的逆转录活性提高2 - 3倍。然而，两种Tat在400 nM时均为RT活性的有效抑制剂。较低浓度的Tat通过促进多轮DNA合成提高了逆转录效率，而当使用具有多个突变半胱氨酸或碱性结构域的Tat蛋白时，这种提高要么未出现，要么有所降低。Tat增强的逆转录以不依赖RNA的方式发生，并且需要形成Tat-RT复合物。下拉实验和免疫沉淀实验证实Tat可与RT p51亚基相互作用，哺乳动物双杂交实验表明Tat与p51和p66亚基均有相互作用。这些结果共同提供了证据，表明Tat可通过与RT相互作用来刺激逆转录。
