Nouhin Janin, Tzou Philip L, Rhee Soo-Yon, Sahoo Malaya K, Pinsky Benjamin A, Krupkin Miri, Puglisi Joseph D, Puglisi Elisabetta V, Shafer Robert W
Division of Infectious Diseases, Stanford University, Stanford, CA, USA and Virology Unit, Institut Pasteur du Cambodge, Pasteur Network, Phnom Penh, Cambodia.
Division of Infectious Diseases, Stanford University, Stanford, CA, USA.
medRxiv. 2023 Aug 29:2023.06.04.23290942. doi: 10.1101/2023.06.04.23290942.
HIV-1 RT initiation depends on interaction between viral 5'-leader RNA, RT, and host tRNA3. We therefore sought to identify co-evolutionary changes between the 5'-leader and RT in viruses developing RT-inhibitor resistance mutations.
We sequenced 5'-leader positions 37-356 of paired plasma virus samples from 29 individuals developing the NRTI-resistance mutation M184V, 19 developing an NNRTI-resistance mutation, and 32 untreated controls. 5'-leader variants were defined as positions where ≥20% of NGS reads differed from the HXB2 sequence. Emergent mutations were defined as nucleotides undergoing ≥4-fold change in proportion between baseline and follow-up. Mixtures were defined as positions containing ≥2 nucleotides each present in ≥20% of NGS reads.
Among 80 baseline sequences, 87 positions (27.2%) contained a variant; 52 contained a mixture. Position 201 was the only position more likely to develop a mutation in the M184V (9/29 vs. 0/32; p=0.0006) or NNRTI-resistance (4/19 vs. 0/32; p=0.02; Fisher's Exact Test) groups than the control group. Mixtures at positions 200 and 201 occurred in 45.0% and 28.8%, respectively, of baseline samples. Because of the high proportion of mixtures at these positions, we analyzed 5'-leader mixture frequencies in two additional datasets: five publications reporting 294 dideoxyterminator clonal GenBank sequences from 42 individuals and six NCBI BioProjects reporting NGS datasets from 295 individuals. These analyses demonstrated position 200 and 201 mixtures at proportions similar to those in our samples and at frequencies several times higher than at all other 5'-leader positions.
Although we did not convincingly document co-evolutionary changes between RT and 5'-leader sequences, we identified a novel phenomenon, wherein positions 200 and 201, immediately downstream of the HIV-1 primer binding site exhibited an extraordinarily high likelihood of containing a nucleotide mixture. Possible explanations for the high mixture rates are that these positions are particularly error-prone or provide a viral fitness advantage.
HIV-1逆转录酶（RT）的起始依赖于病毒5'-前导RNA、RT和宿主tRNA3之间的相互作用。因此，我们试图确定在出现RT抑制剂耐药性突变的病毒中，5'-前导序列和RT之间的共同进化变化。
我们对来自29名出现核苷类逆转录酶抑制剂（NRTI）耐药性突变M184V的个体、19名出现非核苷类逆转录酶抑制剂（NNRTI）耐药性突变的个体以及32名未接受治疗的对照者的配对血浆病毒样本的5'-前导序列37-356位进行了测序。5'-前导序列变异定义为下一代测序（NGS）读数中≥20%与HXB2序列不同的位置。新发突变定义为在基线和随访之间比例变化≥4倍的核苷酸。混合物定义为在NGS读数中≥20%的读数中各自存在≥2种核苷酸的位置。
在80个基线序列中，87个位置（27.2%）含有变异；52个位置含有混合物。201位是在M184V组（9/29 vs. 0/32；p=0.0006）或NNRTI耐药组（4/19 vs. 0/32；p=0.02；Fisher精确检验）中比对照组更有可能发生突变的唯一位置。200位和201位的混合物分别出现在45.0%和28.8%的基线样本中。由于这些位置混合物的比例很高，我们在另外两个数据集中分析了5'-前导序列混合物频率：五篇报告来自42名个体的294个双脱氧终止克隆GenBank序列的出版物，以及六篇报告来自295名个体的NGS数据集的NCBI生物项目。这些分析表明，200位和201位的混合物比例与我们样本中的比例相似，且频率比所有其他5'-前导序列位置高几倍。
虽然我们没有令人信服地证明RT和5'-前导序列之间的共同进化变化，但我们发现了一种新现象，即HIV-1引物结合位点下游紧邻的200位和201位含有核苷酸混合物的可能性极高。混合物比例高的可能解释是这些位置特别容易出错或提供了病毒适应性优势。
