Ni Na, Morrow Casey D
Department of Cell Biology, University of Alabama at Birmingham, 802 Kaul Building, 720 20th Street South, Birmingham, AL 35294-0024, USA.
Virus Res. 2007 Mar;124(1-2):29-37. doi: 10.1016/j.virusres.2006.09.009. Epub 2006 Oct 30.
Human immunodeficiency virus (HIV-1) exclusively selects tRNA(Lys,3) as the primer for initiation of reverse transcription. How and why HIV-1 selects the tRNA is unresolved. To address this issue, we have generated HIV-1 in which the PBS was changed to be complementary to alternative tRNAs. In this study, we report on HIV-1 that have the PBS mutated to be complementary to tRNA(Thr), tRNA(Phe), tRNA(Ser) and tRNA(Tyr). Virus with a PBS complementary to tRNA(Thr) grew slightly slower than the wild type virus and maintained the PBS for an extended culture period before finally reverting back to utilize tRNA(Lys,3). In contrast, viruses with a PBS complementary to tRNA(Phe) or tRNA(Ser) rapidly reverted to utilize tRNA(Lys,3) following limited in vitro replication, while a virus with a PBS complementary to tRNA(Tyr) had severely compromised infectivity and did not productively infect a continuous T cell line (SupT1) or human peripheral blood mononuclear cells (PBMC). Modification of the A-loop region to be complementary to tRNA(Thr) with the mutation in the PBS to be complementary to tRNA(Thr) resulted in a virus that could stably utilize this tRNA while the modification of the A-loop to be complementary to the anticodon of tRNA(Ser) did not allow the virus to stably utilize tRNA(Ser). Modification of the A-loop region to be complementary to the anticodon of tRNA(Phe) severely impacted the replication of this virus. Finally, the modification of the A-loop region to be complementary to tRNA(Tyr) did not rescue the virus with a PBS complementary to tRNA(Tyr). The results of these studies demonstrate the diverse effects that alteration of the PBS to force selection of alternative primers have on HIV-1 replication and provide a framework to understand the dynamics of primer selection.
人类免疫缺陷病毒1型（HIV-1）专门选择tRNA（Lys，3）作为逆转录起始的引物。HIV-1如何以及为何选择这种tRNA尚未明确。为解决这一问题，我们构建了PBS与其他tRNA互补的HIV-1。在本研究中，我们报道了PBS发生突变以与tRNA（Thr）、tRNA（Phe）、tRNA（Ser）和tRNA（Tyr）互补的HIV-1。PBS与tRNA（Thr）互补的病毒生长速度略慢于野生型病毒，并且在延长的培养期内维持该PBS，最终才恢复使用tRNA（Lys，3）。相反，PBS与tRNA（Phe）或tRNA（Ser）互补的病毒在有限的体外复制后迅速恢复使用tRNA（Lys，3），而PBS与tRNA（Tyr）互补的病毒感染性严重受损，无法有效感染连续T细胞系（SupT1）或人外周血单个核细胞（PBMC）。将A环区域修饰为与tRNA（Thr）互补且PBS突变以与tRNA（Thr）互补，产生了一种能够稳定使用这种tRNA的病毒，而将A环修饰为与tRNA（Ser）的反密码子互补则不允许病毒稳定使用tRNA（Ser）。将A环区域修饰为与tRNA（Phe）的反密码子互补严重影响了该病毒的复制。最后，将A环区域修饰为与tRNA（Tyr）互补并不能挽救PBS与tRNA（Tyr）互补的病毒。这些研究结果表明，改变PBS以强制选择替代引物对HIV-1复制具有多种影响，并为理解引物选择的动态过程提供了一个框架。
