Ni Na, Xu Wenqin, Morrow Casey D
Department of Cell Biology, University of Alabama at Birmingham, Birmingham, AL 35294-0024, USA.
Virol J. 2007 Jan 10;4:4. doi: 10.1186/1743-422X-4-4.
Human immunodeficiency virus (HIV-1) preferentially selects tRNALys,3 as the primer for reverse transcription. HIV-1 can be forced to select alternative tRNAs through mutation in the primer-binding site (PBS) and a region upstream of the PBS designated as the A-loop. Alteration of the PBS and A-loop to be complementary to the 3' terminal nucleotides and anticodon of tRNAHis results in HIV-1 that can stably utilize this tRNA for replication.
In the current study, we have investigated the effect that mutations within the A-loop have on the stability of HIV-1 with a PBS complementary to tRNAHis. For these studies, we have altered the A-loop to be complementary to tRNAMet, tRNAGln, tRNAIle, tRNAThr and tRNASer. All substitutions of the A-loops with the PBS complementary to tRNAHis resulted in a reduction of infectious virus obtained following transfection of proviral genomes in the 293T cells. Virus replication in SupT1 cells was also impaired as a result of the alteration of the A-loop. Viruses with the A-loop complementary to tRNALys,3 and tRNASer reverted to utilize tRNALys,3 following in vitro replication. In contrast, viruses with the A-loop complementary to the other tRNAs remained stable and continued to use tRNAHis. RNA modeling of the stem-loop structure revealed that nucleotides were displayed on the loop region that could potentially interact with the anticodon of tRNAHis. To further explore the effects of the A-loop mutations on virus replication, the A-loops complementary to tRNASer or tRNAHis were cloned into the wild type genome with the PBS complementary to tRNALys,3. Transfection of proviral genomes which contained the wild type PBS and A-loops complementary to tRNASer or tRNAHis into 293 T cells did not impact on the production of viruses as measured by p24 antigen ELISA. However, viruses with the A-loop complementary to tRNAHis had greatly reduced infectivity and replicated poorly in SupT1 compared to the wild type or viruses with the A-loop complementary to tRNASer.
These studies demonstrate that complementarity of A-loop region with the anticodon of tRNAHis has a pronounced effect on the capacity of HIV-1 to utilize tRNAHis as the primer for reverse transcription. Complementarity between A-loop and anticodon of the tRNA then is important for the selection of the tRNA primer used for reverse transcription.
人类免疫缺陷病毒1型（HIV-1）优先选择tRNALys,3作为逆转录引物。通过在引物结合位点（PBS）以及PBS上游一个称为A环的区域进行突变，可以迫使HIV-1选择其他tRNA。将PBS和A环改变为与tRNAHis的3'末端核苷酸和反密码子互补，会产生能够稳定利用该tRNA进行复制的HIV-1。
在本研究中，我们调查了A环内的突变对具有与tRNAHis互补的PBS的HIV-1稳定性的影响。对于这些研究，我们将A环改变为与tRNAMet、tRNAGln、tRNAIle、tRNAThr和tRNASer互补。所有将A环替换为与tRNAHis互补的PBS的操作，都导致在293T细胞中转染前病毒基因组后获得的感染性病毒减少。由于A环的改变，SupT1细胞中的病毒复制也受到损害。具有与tRNALys,3和tRNASer互补的A环的病毒在体外复制后恢复为利用tRNALys,3。相比之下，具有与其他tRNA互补的A环的病毒保持稳定并继续使用tRNAHis。茎环结构的RNA建模显示，环区域上展示的核苷酸可能与tRNAHis的反密码子相互作用。为了进一步探索A环突变对病毒复制的影响，将与tRNASer或tRNAHis互补的A环克隆到具有与tRNALys,3互补的PBS的野生型基因组中。通过p24抗原ELISA检测，将含有野生型PBS和与tRNASer或tRNAHis互补的A环的前病毒基因组转染到293T细胞中，对病毒产生没有影响。然而，与野生型或具有与tRNASer互补的A环的病毒相比，具有与tRNAHis互补的A环的病毒感染性大大降低，并且在SupT1中的复制较差。
这些研究表明，A环区域与tRNAHis反密码子的互补性对HIV-1利用tRNAHis作为逆转录引物的能力有显著影响。因此，A环与tRNA反密码子之间的互补性对于选择用于逆转录的tRNA引物很重要。
