限制性内切酶克隆方法，以评估<em>在体外</em> HIV-1 C亚型的Gag-MJ4嵌合病毒的复制能力
概要
HIV-1 pathogenesis is defined by both viral characteristics and host genetic factors. Here we describe a robust method that allows for reproducible measurements to assess the impact of the gag gene sequence variation on the in vitro replication capacity of the virus.
Abstract
The protective effect of many HLA class I alleles on HIV-1 pathogenesis and disease progression is, in part, attributed to their ability to target conserved portions of the HIV-1 genome that escape with difficulty. Sequence changes attributed to cellular immune pressure arise across the genome during infection, and if found within conserved regions of the genome such as Gag, can affect the ability of the virus to replicate in vitro. Transmission of HLA-linked polymorphisms in Gag to HLA-mismatched recipients has been associated with reduced set point viral loads. We hypothesized this may be due to a reduced replication capacity of the virus. Here we present a novel method for assessing the in vitro replication of HIV-1 as influenced by the gag gene isolated from acute time points from subtype C infected Zambians. This method uses restriction enzyme based cloning to insert the gag gene into a common subtype C HIV-1 proviral backbone, MJ4. This makes it more appropriate to the study of subtype C sequences than previous recombination based methods that have assessed the in vitro replication of chronically derived gag-pro sequences. Nevertheless, the protocol could be readily modified for studies of viruses from other subtypes. Moreover, this protocol details a robust and reproducible method for assessing the replication capacity of the Gag-MJ4 chimeric viruses on a CEM-based T cell line. This method was utilized for the study of Gag-MJ4 chimeric viruses derived from 149 subtype C acutely infected Zambians, and has allowed for the identification of residues in Gag that affect replication. More importantly, the implementation of this technique has facilitated a deeper understanding of how viral replication defines parameters of early HIV-1 pathogenesis such as set point viral load and longitudinal CD4+ T cell decline.
Introduction
确定主机和影响HIV-1发病机理和疾病进展是至关重要的合理的疫苗设计的病毒特征。细胞免疫应答是HIV-1感染人体免疫反应的重要组成部分。细胞毒性T淋巴细胞（CTL）所必需的急性病毒血症的初始控制，并允许主机建立一个稳定的状态（设定点）的病毒载量1,2。这些效应细胞的实验耗尽导致的病毒控制3,4的损失。尽管这样，逃避突变发生在病毒基因组的颠覆CTL识别病毒感染的细胞5-9的范围内。
某些HLA等位基因已低病毒载量和较慢的疾病进展，包括B * 57，B * 27和B * 81 10-15关联。的HLA I类等位基因的保护益处部分可以归因于它们的目标基因组的功能约束区域，例如Gag的事实并选择该减少病毒在体外 16-21复制的能力逃逸突变。虽然从细胞免疫系统逃逸，是在选择中的HLA I类等位基因的上下文中的病毒有益的，这些突变的影响可以具有用于发送时主机差动后果与HLA-错配的个体22,23。因此，了解病毒复制能力传输的HLA相关逃逸突变的影响，以促进我们的早期HIV-1发病机制的理解将是非常重要的。
尽管已经取得了很大的进展，以识别和描述与具体的HLA-I类等位基因24-29相关个人逃逸突变的适应度的缺陷，自然产生的HIV-1毒株的HLA相关基因多态性的独特而复杂的脚印，而产生的人类白细胞抗原可能不同的免疫遗传背景的30介导的免疫压力。在APrevious分析，格普费特等 。结果显示，从88急性感染赞比亚得到的传输加格序列的HLA相关的基因突变的积累与设定点病毒载量的31减少有关。这个建议的有害逃逸突变的传输，特别是在加格，对HLA不匹配的收件人提供临床益处，并且可能是由于减毒病毒复制。展望未来，就必须研究如何复杂的自然产生的菌株中的Gag基因多态性的组合协同工作来定义传播病毒的特性，例如复制能力，以及如何早期复制可能反过来影响HIV-1的临床指标和晚期发病。
布罗克曼等人首次证实GAG-Pro序列的过程中慢性期感染和病毒载量在这两个亚型C和B感染隔离复制能力之间的联系32-35。实验方法在这些研究中提出，虽然适当的审查由慢性感染个体的序列的体外复制能力，有几个技术上的警告和限制，使学习在C亚型HIV-1的复制能力急性感染的个体困难。此方法依赖于种群基于PCR扩增的序列引入B亚型NL4-3原病毒，这是来自部分来自LAV的重组，实验室适于病毒原液36。病毒的产生是完成通过共转染在CEM基于T细胞系37与PCR产物的消化Δ-GAG-亲 NL4-3的DNA。这种方法需要病毒在一段周的向外生长到几个月，这可能歪斜回收的病毒原液的性质有关的病毒准种的体内 ，并因此改变的体外复制能力的测定。这种方法Iš更适合于研究慢性感染的个体，它有效地用于病毒选择具有最高的复制能力，并在那里从大量的慢性感染个体的克隆许多不同的病毒变异体是相当劳力密集的，因此是不可行的。然而，急性感染个体中，有一般一到两个变体存在，并且因此，可以消除通过在体外选择压力偏斜所回收的病毒原液的性质的风险，使得在体外复制能力的更精确的评估。其次，这个方法需要重组C亚型的gag-Pro序列成B亚型来自骨干，并且可以引入骨干不相容偏压入分析。由于这些限制，大量的序列必须以克服引入的任何可能存在的偏差进行分析。
在这里，我们描述了一个另类的实验Appro公司ACH适合学习从C亚型急性感染个体的序列。我们用限制性内切酶克隆策略，引进源自HIV-1 C亚型感染者急性感染时间点进入C亚型前病毒的骨干，MJ4 gag基因。使用MJ4的为其中克隆的gag基因的共同骨架为C亚型的序列的分析至关重要。 MJ4从初级分离38演变而来，因此也不太可能引入偏倚由于骨干网和gag基因的亚型不兼容。另外，使用酶和限制性克隆的方法允许在原病毒构建体直接转染到293T细胞中，并在一个克隆的病毒原液的恢复相同的克隆的gag序列。
下面介绍的方法是一种高通量的方法来评估C亚型的复制能力得出的Gag-MJ4嵌合病毒。转染到293T细胞中是直接的和病毒的回收率只需要三天。 体外复制能力的测定在由布罗克曼等人创建的相同的CEM-CCR5的基于T细胞系37中 ，除了使用所必需的成功复制重要协议的修改的C亚型MJ4嵌合病毒。使用合适的T细胞系，而不是外周血单个核细胞的允许大量的C亚型MJ4-嵌合病毒对以高测定再现性测试。最后，使用放射性标记的逆转录酶测定法对病毒的上清液中定量更具成本比使用市售的p24 ELISA试剂盒有效。这也给了一个更高的动态范围，这是用于检测相同的检测中均不佳，高病毒复制和用于检测菌株之间的细微差别复制很重要。
总之，这里提出的方法允许对从HIV-1 C亚型来源的插科打诨序列的复制能力的深入研究急性赞比亚感染者，并作为书面，也可以扩展到其他学习C亚型病毒感染的人群。观察高度变异在不同的Gag菌株之间的复制能力。此外，我们能够显示发送加格的复制能力和设定点的病毒载量的CD4 +下降之间以及与统计协会在3年的时间39。这样的结果凸显学习如何传播的病毒特征，比如复制能力的重要性，与宿主免疫系统在感染早期，影响发病机制相互作用，将整体开发有效的疫苗干预以及治疗。
Protocol
Representative Results
Discussion
由于长度和这个协议的技术性质，有几个步骤，这对嵌合的Gag-MJ4质粒均构建成功以及对病毒复制的能力的定量的关键。虽然限制性内切酶克隆的策略引进国外GAG基因导入MJ4本协议所列拥有超过以前使用重组为基础的方法具有许多优点，该协议可以是技术上的挑战，如果不采取关键步骤精确。
首先，这是绝对必要的使用已在主管菌株缺乏DCM和大坝的DNA甲基化酶生成MJ4质?…
開示
The authors have nothing to disclose.
Acknowledgements
The investigators thank all the volunteers in Zambia who participated in this study and all the staff at the Zambia Emory HIV Research Project in Lusaka who made this study possible. The investigators would like to thank Jon Allen, Smita Chavan, and Mackenzie Hurlston for technical assistance and sample management. We would also like to thank Dr. Mark Brockman for his discussions and generous donation of the GXR25 cells.
This study was funded by R01 AI64060 and R37 AI51231 (EH) and the International AIDS Vaccine Initiative. This work was made possible in part by the generous support of the American people through the United States Agency for International Development (USAID). The contents are the responsibility of the study authors and do not necessarily reflect the views of USAID or the United States Government. This work also was supported, in part, by the Virology Core at the Emory Center for AIDS Research (Grant P30 AI050409). DC and JP were supported in part by Action Cycling Fellowships. This work was supported in part by the Yerkes National Primate Research Center base grant (2P51RR000165-51). This project was also funded in part by the National Center for Research Resources P51RR165 and is currently supported by the Office of Research Infrastructure Programs/OD P51OD11132.
Materials
| Name of the Reagent | Company | Catalogue number | コメント |
| PCR reagents | |||
| GOF: 5' ATTTGACTAGCGGAGGCTAGAA 3' | IDT DNA | Custom Oligo | 25nmol, standard desalt |
| VifOR: 5' TTCTACGGAGACTCCATGACCC 3' | IDT DNA | Custom Oligo | 25nmol, standard desalt |
| GagInnerF1: 5' AGGCTAGAAGGAGAGAGATG 3' |
IDT DNA | Custom Oligo | 25nmol, standard desalt |
| BclIDegRev2: 5' AGTATTTGATCATAYTGYYTYACTTTR 3' |
IDT DNA | Custom Oligo | 25nmol, standard desalt |
| MJ4For1b: 5' CGAAATCGGCAAAATCCC 3' | IDT DNA | Custom Oligo | 25nmol, standard desalt |
| MJ4Rev: 5' CCCATCTCTCTCCTTCTAGC 3' | IDT DNA | Custom Oligo | 25nmol, standard desalt |
| BclIRev: 5' TCTATAAGTATTTGATCATACTGTCTT 3' | IDT DNA | Custom Oligo | 25nmol, standard desalt |
| GagF2: 5' GGGACATCAAGCAGCCAT 3' | IDT DNA | Custom Oligo | 25nmol, standard desalt |
| For3: 5' CTAGGAAAAAGGGCTGTTGGAAATG 3' | IDT DNA | Custom Oligo | 25nmol, standard desalt |
| GagR6: 5' CTGTATCATCTGCTCCTG 3' | IDT DNA | Custom Oligo | 25nmol, standard desalt |
| Rev3: 5' GACAGGGCTATACATTCTTACTAT 3' | IDT DNA | Custom Oligo | 25nmol, standard desalt |
| Rev1: 5' AATTTTTCCAGCTCCCTGCTTGCCCA 3' | IDT DNA | Custom Oligo | 25nmol, standard desalt |
| CoolRack PCR 96 XT | Biocision | BCS-529 | |
| CoolRack M15 | Biocision | BCS-125 | |
| Nuclease free water | Fisher | SH30538FS | Manufactured by Hyclone |
| QIAamp Viral RNA Mini Kit | Qiagen | 52906 | |
| Simport PCR 8 Strip Tubes, Blue (Flat Cap) | Daigger | EF3647BX | |
| SuperScript III one-step RT-PCR system | Life Technologies/Invitrogen | 12574035 | |
| Phusion Hot-start II DNA polymerase | Fisher | F-549L | |
| PCR Nucleotide Mix | Roche | 4638956001 | |
| Agarose, high gel strength | Fisher | 50-213-128 | |
| TAE 10X | Life Technologies/Invitrogen | AM9869 | |
| Promega 1kb DNA ladder | Fisher | PRG5711 | Manufactured by Promega |
| Sybr Safe DNA Gel Stain, 10000x | Life Technologies/Invitrogen | S33102 | |
| Wizard SV Gel and PCR Clean-Up System | Promega | A9282 | |
| Razor blades, single-edged | Fisher | 12-640 | Manufactured by Surgical Design |
| Thermocycler, PTC-200 | MJ Research | ||
| Microbiology & Cloning reagents | |||
| LB Agar, Miller | Fisher | BP1425-2 | |
| LB Broth, Lennox | Fisher | BP1427-2 | |
| Sterile 100mm x 15mm polystyrene petri dishes | Fisher | 08-757-12 | |
| Ampicillin sodium salt | Sigma-Aldrich | A9518-5G | |
| Falcon 14ml Polypropylene round-bottom tubes | BD Biosciences | 352059 | |
| NgoMIV restriction endonuclease | New England BioLabs | R0564L | |
| BclI restriction endonuclease | New England BioLabs | R0160L | |
| HpaI restriction endonuclease | New England BioLabs | R0105L | |
| T4 DNA Ligase, 5U/μL | Roche | 10799009001 | |
| JM109 competent cells, >10^8 cfu/μg | Promega | L2001 | |
| PureYield plasmid miniprep system | Promega | A1222 | |
| Safe Imager 2.0 Blue Light Transilluminator | Invitrogen | G6600 | |
| Microfuge 18 centrifuge | Beckman Coulter | 367160 | |
| Cell culture reagents | |||
| Amphyl cleaner/disinfectant | Fisher | 22-030-394 | |
| Fugene HD, 1 mL | VWR | PAE2311 | Manufactured by Promega |
| Hexadimethrine bromide (Polybrene) | Sigma-Aldrich | H9268-5G | |
| Costar Plates, 6-well, flat | Fisher | 07-200-83 | Manufactured by Corning Life |
| Costar Plates, 24-well, flat | Fisher | 07-200-84 | Manufactured by Corning Life |
| Costar Plates, 96-well, round | Fisher | 07-200-95 | Manufactured by Corning Life |
| Flasks, Corning filter top/canted neck, 75 cm^2 | Fisher | 10-126-37 | |
| Flasks, Corning filter top/canted neck, 150 cm^2 | Fisher | 10-126-34 | Manufactured by Corning Life |
| Conical Tubes, 50ml, blue cap | Fisher | 14-432-22 | Manufactured by BD Biosciences |
| Conical Tubes, 15ml, blue cap | Fisher | 14-959-70C | Manufactured by BD Biosciences |
| Trypsin-EDTA | Fisher | MT25052CI | Manufactured by Mediatech |
| RPMI, 500 ml | Life Technologies/Invitrogen | 11875-119 | |
| DMEM, 500 ml | Life Technologies/Invitrogen | 11965-118 | |
| Penicillin/Streptomycin/Glutamine, 100X | Life Technologies/Invitrogen | 10378-016 | |
| PBS with magnesium and calcium, 500ml | Life Technologies/Invitrogen | 14040-133 | |
| PBS without magnesium and calcium | Life Technologies/Invitrogen | 20012-050 | |
| Sarstedt tubes, assorted colors | Sarstedt | 72.694.996 | |
| Reservoir Trays for Multichannel, 55ml | Fisher | 13-681-501 | |
| DEAE-Dextran | Fisher | NC9691007 | |
| Corning 96 well clear V bottom tissue culture treated microplate | Fisher | 07-200-96 | Manufactured by Corning Life |
| HEPES, 1M Buffer Solution | Life Technologies/Invitrogen | 15630-080 | |
| FBS, Defined, 500 ml | Fisher | SH30070 03 | |
| X-gal | VWR | PAV3941 | Manufactured by Promega |
| Glutaraldehyde, Grade II, 25% in H2O | Sigma-Aldrich | G6257-100ML | |
| 1M Magnesium chloride solution | Sigma-Aldrich | M1028-100ML | |
| Formaldehyde solution, for molecular biology, 36.5% | Sigma-Aldrich | F8775-500ML | |
| Potassium hexacyanoferrate(II) trihydrate | Sigma-Aldrich | P9387-100G | |
| Potassium hexacyanoferrate(III) | Sigma-Aldrich | P8131-100G | |
| Allegra X15-R centrifuge | Beckman Coulter | 392932 | |
| TC10 automated cell counter | Bio-Rad | 1450001 | |
| VistaVision inverted microscope | VWR | ||
| Reverse-Transcriptase Quantification Assay reagents | |||
| dTTP, [α-33P]- 3000Ci/mmol, 10mCi/ml, 1 mCi | Perkin-Elmer | NEG605H001MC | |
| 1M Tris-Cl, pH 8.0 | Life Technologies/Invitrogen | 15568025 | Must be adjusted to pH 7.8 with KOH |
| 2M potassium chloride (KCl) | Life Technologies/Invitrogen | AM9640G | Adjust to 1M solution |
| 0.5M EDTA | Life Technologies/Invitrogen | 15575-020 | |
| Nonidet P40 | Roche | 11333941103 | |
| Polyadenylic acid (Poly rA) potassium salt | Midland Reagent Co. | P-3001 | |
| Oligo d(T) primer | Life Technologies/Invitrogen | 18418-012 | |
| Dithiothreitol (DTT) | Sigma-Aldrich | 43815-1G | |
| SR, Super Resolution Phosphor Screen, Small | Perkin-Elmer | 7001485 | |
| Corning Costar Thermowell 96 well plate model (M) Polycarbonate | Fisher | 07-200-245 | Manufactured by Corning Life |
| Corning 96 Well Microplate Aluminum Sealing Tape, Nonsterile | Fisher | 07-200-684 | Manufactured by Corning Life |
| DE-81 anion exchange paper | Whatman | 3658-915 | |
| Trisodium citrate dihydrate | Sigma-Aldrich | S1804-1KG | |
| Sodium Chloride | Fisher | S671-3 | |
| Autoradiography cassette | Fisher | FB-CA-810 | |
| Cyclone storage phoshpor screen | Packard |
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