Javier Martínez del Río , Estrella Frutos-Beltrán , Alba Sebastián-Martín , Fátima Lasala , Kiyoshi Yasukawa , Rafael Delgado , Luis Menéndez-Arias
{"title":"HIV-1 逆转录酶错误率和转录阈值,基于对来自体外转录细胞和 HIV 感染细胞的目标 RNA 的单链共识测序。","authors":"Javier Martínez del Río , Estrella Frutos-Beltrán , Alba Sebastián-Martín , Fátima Lasala , Kiyoshi Yasukawa , Rafael Delgado , Luis Menéndez-Arias","doi":"10.1016/j.jmb.2024.168815","DOIUrl":null,"url":null,"abstract":"<div><div>Nucleotide incorporation and <em>lacZ</em>-based forward mutation assays have been widely used to determine the accuracy of reverse transcriptases (RTs) in RNA-dependent DNA polymerization reactions. However, they involve quite complex and laborious procedures, and cannot provide accurate error rates. Recently, NGS-based methods using barcodes opened the possibility of detecting all errors introduced by the RT, although their widespread use is limited by cost, due to the large size of libraries to be sequenced. In this study, we describe a novel and relatively simple NGS assay based on single-strand consensus sequencing that provides robust results with a relatively small number of raw sequences (around 60 Mb). The method has been validated by determining the error rate of HIV-1 (BH10 strain) RT using the HIV-1 protease-coding sequence as target. HIV-1 reverse transcription error rates in standard conditions (37 °C/3 mM Mg<sup>2+</sup>) using an <em>in vitro</em>-transcribed RNA were around 7.3 × 10<sup>−5</sup>. In agreement with previous reports, an 8-fold increase in RT’s accuracy was observed after reducing Mg<sup>2+</sup> concentration to 0.5 mM. The fidelity of HIV-1 RT was also higher at 50 °C than at 37 °C (error rate 1.5 × 10<sup>−5</sup>). Interestingly, error rates obtained with HIV-1 RNA from infected cells as template of the reverse transcription at 3 mM Mg<sup>2+</sup> (7.4 × 10<sup>−5</sup>) were similar to those determined with the <em>in vitro</em>-transcribed RNA, and were reduced to 1.8 × 10<sup>−5</sup> in the presence of 0.5 mM Mg<sup>2+</sup>. Values obtained at low magnesium concentrations were modestly higher than the transcription error rates calculated for human cells, thereby suggesting a realistic transcriptional threshold for our NGS-based error rate determinations.</div></div>","PeriodicalId":369,"journal":{"name":"Journal of Molecular Biology","volume":null,"pages":null},"PeriodicalIF":4.7000,"publicationDate":"2024-10-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"HIV-1 Reverse Transcriptase Error Rates and Transcriptional Thresholds Based on Single-strand Consensus Sequencing of Target RNA Derived From In Vitro-transcription and HIV-infected Cells\",\"authors\":\"Javier Martínez del Río , Estrella Frutos-Beltrán , Alba Sebastián-Martín , Fátima Lasala , Kiyoshi Yasukawa , Rafael Delgado , Luis Menéndez-Arias\",\"doi\":\"10.1016/j.jmb.2024.168815\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<div><div>Nucleotide incorporation and <em>lacZ</em>-based forward mutation assays have been widely used to determine the accuracy of reverse transcriptases (RTs) in RNA-dependent DNA polymerization reactions. However, they involve quite complex and laborious procedures, and cannot provide accurate error rates. Recently, NGS-based methods using barcodes opened the possibility of detecting all errors introduced by the RT, although their widespread use is limited by cost, due to the large size of libraries to be sequenced. In this study, we describe a novel and relatively simple NGS assay based on single-strand consensus sequencing that provides robust results with a relatively small number of raw sequences (around 60 Mb). The method has been validated by determining the error rate of HIV-1 (BH10 strain) RT using the HIV-1 protease-coding sequence as target. HIV-1 reverse transcription error rates in standard conditions (37 °C/3 mM Mg<sup>2+</sup>) using an <em>in vitro</em>-transcribed RNA were around 7.3 × 10<sup>−5</sup>. In agreement with previous reports, an 8-fold increase in RT’s accuracy was observed after reducing Mg<sup>2+</sup> concentration to 0.5 mM. The fidelity of HIV-1 RT was also higher at 50 °C than at 37 °C (error rate 1.5 × 10<sup>−5</sup>). Interestingly, error rates obtained with HIV-1 RNA from infected cells as template of the reverse transcription at 3 mM Mg<sup>2+</sup> (7.4 × 10<sup>−5</sup>) were similar to those determined with the <em>in vitro</em>-transcribed RNA, and were reduced to 1.8 × 10<sup>−5</sup> in the presence of 0.5 mM Mg<sup>2+</sup>. Values obtained at low magnesium concentrations were modestly higher than the transcription error rates calculated for human cells, thereby suggesting a realistic transcriptional threshold for our NGS-based error rate determinations.</div></div>\",\"PeriodicalId\":369,\"journal\":{\"name\":\"Journal of Molecular Biology\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":4.7000,\"publicationDate\":\"2024-10-09\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Journal of Molecular Biology\",\"FirstCategoryId\":\"99\",\"ListUrlMain\":\"https://www.sciencedirect.com/science/article/pii/S0022283624004376\",\"RegionNum\":2,\"RegionCategory\":\"生物学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q1\",\"JCRName\":\"BIOCHEMISTRY & MOLECULAR BIOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Journal of Molecular Biology","FirstCategoryId":"99","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S0022283624004376","RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"BIOCHEMISTRY & MOLECULAR BIOLOGY","Score":null,"Total":0}
HIV-1 Reverse Transcriptase Error Rates and Transcriptional Thresholds Based on Single-strand Consensus Sequencing of Target RNA Derived From In Vitro-transcription and HIV-infected Cells
Nucleotide incorporation and lacZ-based forward mutation assays have been widely used to determine the accuracy of reverse transcriptases (RTs) in RNA-dependent DNA polymerization reactions. However, they involve quite complex and laborious procedures, and cannot provide accurate error rates. Recently, NGS-based methods using barcodes opened the possibility of detecting all errors introduced by the RT, although their widespread use is limited by cost, due to the large size of libraries to be sequenced. In this study, we describe a novel and relatively simple NGS assay based on single-strand consensus sequencing that provides robust results with a relatively small number of raw sequences (around 60 Mb). The method has been validated by determining the error rate of HIV-1 (BH10 strain) RT using the HIV-1 protease-coding sequence as target. HIV-1 reverse transcription error rates in standard conditions (37 °C/3 mM Mg2+) using an in vitro-transcribed RNA were around 7.3 × 10−5. In agreement with previous reports, an 8-fold increase in RT’s accuracy was observed after reducing Mg2+ concentration to 0.5 mM. The fidelity of HIV-1 RT was also higher at 50 °C than at 37 °C (error rate 1.5 × 10−5). Interestingly, error rates obtained with HIV-1 RNA from infected cells as template of the reverse transcription at 3 mM Mg2+ (7.4 × 10−5) were similar to those determined with the in vitro-transcribed RNA, and were reduced to 1.8 × 10−5 in the presence of 0.5 mM Mg2+. Values obtained at low magnesium concentrations were modestly higher than the transcription error rates calculated for human cells, thereby suggesting a realistic transcriptional threshold for our NGS-based error rate determinations.
期刊介绍:
Journal of Molecular Biology (JMB) provides high quality, comprehensive and broad coverage in all areas of molecular biology. The journal publishes original scientific research papers that provide mechanistic and functional insights and report a significant advance to the field. The journal encourages the submission of multidisciplinary studies that use complementary experimental and computational approaches to address challenging biological questions.
Research areas include but are not limited to: Biomolecular interactions, signaling networks, systems biology; Cell cycle, cell growth, cell differentiation; Cell death, autophagy; Cell signaling and regulation; Chemical biology; Computational biology, in combination with experimental studies; DNA replication, repair, and recombination; Development, regenerative biology, mechanistic and functional studies of stem cells; Epigenetics, chromatin structure and function; Gene expression; Membrane processes, cell surface proteins and cell-cell interactions; Methodological advances, both experimental and theoretical, including databases; Microbiology, virology, and interactions with the host or environment; Microbiota mechanistic and functional studies; Nuclear organization; Post-translational modifications, proteomics; Processing and function of biologically important macromolecules and complexes; Molecular basis of disease; RNA processing, structure and functions of non-coding RNAs, transcription; Sorting, spatiotemporal organization, trafficking; Structural biology; Synthetic biology; Translation, protein folding, chaperones, protein degradation and quality control.