HIV-1 Leader Mutations During RT Inhibitor Therapy

项目摘要 HIV-1 356 bp 5'非翻译前导序列含有多种生物学功能，包括HIV-1基因表达所需的元件。 启动前病毒DNA转录、病毒RNA逆转录、病毒剪接、病毒二聚化和病毒 包装.特别是逆转录起始是HIV-1复制的主要瓶颈， 被用于抗逆转录病毒（ARV）的开发。斯坦福大学的研究人员，包括一位合作者， 该提议的研究者已经公布了逆转录酶（RT）起始的冷冻-EM结构 包含HIV-1 RT、tRNA 3Lys和包含5 ′-前导序列的101个核苷酸的病毒RNA片段的复合物 位置123到223。结构显示tRNA 3Lys重折叠并堆叠在病毒RNA引物结合位点上 以在RT裂缝中形成双链螺旋结构。病毒RNA-tRNA复合物的庞大 将RT酶转化为必须导航高度结构化的5 ′-前导序列的次优活性构象。 确定5 '-前导区是否处于RT抑制剂选择性药物压力下， 治疗HIV-1和HIV-2，以及完善我们对逆转录酶抑制剂耐药性的遗传障碍的理解。 然而，还没有发表的研究配对序列的患者之前和之后的抗逆转录病毒治疗。 我们推测，在接受RT抑制剂的患者中，HIV-1可能需要发展5 '前导序列， 突变以适应已知核苷RT抑制剂（NRTI）或非核苷RT抑制剂的作用 （NNRTI）-耐药突变。我们已经对配对的DNA序列的5 '-前导序列位置47至356进行了桑格测序。 来自11名患者的病毒样本在发生胞嘧啶类似物耐药突变M184 V之前和之后 和/或胸苷类似物抗性突变T215 Y。我们在11个测序的病毒中的7个中鉴定出22个突变 在跨越位置123至223的14个核苷酸处成对。这些突变不是随机分布的： 发生在位置200和201，多态性核苷酸邻近引物结合位点和七个 发生在核苷酸207至216之间的多态环中。 关键NRTI和NNRTI耐药性产生前后其他成对病毒的序列 需要突变来确认RT和5 '-前导突变之间的关联。我们计划执行Illumina 对105个配对样品的包含位置47至356的5 '-前导序列进行下一代测序（NGS） 包括（i）20个开发M184 V/I;（ii）15个开发T215 Y/F;（iii）20个开发K65 R、L74 V/I、K70 Q/E/N/T， 和Q151 M;（iv）20例发生NNRTI耐药突变;和（v）30例未治疗的对照患者， 不发生RT PCR抗性突变的病毒。NGS将使识别共变的5 '-前导序列成为可能 通过桑格测序，用电泳混合物在位置处检测核苷酸，并获得高质量的序列 数据，即使当病毒准种显示长度异质性。如果这项研究表明，5 '-leader 在选择性RT抑制剂的压力下，这将影响RT抑制剂的开发方式以及它们的 确定了耐药性的遗传障碍。

PROJECT SUMMARY The HIV-1 356 bp 5’ untranslated leader contains multiple biological functions including elements required for initiating proviral DNA transcription, viral RNA reverse transcription, viral splicing, viral dimerization, and viral packaging. Reverse transcription initiation, in particular, is a major bottleneck to HIV-1 replication that has not been exploited for antiretroviral (ARV) development. Researchers at Stanford University, including a co- investigator of this proposal have published a cryo-EM structure of the reverse transcriptase (RT) initiation complex comprising HIV-1 RT, tRNA3Lys, and a 101-nucleotide viral RNA fragment encompassing 5’-leader positions 123 to 223. The structure shows that tRNA3Lys refolds and stacks onto the viral RNA primer binding site to form a double-stranded helical structure in the RT cleft. The bulkiness of the viral RNA-tRNA complex forces the RT enzyme into a suboptimally active conformation that must navigate the highly structured 5’-leader. Determining whether the 5’-leader region is under RT inhibitor selective drug pressure has implications for treating HIV-1 and HIV-2, and for refining our understanding of the genetic barriers to RT inhibitor resistance. However, there have been no published studies of paired sequences from patients before and after ARV therapy. We hypothesize that in patients receiving RT inhibitors, it may be necessary for HIV-1 to develop 5’-leader mutations to accommodate the effects of known nucleoside RT inhibitor (NRTI)- or nonnucleoside RT inhibitor (NNRTI)-resistance mutations. We have performed Sanger sequencing of 5’-leader positions 47 to 356 of paired virus samples from 11 patients before and after developing the cytosine analog resistance mutation M184V and/or the thymidine analog resistance mutation T215Y. We identified 22 mutations in 7 of 11 sequenced virus pairs at 14 nucleotides spanning positions 123 to 223. These mutations were not distributed randomly: seven occurred at positions 200 and 201, polymorphic nucleotides adjacent to the primer binding site and seven occurred in a polymorphic loop between nucleotides 207 to 216. Sequences of additional paired viruses before and after the development of key NRTI- and NNRTI-resistance mutations are needed to confirm an association between RT and 5’-leader mutations. We plan to perform Illumina next-generation sequencing (NGS) of the 5’-leader encompassing positions 47 to 356 for 105 paired samples including (i) 20 developing M184V/I; (ii) 15 developing T215Y/F; (iii) 20 developing K65R, L74V/I, K70Q/E/N/T, and Q151M; (iv) 20 developing an NNRTI-resistance mutation; and (v) 30 untreated control patients with paired viruses not developing RT inhibitor-resistance mutations. NGS will make it possible to identify covarying 5’-leader nucleotides at positions with electrophoretic mixtures by Sanger sequencing and to obtain high quality sequence data even when viral quasispecies display length heterogeneity. Should this study demonstrate that the 5’-leader is under selective RT inhibitor pressure, this would influence how RT inhibitors are developed and how their genetic barriers to drug resistance are defined.