Li Y, Kang S M, Morrow C D
Department of Microbiology, University of Alabama at Birmingham 35294, USA.
AIDS Res Hum Retroviruses. 1997 Feb 10;13(3):253-62. doi: 10.1089/aid.1997.13.253.
The initiation of human immunodeficiency virus type 1 (HIV-1) reverse transcription occurs by the extension of a tRNALys,3 positioned at an 18-nucleotide sequence in the RNA genome referred to as the primer-binding site (PBS). We have found that mutations within the PBS and a region upstream in U5, designated the A loop, influenced the selection of the tRNA primer used to initiate reverse transcription. Surprisingly, a proviral genome that contained a PBS and A loop complementary to tRNAPro resulted in the generation of viruses that contained two PBSs within the same genome: one of the PBSs in the virus was complementary to tRNALys,3 while the second PBS was complementary to tRNAIle, tRNAPro, or tRNALys,3. There were 14 nucleotides separating the two PBSs in the viral genome. In the current study, DNA encompassing U5 and the dual PBS complementary to the different tRNAs were amplified by PCR and exchanged for the corresponding region in an infectious HIV-1 clone, HXB2. Transfection of the different proviruses into cells resulted in the production of viruses that were infectious as determined by coculture with SupT1 cells. PCR was used to amplify the PBS regions from the different proviral DNAs followed by DNA sequencing of individual PCR clones. Proviruses containing the dual PBS complementary to tRNALys,3 and tRNAIle stably maintained the dual PBS complementary to both of these tRNAs following in vitro culture, although we noted consistent G-to-T and AA-to-GG substitutions in the 14-nucleotide region between the PBSs. The viruses derived from genomes that contained the dual PBS complementary to tRNALys,3 and tRNAPro also maintained both PBSs following in vitro culture; a single mutation was noted after in vitro culture in the 14-nucleotide region between the PBSs, which changed a consensus integration site (CA dinucleotide) prior to the PBS complementary to tRNAPro. In contrast, the proviral genomes containing the dual PBS complementary to tRNALys,3 were not stable and reverted back to a single PBS complementary to tRNALys,3. The results of our studies suggest that only the 5'-proximal PBS has been used to initiate reverse transcription. On the basis of our results, a mechanism is proposed for the generation of a dual PBS, which provides new insights into HIV-1 reverse transcription.
1型人类免疫缺陷病毒（HIV-1）逆转录的起始是通过位于RNA基因组中一个18核苷酸序列（称为引物结合位点，PBS）处的tRNALys,3的延伸来实现的。我们发现，PBS内以及U5上游一个称为A环的区域内的突变会影响用于起始逆转录的tRNA引物的选择。令人惊讶的是，一个包含与tRNAPro互补的PBS和A环的前病毒基因组导致产生了在同一基因组内含有两个PBS的病毒：病毒中的一个PBS与tRNALys,3互补，而第二个PBS与tRNAIle、tRNAPro或tRNALys,3互补。在病毒基因组中，两个PBS之间相隔14个核苷酸。在当前研究中，通过PCR扩增包含U5和与不同tRNA互补的双PBS的DNA，并将其与感染性HIV-1克隆HXB2中的相应区域进行交换。将不同的前病毒转染到细胞中，通过与SupT1细胞共培养确定产生了具有感染性的病毒。使用PCR从不同的前病毒DNA中扩增PBS区域，随后对单个PCR克隆进行DNA测序。含有与tRNALys,3和tRNAIle互补的双PBS的前病毒在体外培养后稳定地维持了与这两种tRNA互补的双PBS，尽管我们注意到在PBS之间的14核苷酸区域存在一致的G到T以及AA到GG的替换。源自包含与tRNALys,3和tRNAPro互补的双PBS的基因组的病毒在体外培养后也维持了两个PBS；在体外培养后，在PBS之间的14核苷酸区域发现了一个单一突变，该突变改变了与tRNAPro互补的PBS之前的一个共有整合位点（CA二核苷酸）。相比之下，包含与tRNALys,3互补的双PBS的前病毒基因组不稳定，会回复到与tRNALys,3互补的单一PBS。我们的研究结果表明，只有5'-近端PBS被用于起始逆转录。基于我们的结果，提出了一种双PBS产生的机制，这为HIV-1逆转录提供了新的见解。
