简化反向遗传学方法，恢复重组性轮状病毒表达报告蛋白
Summary
从质粒DNA生成重组性轮状病毒为研究轮状病毒复制和发病机制以及发展轮状病毒表达载体和疫苗提供了重要工具。在这里，我们描述了一种简化的反向遗传学方法来产生重组性轮状病毒，包括表达荧光报告蛋白的菌株。
Abstract
轮状病毒是一大批正在进化的分段双链RNA病毒，在许多哺乳动物和鸟类宿主物种（包括人类）幼年时引起严重的肠胃炎。随着轮状病毒反向遗传学系统的出现，利用定向诱变探索轮状病毒生物学，修改和优化现有的轮状病毒疫苗，开发轮状病毒多目标疫苗载体成为可能。在本报告中，我们描述了一个简化的反向遗传学系统，该系统允许有效和可靠地恢复重组性轮状病毒。该系统基于T7转录载体的共同转染，表达全长轮状病毒（+）RNA和CMV载体编码RNA封盖酶到BHK细胞中，产生T7RNA聚合酶（BHK-T7）。重组轮状病毒通过监督带有MA104细胞的转染BHK-T7细胞进行扩增，这是一种对病毒生长高度宽松的猴子肾细胞系。在本报告中，我们还描述了一种生成重组轮状病毒的方法，该方法通过在基因组段 7 （NSP3） 中引入 2A 转化停止重启元件来表达单独的荧光报告蛋白。这种方法避免删除或修改任何病毒开放阅读框架，从而允许产生重组轮状病毒，在表达荧光蛋白的同时保留功能齐全的病毒蛋白。
Introduction
轮状病毒是婴儿和幼儿以及许多其他哺乳动物和鸟类1幼虫严重肠胃炎的主要原因。作为Reoviridae家族的成员，轮状病毒具有分段的双链RNA（dsRNA）基因组。基因组部分包含在由三个同心蛋白质2层形成的非包络的二体体上。根据基因组片段的测序和植物遗传学分析，确定了9种轮状病毒（A+D、F+J）3个。3那些由轮状病毒A类组成的菌株是造成绝大多数人类疾病的罪魁祸首。从过去十年开始，将轮状病毒疫苗引入儿童免疫计划与轮状病毒死亡率和发病率的显著降低有关。最值得注意的是，与轮状病毒相关的儿童死亡人数从2000年的约528，000人下降到2016年的128，500人，,5。轮状病毒疫苗是从病毒的活衰减菌株中配制的，在6个月大时给儿童施用2至3剂。大量基因多样化的轮状病毒株在人类和其他哺乳动物物种中循环，加上它们通过诱变和重新分类迅速进化的能力，可能导致感染儿童66、7、87,8的轮状病毒类型的抗原变化。这种变化可能损害现有疫苗的功效，需要更换或修改。
完全基于质粒的反向遗传学系统的发展，使操纵11个轮状病毒基因组片段中的任何一个，直到最近才达到9个。随着这些系统的提供，有可能解开轮状病毒复制和发病机制的分子细节，开发改进的抗轮状病毒化合物的高通量筛选方法，并创造新的可能更有效的轮状病毒疫苗类别。在轮状病毒复制期间，封盖病毒（+）RNA不仅指导病毒蛋白的合成，而且还作为后代dsRNA基因组片段10，11,11的合成模板。迄今描述的所有轮状病毒反向遗传学系统都依赖于T7转录载体的转染到哺乳动物细胞系中，作为cDNA衍生（+）RNA的来源，用于恢复重组病毒9,9、12、13。,13在转录载体中，全长病毒cDNA位于上游T7促进剂和下游肝炎三角洲病毒（HDV）核糖核素之间，因此病毒（+）RNA由含有正宗5’和3’termini的T7RNA聚合酶合成（图1A）。在第一代反向遗传学系统中，重组病毒是通过用11 T7（pT7）转录婴儿仓鼠肾细胞，表达T7RNA聚合酶（BHK-T7）的重组病毒， 分别对西米亚SA11病毒株的一种独特的（+）RNA和三个CMV促进驱动表达质粒进行定向合成，一个编码禽流感病毒p10FAST融合蛋白和两个编码子单元的病毒D1R-D12L封盖酶复合物9。在转染的BHK-T7细胞中产生的重组SA11病毒通过用MA104细胞进行监督而放大，MA104细胞是一种细胞系，允许轮状病毒生长。第一代反向遗传学系统的一个修改版本被描述为不再使用支持质粒12。相反，经过修改的系统仅仅通过用11 SA11 T7转录载体转染BHK-T7细胞，就成功地生成重组轮状病毒，并警告病毒工厂（病毒质）构建基块（非结构蛋白NSP2和NSP5）的载体添加水平比其他载体14、15,15高3倍。反向遗传学系统的改性版本也已经开发，以支持恢复人类KU和Odelia的轮状病毒菌株16，17。16,轮状病毒基因组非常适应反向遗传学的操纵，重组病毒生成至今，突变引入VP418、NSP19、NSP219、NSP320、2120,21和NSP522、23。922,2319迄今产生的最有用的病毒包括那些被设计成表达荧光报告蛋白（FPs）的病毒9、12、21、24、25。,12,21,24,25
在本出版物中，我们为在实验室中用于生成SA11轮状病毒重组菌株的反向遗传学系统提供了协议。我们协议的主要特征是与11个pT7转录载体共同转染BHK-T7细胞（修改为包括pT7/NSP2SA11和pT7/NSP5SA11载体的3倍水平）和一个CMV表达载体编码非洲猪瘟病毒（ASFV）NP868R封盖酶21（图2）。在我们手中，NP868R质粒的存在导致通过转染BHK-T7细胞产生更高的重组病毒的结节。在本出版物中，我们还提供了修改 pT7/NSP3SA11 质粒的协议，以便生成重组病毒，不仅表达第 7 段蛋白产品 NSP3，还表示单独的 FP。这是通过重新设计pT7/NSP3SA11质粒中的NSP3开放读取帧（ORF）来包含下游的2A平移停止重启元件，然后是FP ORF（图1B）24，26。24,26通过这种方法，我们产生了反应重组轮状病毒，表达各种FP：UnaG（绿色）、mKate（远红色）、mRuby（红色）、TagBFP（蓝色）、CFP（青色）和YFP（黄色）24、27、28 。24,27,28这些FP表达轮状病毒是在不删除NSP3 ORF的情况下制造的，从而产生病毒，这些病毒有望编码全功能病毒蛋白。
Protocol
Representative Results
Discussion
在我们的实验室中，我们通常依靠本文描述的反向遗传学协议来产生重组SA11轮状病毒。使用这种方法，在分子生物学技术或使用轮状病毒方面经验不足的个人，即使在第一次尝试时，也能恢复重组病毒。我们按照此协议生成了近 100 种重组病毒，包括那些经过重新设计以表达外来蛋白质（例如 FPs）且包含序列添加、删除和点突变的基因组病毒。
该协议给出的条件和潜伏时间?…
Declarações
The authors have nothing to disclose.
Acknowledgements
这项工作得到了NIH赠款R03 AI131072和R21 AI144881、印第安纳大学启动基金和劳伦斯·布拉特捐赠基金的支持。我们感谢IU罗塔霍西尔实验室、乌尔里希·德塞尔贝格尔和吉多·帕帕的成员在制定反向遗传学方案方面所作的许多贡献和建议。
Materials
| Baby Hamster Kidney – T7 RdRP (BHK-T7) Cells | Contact: ubuchholz@niaid.nih.gov | ||
| Bio-Rad 8-16% Tris-Glycine Polyacrylamide Mini-Gel | Bio-Rad | 45608105 | |
| Cellometer AutoT4 viable cell counter | Nexcelom | ||
| ChemiDoc MP Gel Imaging System | Bio-Rad | ||
| Chloroform | MP | 194002 | |
| Clarity Western Enhanced Chemiluminescence (ECL) Substrate | Bio-Rad | 170-5060 | |
| Competent E.coli DH5alpha Bacteria | Lucigen | 60602-2 | |
| Complete Protease Inhibitor | Pierce | A32965 | |
| Disposable Transfer Pipettes, Ultrafine Extended Tips | MTC Bio | P4113-11 | |
| Dulbecco's Modified Eagle Medium (DMEM) | Lonza | 12-604F | |
| Eagle's Minimal Essential Medium, 2x (2xEMEM) | Quality Biological | 115-073-101 | |
| Ethanol, Absolute (200 proof) | Fisher Bioreagents | BP2818-500 | |
| Ethidium Bromide Solution (10 mg/ml) | Invitrogen | 15585-011 | |
| Fetal Bovine Serum (FBS) | Corning | 35-010-CV | |
| Fetal Bovine Serum (FBS), Heat Inactivated | Corning | 35-011-CV | |
| Flag M2 Antibody, Mouse Monoclonal | Sigma-Aldrich | F1804 | |
| GenEluate HP Plasmid Midiprep Kit | Sigma | NA0200-1KT | |
| Geneticin (G-418) | Invitrogen | 10131-027 | |
| Gibco FluroBrite DMEM | ThermoFisher | A1896701 | DMEM with low background fluorescence |
| Glasgow Minimal Essential Medium (GMEM) | Gibco | 11710-035 | |
| Goat Anti-Rabbit IgG, Horseradish Peroxidase (HRP) Conjugated | Cell-Signaling Technology | 7074S | |
| Guinea Pig Anti-NSP3 Antiserum | Patton lab | lot 55068 | |
| Guinea Pig Anti-VP6 Antierum | Patton lab | lot 53963 | |
| Horse Anti-Guinea Pig IgG, Horseradish Peroxidase (HRP) Conjugated | KPL | 5220-0366 | |
| Horse Anti-Mouse IgG, Horseradish eroxidase (HRP) Conjugated | Cell-Signaling Technology | 7076S | |
| iNtRON Biotechnology e-Myco Mycoplasma PCR Detection Kit | JH Science | 25235 | |
| Isopropyl alcohol | Macron | 3032-02 | |
| L-glutamine Solution (100x) | Gibco | 25030-081 | |
| Luria Agar Powder (Miller's LB Agar) | RPI research products | L24020-2000.0 | |
| Medium 199 (M199) Culture Medium | Hyclone | Sh30253.01 | |
| Minimal Essential Medium -Eagle Joklik's Forumation (SMEM) | Lonza | 04-719Q | |
| Monkey Kidney (MA104) Cells | ATCC | ATCC CRL-2378.1 | |
| NanoDrop One Spectrophotometer | ThermoScientific | ||
| Neutral Red Solution (0.33%) | Sigma-Aldrich | N2889-100ml | |
| Non-Essential Amino Acid Solution (100x) | Gibco | 11140-050 | |
| Novex 10% Tris-Glycine Polyacrylamide Mini-Gel | Invitrogen | XP00102BOX | |
| Nuclease-Free Molecular Biology Grade Water | Invitrogen | 10977-015 | |
| NucleoSpin Gel and PCR Clean-Up Kit | Takara | 740609.25 | |
| Opti-MEM Reduced Serum Medium | Gibco | 31985-070 | |
| Pellet pestle (RNase-free, disposable) | Fisher | 12-141-368 | |
| Penicillin-Streptomycin Solution, (100x penn-strep) | Corning | 30-002-Cl | |
| Phosphate Buffered Saline (PBS), 10x | Fisher Bioreagents | BP399-20 | |
| Porcine Trypsin, Type IX-S | Sigma-Aldrich | T0303 | |
| PureYield Plasmid Miniprep System | Promega | A1223 | |
| Qiagen Plasmid Maxi Kit | Qiagen | 12162 | |
| Qiagen Plasmid Midi Kit | Qiagen | 12143 | |
| QIAprep Spin Miniprep Kit | Qiagen | 27104 | |
| SA11 pT7 Transcription Vectors | Addgene | 89162-89172 | |
| SA11 pT7/NSP3 Transcription Vectors Expressing Fluorescent Proteins | Contact: jtpatton@iu.edu | ||
| SeaKem LE Agarose | Lonza | 50000 | For gel electrophoresis |
| SeaPlaque agarose | Lonza | 50100 | For plaque assay |
| Superscript III One-Step RT-PCR kit | Invitrogen | 12574-035 | |
| Trans-Blot Turbo Nitrocellulose Transfer Kit | Bio-Rad | 170-4270 | |
| Trans-Llot Turbo Transfer System | Bio-Rad | ||
| TransIT-LTI Transfection Reagent | Mirus | MIR2306 | |
| Tris-Glycine-SDS Gel Running Buffer (10x) | Bio-Rad | 161-0772 | |
| Triton X 100 | Fisher Bioreagents | BP151-500 | |
| Trizol RNA Extraction Reagent | Ambion | 15596026 | |
| Trypan blue | Corning | 25-900-CI | |
| Trypsin (0.05%)-EDTA (0.1%) Cell Dissociation Solution | Quality Biological | 118-087-721 | |
| Tryptose Phosphate Broth | Gibco | 18050-039 | |
| Tween-20 | VWR | 0777-1L | |
| Vertrel VF solvent | Zoro | G0707178 | |
| Zoe Fluorescent Live Cell Imager | Bio-Rad |
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