Mutations in both the U5 region and the primer-binding site influence the selection of the tRNA used for the initiation of HIV-1 reverse transcription.
Mutations in both the U5 region and the primer-binding site influence the selection of the tRNA used for the initiation of HIV-1 reverse transcription.
复制标题
U5 区域和引物结合位点的突变都会影响用于启动 HIV-1 逆转录的 tRNA 的选择。
DOI：
10.1006/viro.1996.0437
复制
发表时间：
1996
期刊：
影响因子：
--
通讯作者：
Morrow,CD
中科院分区：
文献类型：
--
作者：
Kang,SM;Wakefield,JK;Morrow,CD
HIV-1逆转录的起始是由细胞tRNALys，3分子引发的，该分子与病毒RNA基因组5 '端附近的互补序列结合，被指定为引物结合位点（PBS）。最近的研究表明，U5内PBS上游由一段腺嘌呤核苷酸（称为a环）组成的序列可能对tRNALys的选择和定位很重要，tRNALys是用于启动逆转录的引物。为了进一步探索a环在逆转录中的作用，我们构建了原病毒基因组，其中PBS被改变以与tRNAIle、tRNAPro或tRNATrp的3 '端18个核苷酸互补[分别为pHXB（Ile）、pHXB（Pro）或pHXB(Trp)]；构建了包含额外突变的第二组原病毒基因组，使a环区域与tRNAIle[pHXB(Ile-AC)]、tRNAPro[pHXB(Pro-AC)]或tRNATrp[pHXB(Trp-AC)]的反密码子区域互补。将原病毒转染到COS-1细胞中，然后与SupT1细胞共培养，产生感染性病毒。用PCR扩增PBS片段，亚克隆到M13mp18上，进行DNA序列分析。短期培养后，pHXB（Ile）、pHXB（Pro）和pHXB（Trp）衍生的原病毒的PBSs恢复为tRNALys的互补，3。从pHXB衍生的病毒（Ile-AC）的PBSs也恢复为与tRNALys互补，3；A-loop区域仍与tRNAIle互补。相比之下，转染pHXB（Pro-AC）衍生的病毒最初维持与tRNAPro互补的PBS。通过扩展培养，我们鉴定出含有PBSs的原病毒，这些PBSs与另外两个trna互补：trnaile和tRNALys，3。此外，我们发现在同一基因组中含有两个PBSs的原病毒：一个与tRNALys，3互补，另一个与tRNAProor tRNAIle互补。转染pHXB衍生的病毒（Trp-AC）在转染后外观延迟最多。PBS分析显示，转染后早期，大多数PBS与tRNATrp互补。在进一步的体外培养后，用与新tRNA tRNAMet互补的PBS鉴定原病毒。最后，经过长时间培养，转染pHXB（il - ac）、pHXB（Pro-AC）和pHXB（Trp-AC）的病毒在U5中含有来自PBS上游的突变，产生了一段3个腺嘌呤核苷酸。这些研究的结果强调了在选择用于启动HIV-1逆转录的tRNA引物方面存在的灵活性。
The initiation of HIV-1 reverse transcription is primed by a cellular tRNALys,3molecule which is bound to a complementary sequence near the 5′ end of the viral RNA genome designated as the primer-binding site (PBS). Recent studies have suggested that sequences upstream of the PBS within U5 consisting of a stretch of adenine nucleotides (referred to as the A-loop) might be important in the selection and positioning of tRNALys,3primer used to initiate reverse transcription. To further explore the role that the A-loop plays in reverse transcription, we have constructed proviral genomes in which the PBS was changed so as to be complementary to the 3′-terminal 18 nucleotides of tRNAIle, tRNAPro, or tRNATrp[pHXB(Ile), pHXB(Pro), or pHXB(Trp), respectively]; a second set of proviral genomes was constructed which contained additional mutations so that the A-loop regions were complementary to the anticodon region of tRNAIle[pHXB(Ile-AC)], tRNAPro[pHXB(Pro-AC)], or tRNATrp[pHXB(Trp-AC)]. Transfection of the proviruses into COS-1 cells followed by coculture with SupT1 cells resulted in production of infectious virus. PCR was used to amplify the PBS regions which were subcloned into M13mp18 followed by DNA sequence analysis. After short-term culture, the PBSs of proviruses derived from pHXB(Ile), pHXB(Pro), and pHXB(Trp) reverted to be complementary to tRNALys,3. The PBSs of the viruses derived from pHXB(Ile-AC) also reverted to be complementary to tRNALys,3; the A-loop region was still complementary to tRNAIle. In contrast, viruses derived from transfection of pHXB(Pro-AC) initially maintained a PBS complementary to tRNAPro. Upon extended culture, we identified proviruses which contained PBSs complementary to two additional tRNAs: tRNAIleand tRNALys,3. Furthermore, we found proviruses which contain two PBSs within the same genome: one complementary to tRNALys,3and a second complementary to tRNAProor tRNAIle. Viruses derived from transfection of pHXB(Trp-AC) were the most delayed in appearance following transfection. Analysis of the PBS revealed that early after transfection, the majority of the PBSs were complementary to tRNATrp. After furtherin vitroculture, proviruses were identified with a PBS complementary to a new tRNA, tRNAMet. Finally, upon extended culture, the viruses derived from the transfection of pHXB(Ile-AC), pHXB(Pro-AC), and pHXB(Trp-AC) contained mutations upstream from the PBS in U5 that created a stretch of 3 adenine nucleotides. The results of these studies then highlight the flexibility that exists with respect to the selection of the tRNA primer used to initiate HIV-1 reverse transcription.