简单且可靠的 in vivo 与 in vitro 研究病毒组装的方法

摘要

一种简单、高效且稳健的方法，通过同步递送多种病毒组分至植物细胞 农杆菌介导的瞬时表达方法被描述。该方法适用于研究复制、衣壳化，随后的 体外 非病毒组分的重装配，形成基因组耗尽的光学病毒幽灵，适用于生物医学应用。

摘要

在感染人类、动物和植物的正链RNA病毒中，子代病毒颗粒被包裹形成成熟且稳定的病毒粒子是其在特定宿主中建立感染的关键阶段。因此，对包裹过程的研究有助于揭示调控病毒组装以形成具有感染性病毒粒子的机制。这类信息对于制定抑制病毒传播和控制疾病的新方法至关重要。病毒包裹过程可被研究 体内 和 体外基因组包装 体内 是一个高度调控的、选择性过程，涉及大分子相互作用和亚细胞区室化。因此，研究病毒衣壳化所涵盖的事件至关重要 体内 将为理解病毒如何增殖和组装提供基础知识。近期 体外 衣壳化已被广泛应用于生物医学成像及治疗领域的研究。在这一探索进程中，非包膜植物病毒处于领先地位 体外 负电荷外源物质的衣壳化。麦芽花叶病毒（Brome mosaic virus, BMV）是一种对植物具有致病性的无包膜多组分RNA病毒，已被用作研究基因组包装的模型系统。 体内 和 体外用于包封实验的 烟草本氏 植物， 农杆菌 介导的瞬时表达，称为农杆菌浸润（agroinfiltration），是一种高效且可靠的同步递送多个组分并在同一细胞中表达的技术。该方法中，将含有相应组分的农杆菌悬浮液 Agrobacterium tumefaciens 将含有目标病毒mRNA cDNA的双元质粒载体导入农杆菌，再利用1 ml一次性注射器（无针头）将其注入植物叶片的细胞间隙。该过程可将DNA插入片段转移至植物细胞内；T-DNA插入片段在细胞核中短暂存在，并由宿主RNA聚合酶II转录，从而实现瞬时表达。所产生的mRNA转录本（具有5'端帽子结构和3'端多聚腺苷酸尾）随后被转运至细胞质中进行翻译。在侵染后约24至48小时，可取侵染区域的叶片组织用于显微观察或生化分析。农杆菌介导的瞬时转化可实现大量细胞（数百至数千个）的同步转染。对于体外衣壳化实验，首先通过透析含有氯化钙的解离缓冲液，将纯化的苜蓿花叶病毒（BMV）病毒颗粒解离为衣壳蛋白，再经离心去除RNA及未完全解离的病毒颗粒。随后，基因组缺失的衣壳蛋白亚基可与目标病毒基因组组分或非病毒组分（如吲哚菁染料）重新组装形成新的病毒样颗粒。

方案

1. Plant material

1. Nicotiana benthamiana plants to be used in the encapsidation assay should be at 4 leaves stage (approximately 3-4 week old plants).

2. Delivery and expression of functional viral genome components to plant cells by agroinfiltration

1. Day 1: The Agrobacterium strain (eg. EH 105 or GV 3101) harboring the pCass- BMV RNA 1, BMV RNA 2 and BMV RNA 3^1,2 are grown on LB agar plates supplemented with 50 mg/ml of kanamycin (selects for the pCASS vector) and 100mg/ml of rifampicin (selects for the Agrobacterium). Incubation is being carried out at 28 °C for two days.
2. Day 3: Inoculate single colony from the LB agar plate into 2 ml of LB broth containing 50 mg/ml of kanamycin and 100 mg/ml of rifampicin for 1 day at 28 °C in orbital shaker set at 250 RPM.
3. Day 4: Inoculate 50 ml of the LB broth supplemented with 50 mg/ml of kanamycin and 100 mg/ml of rifampicin, 10 mM MES pH 5.6 and 100 mM acetosyringone with one ml of culture in a 250 ml of Erlenmeyer flask for 16 hours at 28 °C on orbital shaker at 250 RPM.
4. Day 5: Transfer the culture (when OD₆₀₀ reached 1.0) to an oak ridge tube or sterile Falcon tube and centrifuge for 10 minutes at 5000 rpm at 4 °C.
5. Dissolve the pellet in 10 ml of 10 mM MgCl₂ and centrifuge for 10 minutes at 5000 RPM at 4 °C, Repeat this step one more time to ensure complete removal of the antibiotic.
6. Suspend the pellet in 10 ml buffer containing 10 mM MgCl₂ and 10 mM MES (pH 5.6).
7. Measure the OD₆₀₀ for each culture of BMV RNA 1, RNA 2 and RNA 3 and adjust to 0.1 OD₆₀₀ using 10 mM MgCl₂ and 10mM MES (pH 5.6).
8. Mix 1 ml each all three 0.1 OD₆₀₀ culture suspensions.
9. Add 100 mM of acetosyringone, mix gently and keep the mixture undisturbed at room temperature for 3 hours.
10. Draw the culture suspension into 1 ml. syringe without needle.
11. Infiltrate the above culture suspension into abaxial side of 2-3 well-expanded leaves of N. benthamiana (5 leaves stage, 3-4 week old plants) by gently pressing the syringe to one half of the abaxial surface of leaf.
12. Repeat this infiltration procedure to other leaves.
13. Keep infiltrated plants in the green house with 24 °C.
14. Harvest infiltrated leaves 3 to 4 days post infiltration (dpi).

3. Purification of BMV virions

1. Collect N. benthamiana leaves agroinfiltrated with a mixture containing all three wild type BMV agrotransformants.
2. Grind leaves in a sterile pestle and mortar with equal volume (w/v) of BMV extraction buffer (0.5 M NaAc; 0.08 M MgAc, pH 4.5 and 1/100 volume of β-mercaptoethanol which is to be added just before use)³. To maximize virus yield, it is recommended to add acid washed sand (~0.5-1 g) that facilitates easy grinding and efficient cell disruption.
3. Filter the leaf extract through 2-3 layers of muslin cloth and collect the flurry.
4. Again grind the retained portion on muslin cloth with equal volume of BMV extraction buffer to the initial weight of leaf material.
5. Repeat the filtration procedure using muslin cloth. These steps should be carried out at 4 °C.
6. Transfer the filtrate solution to a centrifuge tube and add equal volume of pre-chilled chloroform and vortex (or shake) for 5 minutes at the room temperature till the color of the suspension turns light green.
7. Centrifuge the emulsified solution for 15 minutes at 12,000 RPM at 4 °C.
8. Collect the aqueous phase and stir for 30 minutes on magnetic stirrer to remove any residual chloroform.
9. Transfer the suspension to a sterile ultracentrifuge tube.
10. Underlay 1 ml. 10% sucrose (prepared in virus extraction buffer) as a cushion³.
11. Centrifuge at 30,000 rpm for 3 hours in a Beckman ultracentrifuge at 4 °C.
12. Discard the supernatant and suspend pellet in desired volume of BMV suspension buffer (to prepare suspension buffer dilute BMV extraction buffer to 1/10 with sterile distilled water).
13. The partially purified virion preparation from above step is subjected to 10-40% sucrose density gradient centrifugation for 150 min at 28000 rpm at 4 °C.
14. Collect the virus band either manually or by a fractionator. The virus band will appear blue under white light illumination.
15. Dilute the sucrose solution containing virus sample at least 50% with virus suspension buffer.
16. Centrifuge diluted virus containing sucrose solution for 3 hours at 30,000 rpm in a Beckman ultracentrifuge at 4 °C.
17. Finally suspend the highly purified virus pellet in a desired volume of virus suspension buffer.
18. Measure the concentration of the virion (mg/ml) using spectrophotometer at OD₂₆₀.

Note: Based on RNA content, the extinction coefficient for BMV is 5⁴.

19. Verify the purity of virions by viewing under TEM (Fig. A)

4. Preparation of capsid protein subunits for in vitro assembly

1. Prepare 1 Liter of 1x virus dissociation buffer (0.5 M CaCl₂; 50 mM Tris HCl, pH 7.5; 1.0 mM EDTA; 1.0 mM DTT and 0.5 mM PMSF)⁵.
2. Prepare dialysis membrane according to Sambrook et al ⁶.
3. Dispense required concentration of purified virus into a dialysis bag.
4. Place the virus containing dialysis bag into a beaker containing the 1x virus dissociation buffer.
5. Dialyze 24 hours at 4°C while stirring.
6. Collect the solution from the dialysis bag after 24 hours and centrifuge at 12,000x g for 15 min at 4 °C. This will separate the BMV RNA from the dissociated virions.
7. Collect the supernatant and centrifuge at 35,000 rpm in Beckman centrifuge for 3 hours at 4°C to pellet any un-dissociated virus particle.
8. Collect the supernatant.
9. At this stage it is imperative to remove any contaminating virion RNA from dissociated capsid protein subunits. To do this, the supernatant from step 8 should be subjected to another round of over night reassembly of capsid protein subunits without adding any RNA (in RNA 1x assembly buffer, see below) followed by an high speed centrifugation (30,000 rpm for 3 hrs at 4 °C) to pellet any assembled virions. After this reassembly step, the supernatant containing coat protein subunits would be free of any residual contaminating RNA. However, to ensure this, it is advisable to perform an additional in vitro reassembly with only coat protein subunits using RNA 1x assembly buffer. After over night reassembly, the preparation must be free of assembled virions (verify using TEM).
10. Determine the concentration of capsid protein subunits by measuring at OD 254 and 280 nm or by other methods such as Bradford assay.
11. Perform 12-15% SDS-PAGE followed by western blot to determine the integrity of the dissociated capsid protein subunits.

5. In vitro assembly of RNA containing virions

1. Prepare RNA transcripts to be re-assembled into virions and calculate the concentration⁷.
2. Mix capsid protein subunits and RNA transcript at a ratio of 1:5 (wt/wt)⁵.
3. Dispense the above mixture to a dialysis bag and properly secure to avoid any leaks.
4. Prepare 1,000 ml. of RNA 1x assembly buffer (50 mM NaCl; 50 mM Tris-HCl, pH 7.2; 10 mM KCl; 5 mM MgCl₂, 1.0 mM DTT)⁵.
5. Place the dialysis bag containing the reaction mixture against 1x assembly buffer into the beaker and stir.
6. Dialyze the re-assembly reaction at 4 °C for 24 hours by gentle stirring.
7. Collect the mixture from the dialysis bag after 24 hours and add 1.5 ml of RNA assembly buffer.
8. Pass this mixture through a Centricon-100 column by centrifuging at low speed (2,000x g) for 30 minutes.
9. Wash the column once with 1.5 ml of assembly buffer at 4 °C for 30 min.
10. Repeat the above washing step twice.
11. Finally, elute the re-assembled virions by centrifugation at 10,000g at 4 °C for 5 min.
12. Estimate the virion concentration at OD 260 nm.

6. Fabrication of Optical Viral Ghosts (OVGs)

1. Add Indocyanine green (ICG)⁸ to the purified capsid protein at desired concentration ratio(the weight ratio of Indocyanine green and capsid protein used in this report was 1:10) and mix thoroughly by pipetting.
2. Dialyze the capsid protein and ICG solution against the OVG assembly buffer (1 M NaCl; 50 mM NaAc; 1 mM EDTA; and 1 mM DTT, pH4.8) at 4 °C for 24 hours.
3. After 24 hours, collect the solution from the dialysis bag (12 kDa pore size) and centrifuge at 90,000 rpm for 1 hour at 4 °C (Fig. B)
4. Remove the supernatant and suspend the pellet in BMV suspension buffer by vortexing or allowing it to suspend by itself in BMV suspension buffer overnight at 4 °C.
5. Verify the morphology of OVGs by TEM (Fig. C).
6. Measure the absorbance from 240 nm to 950 nm using spectrophotometer (Cary 50, Varian Inc.); the presence of ICG is confirmed by signature absorbance peaks around 700 nm and 790 nm⁸. (Fig. D)
7. The typical ICG fluorescence peaks at ~700 nm and ~800 nm can be seen upon 620 nm excitation of OVG solution (Fig. D) using spectrofluorometer (Fluorolog 3, Jobin-Yvon).

7. Representative Results

Figure A. TEM image of negatively stained BMV virions purified from N. benthamiana plants agroinfiltrated with a mixture of all three wild type BMV agroconstructs (scale bar = 100 nm).

Figure B. Pellet of in vitro assembled ICG containing OVGs following high-speed centrifugation.

Figure C. TEM image of negatively stained ICG containing OVGs (scale bar = 100 nm).

Figure D. Absorbance (Top) and fluorescence (bottom) spectra of OVGs. The excitation wavelength used for OVG emission was 620 nm.

讨论

此处介绍的农杆菌浸润法可广泛适用于多种植物病毒。该方法的一个显著特征是能够将多个农杆菌载体同步递送至同一细胞，从而克服了常规使用的植物病毒机械接种法通常存在的主要缺陷。 在体 和 体外 以溴化花叶病毒为模型的组装研究，若遵循一些技巧，可高效开展。（i）为成功进行浸润 N. benthamiana 叶片，在渗透前24小时内不要给植物浇水；（ii）若在下午4至5点之间进行渗透，农杆菌悬浮培养液将更容易扩散至细胞间隙；（iii）培养液在OD600处的光密度值不应超过1.0₆₀₀高密度农杆菌培养物的侵染已知会引发细胞毒性及叶片衰老^9,10 这可能严重影响病毒复制及后续的病毒粒子形成；（iv）在渗透过程中，应轻轻按压叶片的远轴面，以防止对叶片造成严重损伤，从而避免触发植物的免疫反应；（v）可通过将最高浓度与最低浓度的蔗糖相加后除以2的方法，在离心管中用BMV悬浮缓冲液配制10%至40%的25%蔗糖密度梯度 即，10% + 40% / 2 = 25%），立即于 -80 °C 冻结 2–4 小时，随后将蔗糖溶液直立放置于 4 °C 过夜...

材料

本文使用的材料清单

姓名	公司	目录编号	评论
MES 钠盐	Sigma-Aldrich	M2993
吲哚菁绿	Sigma-Aldrich	I2633
贝克曼超速离心机	Beckman Coulter Inc.	型号：L8-70M
Centricon-100 柱	EMD Millipore	YM-100
分光光度计	Varian, Inc.	部件号 10068900
荧光分光光度计	Fluorolog 3, Jobin-Yvon.	部件号 FL3-21