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Measuring Genetic Robustness in Vesicular Stomatitis Virus
测定水泡性口炎病毒中的基因强性   

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Abstract

Genetic robustness is the ability of a genome to incorporate mutations with the result of no fitness changes. Thus, more robust viruses have an increased neutral mutation rate. This property is particularly important in RNA viruses due to their high mutation rates. The most direct way of measuring robustness in vesicular stomatitis virus (VSV) is to carry out clonal analysis of populations: randomly isolating individual VSV strains (plaques), measuring the fitness of each one and generating fitness distributions (Novella et al., 2010). A second possibility is to carry out multiple replicates of repeated plaque-to-plaque passages, determining fitness in progeny populations and generating fitness distributions (Novella et al., 2010). Depending on the expected differences, the former may require hundreds of determinations, while the latter may require tens of determinations. A third approach consists of increasing the mutation rate of populations under analysis to magnify any differences that may exist and, instead of measuring fitness, measuring survival (Novella et al., 2013). One caveat of this method is that changes in survival can also be explained by changes in polymerase fidelity. For that reason, it is important to perform complementary experiments, in this case quantifying mutant frequency.

Materials and Reagents

  1. Test and reference VSV strains
    Note : The former is the strain under investigation, the latter is the control (typically the progenitor).
  2. Baby hamster kidney cells (BHK-21)
  3. 10x Trypsin/EDTA (Life Technologies, Gibco®, catalog number: 15400 )
  4. I1 Monoclonal antibody (I1Mab) hybridoma (Holland et al., 1991) (ATCC, catalog number: CRL-2700 )
    Note: This antibody recognizes the G glycoprotein of VSV.
  5. I14 Mab (Holland et al., 1991) (https://www.kerafast.com/p-172-hybridoma-ie9f9-i14.aspx)
    Note: This antibody recognizes the G glycoprotein of VSV.
  6. Proteose peptone No. 3 (PP3) (BD DifcoTM, catalog number: 212230 ) (12 g/L in dH2O, autoclaved)
  7. Bovine Calf Serum (BCS) (Life Technologies, Gibco®, catalog number: 16170-078 )
    Note: BCS is a good choice to carry out plaque assays and it is a lot cheaper than FBS.
  8. Fetal Bovine Serum (FBS) (Life Technologies, Gibco®, catalog number: 10437 )
  9. 10% CO2
  10. 5-Fluorouracyl (5-FU) (Sigma-Aldrich, catalog number: F-6627 ) (10 mg/ml in ethanol, filtered)
  11. Agarose (Lonza, SeqplaqueTM GTGTM, catalog number: 50111 ) (40 g/L in dH2O, autoclaved)
  12. 4.2% bicarbonate
  13. Penicillin/streptomycin mixture (Mediatech, Cellgro®, catalog number: 30-004-Cl )
  14. Saline solution (see Recipes)
  15. Minimal Essential Medium with Hank’s salts (MEM-H) (Mediatech, Cellgro®, catalog number: 50-019-PB ) (see Recipes)
  16. Crystal violet solution (see Recipes)

Equipment

  1. T25 plug-seal flasks (CytoOne®, catalog number: CC7682-4325)
  2. 5 ml and 25 ml pipetes
  3. Plugged Pasteur pipetes
  4. 1.5 ml tubes
  5. Type II biosafety hood
  6. 37 °C, CO2 cell culture incubator
  7. Transilluminator (optional)

Procedure

  1. Make I1 Mab stock and titrate I1 and I14 antibodies; use at enough concentration to produce full inhibition of wild type. Alternatively, I1 Mab can be purchased from Kerafast (https://www.kerafast.com/p-171-hybridoma-8g5f11-i1.aspx).
    Notes:
    1. Because of high mutation rates leading to high frequency of antibody-resistance mutants, viral stocks can never be fully neutralized. The goal of Mab titration is to produce plaque assays with less than 10-4 (if viral stocks are neutralized) or less than 10-3 (if the Mab is added to the overlay medium) mutants/wt.
    2. Antibodies other than I14 (https://www.kerafast.com) can be used to calculate mutant frequencies.
  2. BHK-21 cells are washed twice with saline solution, trypsinized and 0.8-1.0 x 105 are seeded in T25 flasks with MEM-H supplemented with 7% BCS and 0.06% PP3. The flasks are gassed for 2 sec with 10% CO2 using a plugged Pasteur pipette. Caps are locked.
  3. The cells are incubated for 24 h to produce monolayers 90% confluent.
  4. On day 2 cell monolayers are treated with no mutagen (mock) or with the mutagen 5-FU at a range of concentrations between 1 and 100 μg/ml for 6 h at 37 °C (5, 10, 25, 50 and 100 μg/ml).
  5. Dilute viral stocks in MEM-H+FBS as needed to produce a 106 plaque forming units (PFU)/ml solution.
    1. Use 200 μl to infect a BHK-21 monolayer for each 5-FU concentration. Incubate 10 min at room temperature (RT) and 40 min at 37 °C.  Add 5 ml of MEM-H+FBS+5-FU (Figure 1, right).
    2. From the 106 PFU/ml solution carry out additional, 10-fold, 100-fold and 1000-fold dilutions. Use each of the three dilutions (105, 104 and 103 PFU/ml solutions) to infect at least 3 BHK-21 monolayers with 200 μl of the dilution to carry out plaque assay in the presence of I1Mab. Do not neutralize the mixture with I14Mab prior to infection, as it will result in incorrect data due to phenotypic mixing and hiding (Valcarcel and Ortin, 1989; Holland et al., 1989). Incubate 10 min at room temperature (RT) and 40 min at 37 °C. Prewarm a mixture of MEM-H+FBS and I14Mab, add agarose to a final concentration of 0.2%, and add 5 ml of mixture to flasks (Figure 1, left).
      Note: Even if the virus stock has a known concentration it is recommended that a titration is done in parallel by triplicate plaque assay Figure 1, top left).


    Figure 1. Flowchart showing the infections needed to calculate the frequency of I1 MARM and the sensitivity to 5-FU. Details are given in the text.

  6. Incubate mutagenized infections and plaque assays for 24 h at 37 °C (Figure 1).
  7. Develop all plaque assays (Figure 1). Because of the low agarose concentration there is no need to fix the cells. Just let the medium overlay slide out and add 2-3 ml of crystal violet solution. Wait for 5 min at RT, discard crystal violet and rinse with tap water.
  8. Count plaques and calculate mutant frequency by dividing Monoclonal antibody resistant mutant (MARM) titers (Figure 1B) by wt titers (Figure 1A).
  9. Check virus replication by examining monolayers under the microscope. When cytopatic effect is > 90%, recover mutagenized populations.
    Notes:
    1. For lower FU concentrations 24 h are usually sufficient, but for FU concentrations over 30 μg/ml it may take up to three days.
    2. Samples may be frozen so titrations can be carried out for the complete set of mutagenized samples in a single assay.
  10. Carry out titrations of control and mutagenized viral yields. To improve statistical significante it is recommended to do duplicate or triplicate plaque assays for each dilution. Allow 24-36 h of incubation.
  11. Develop plaque assays with crystal violet and count claque (Figure 1C).
  12. Robustness can be calculated as the slope of the regression of log transformed normalized viral titers vs. square-root transformed 5-FU concentration (Figure 2). The more robust the strain is the closer to a slope of 1.


    Figure 2. To calculate robustness the log-transformed viral titer is represented against the square root of mutagen concentration. Robustness is the slope of the regression (shown at the top of the graph).

  13. To test whether changes in survival are the result of changes in the overall mutation rate, and not in the neutral mutation rate, mutant frequencies must be compared. When all other environmental parameters are the same, mutant frequencies correlate with mutation rates. If mutant frequency and survival to mutagenesis correlate inversely, the latter may not represent robustness. In such case, a clonal analysis is indicated.

Recipes

  1. Saline solution
    Add 7 g of NaCl to dH2O to 1,000 ml
    Autoclave 20 min
    Stored at RT
  2. MEM-H
    Mix powder with 9.65 L of dH2O
    Add:
    250 ml of 4.2% bicarbonate
    100 ml of penicillin/streptomycin mixture
    Filter and aliquote in 1 L bottles. At this time the medium can be stored in the fridge.
    When ready for use add:
    70 ml of serum (FEB or BCS)
    5 ml of 12% PP3 (if needed)
    Notes:
    1. Making medium is worth it if there is a large volume of cell culture performed in the laboratory.  Ready-to-use MEM-E can also be purchased from several companies.
    2. MEM with Earls salts (MEM-E) is also a good choice, but requires a CO2 incubator. 
  3. Crystal violet solution
    Mix 750 ml H2O, 250 ml ethanol and 5 g crystal violet
    Stir until dissolved and stored at RT

Acknowledgments

This protocol was originally published in Novella et al. (2013). Work was supported by funds from the University of Toledo to ISN.

References

  1. Holland, J. J., de la Torre, J. C., Clarke, D. K. and Duarte, E. (1991). Quantitation of relative fitness and great adaptability of clonal populations of RNA viruses. J Virol 65(6): 2960-2967.
  2. Holland, J. J., de la Torre, J. C., Steinhauer, D. A., Clarke, D., Duarte, E. and Domingo, E. (1989). Virus mutation frequencies can be greatly underestimated by monoclonal antibody neutralization of virions. J Virol 63(12): 5030-5036.
  3. Novella, I. S., Presloid, J. B., Zhou, T., Smith-Tsurkan, S. D., Ebendick-Corpus, B. E., Dutta, R. N., Lust, K. L. and Wilke, C. O. (2010). Genomic evolution of vesicular stomatitis virus strains with differences in adaptability. J Virol 84(10): 4960-4968.
  4. Novella, I. S., Presloid, J. B., Beech, C. and Wilke, C. O. (2013). Congruent evolution of fitness and genetic robustness in vesicular stomatitis virus. J Virol 87(9): 4923-4928.
  5. Valcarcel, J. and Ortin, J. (1989). Phenotypic hiding: the carryover of mutations in RNA viruses as shown by detection of mar mutants in influenza virus. J Virol 63(9): 4107-4109.

简介

遗传稳健性是基因组掺入突变的能力,没有适应度变化的结果。因此,更强壮的病毒具有增加的中性突变率。由于它们的高突变率,这种性质在RNA病毒中特别重要。测量水泡性口炎病毒(VSV)的稳健性的最直接的方法是进行群体的克隆分析:随机分离个体VSV株(噬斑),测量每个VSV株的适合度并产生适应度分布(Novella等人, ,2010)。第二种可能性是进行重复的斑块到斑块通道的多次重复,确定后代群体中的适合度并产生健身分布(Novella等人,2010)。根据预期的差异,前者可能需要数百个测定,而后者可能需要数十次测定。第三种方法包括增加分析中的群体的突变率以扩大可能存在的任何差异,并且代替测量适合度,测量存活率(Novella等人,2013)。该方法的一个警告是生存的变化也可以通过聚合酶保真度的变化来解释。因此,重要的是进行互补实验,在这种情况下量化突变频率。

材料和试剂

  1. 测试和参考VSV毒性
    注意:前者是正在调查的应变,后者是控制(通常是祖先)。
  2. 幼仓鼠肾细胞(BHK-21)
  3. 10x胰蛋白酶/EDTA(Life Technologies,Gibco ,目录号:15400)
  4. I1单克隆抗体(I1Mab)杂交瘤(Holland,et al。,1991)(ATCC,目录号:CRL-2700)
    注意:该抗体识别VSV的G糖蛋白。
  5. I14 Mab(Holland ,1991)( https://www.kerafast.com/p-172-hybridoma-ie9f9-i14.aspx
    注意:该抗体识别VSV的G糖蛋白。
  6. 蛋白胨No.3(PP3)(BD Difco Corp.,目录号:212230)(12g/L,在dH 2 O中,高压灭菌)
  7. 牛小牛血清(BCS)(Life Technologies,Gibco ,目录号:16170-078)
    注意:BCS是进行斑块测定的好选择,并且比FBS便宜。
  8. 胎牛血清(FBS)(Life Technologies,Gibco ,目录号:10437)
  9. 10%CO 2
  10. 5-氟尿嘧啶(5-FU)(Sigma-Aldrich,目录号:F-6627)(10mg/ml,在乙醇中,过滤)
  11. 琼脂糖(Lonza,Seqplaque TM,目录号:50111)(在dH 2 O中为40g/L,高压灭菌)
  12. 4.2%碳酸氢盐
  13. 青霉素/链霉素混合物(Mediatech,Cellgro ,目录号:30-004-Cl)
  14. 盐水溶液(见配方)
  15. 具有汉克氏盐(MEM-H)(Mediatech,Cellgro ,目录号:50-019-PB)的最小必需培养基(参见Recipes)
  16. 水晶紫溶液(见配方)

设备

  1. T25塞密封烧瓶(CytoOne ,目录号:CC7682-4325)
  2. 5ml和25ml移液管
  3. 插入巴斯德吸液管
  4. 1.5 ml管
  5. II型生物安全罩
  6. 37℃,CO 2细胞培养孵化器
  7. 透照器(可选)

程序

  1. 制备I1 Mab原液并滴定I1和I14抗体; 以足够的浓度使用以产生野生型的完全抑制。 或者,I1 Mab可以从Kerafast购买( https://www.kerafast .com/p-171-hybridoma-8g5f11-i1.aspx ) 注意:
    1. 由于高突变率导致抗体 - 抗性突变体的高频率,病毒原种不能完全中和。 Mab滴定的目标是产生具有小于10次的 -4 (如果病毒原种被中和) em> (如果Mab已添加到覆盖媒体)突变/wt。
    2. 除I14以外的抗体( https://www.kerafast.com )可用于计算突变频率。
  2. 将BHK-21细胞用盐水溶液洗涤两次,胰蛋白酶化,并将0.8-1.0×10 5个接种在具有补充有7%BCS和0.06%PP3的MEM-H的T25烧瓶中。 使用塞住的巴斯德移液管将烧瓶用10%CO 2气体鼓泡2秒。 上限已锁定。
  3. 将细胞孵育24小时以产生90%汇合的单层
  4. 在第2天,在37℃下,在无诱变剂(模拟)或用浓度范围为1至100μg/ml的诱变剂5-FU处理细胞单层6小时(5,10,25,50和100μg/ml)
  5. 根据需要稀释MEM-H + FBS中的病毒储液以产生10 6噬斑形成单位(PFU)/ml溶液。
    1. 每个5-FU浓度使用200μl感染BHK-21单层。在室温(RT)孵育10分钟,在37℃孵育40分钟。加入5ml的MEM-H + FBS + 5-FU(图1,右)
    2. 从10 6 PFU/ml溶液进行另外的10倍,100倍和1000倍稀释。使用三种稀释液(10μL,10μL和10μLPFU/ml溶液)中的每一种以感染至少3个BHK-21单层用200μl稀释液在I1Mab存在下进行噬菌斑测定。在感染之前不要用I14Mab中和混合物,因为由于表型混合和隐藏,将导致不正确的数据(Valcarcel和Ortin,1989; Holland等人,1989)。在室温(RT)孵育10分钟,在37℃孵育40分钟。预热MEM-H + FBS和I14Mab的混合物,加入琼脂糖至终浓度为0.2%,并向烧瓶中加入5ml混合物(图1,左)。
      注意:即使病毒原液具有已知浓度,建议通过一式三份噬斑测定(图1,左上)平行进行滴定。


    图1.流程图显示了计算I1 MARM的频率和对5-FU的敏感性所需的感染。详情在文中给出。

  6. 在37°C孵育诱变的感染和空斑测定24小时(图1)
  7. 开发所有斑块测定(图1)。 由于低的琼脂糖浓度,不需要固定细胞。 只要让介质覆盖物滑出并加入2-3ml的结晶紫溶液。 在室温下等待5分钟,丢弃结晶紫并用自来水冲洗
  8. 计数噬菌斑,并通过将单克隆抗体抗性突变体(MARM)滴度(图1B)除以重量滴度(图1A)计算突变频率。
  9. 通过在显微镜下检查单层检查病毒复制。 当细胞病变效应> 90%,恢复突变种群 注意:
    1. 对于较低的FU浓度通常足够24小时,但对于超过30μg/ml的FU浓度,可能需要三天。
    2. 样品可以冷冻,因此可以在一次测定中对完整的诱变样品进行滴定。
  10. 进行滴定的控制和诱变的病毒产量。为了改善统计学显着性,建议对每次稀释进行重复或一式三份的噬斑测定。允许孵育24-36小时。
  11. 用结晶紫和计数斑发展噬斑测定(图1C)
  12. 稳健性可以计算为对数转化的标准化病毒滴度与平方根转化的5-FU浓度的回归的斜率(图2)。越强的应变越接近1的斜率。


    为了计算稳健性,对数转化的病毒滴度代表诱变剂浓度的平方根。 鲁棒性是回归的斜率(显示在图表的顶部)。

  13. 为了测试存活的变化是否是总体突变率的变化的结果,而不是中性突变率,必须比较突变频率。当所有其他环境参数相同时,突变频率与突变率相关。如果突变频率和诱变的存活相反,后者可能不表示鲁棒性。在这种情况下,指示克隆分析

食谱

  1. 盐溶液
    将7g NaCl加入到dH 2 O至1000ml
    中 高压灭菌器20分钟
    存储在RT
  2. MEM-H
    将粉末与9.65L dH 2 O混合 添加:
    250毫升4.2%碳酸氢钠 100ml青霉素/链霉素混合物
    过滤并分装在1升瓶中。 此时,培养基可以储存在冰箱中。
    准备使用时,请添加:
    70ml血清(FEB或BCS)
    5毫升12%PP3(如果需要)
    注意:
    1. 如果在实验室中进行大量的细胞培养,制作培养基是值得的。 准备使用的MEM-E也可以从几家公司购买。
    2. 具有Earls盐的MEM(MEM-E)也是不错的选择,但需要CO
  3. 水晶紫溶液
    混合750ml H 2 O,250ml乙醇和5g结晶紫
    搅拌直至溶解并在RT下贮存

致谢

该协议最初发表于Novella 等人(2013)。工作是由托莱多大学的资金支持的ISN。

参考文献

  1. Holland,J.J.,de la Torre,J.C.,Clarke,D.K。和Duarte,E。(1991)。 定量RNA病毒克隆群体的相对适合度和适应性。 J Virol 65(6):2960-2967。
  2. Holland,J.J.,de la Torre,J.C.,Steinhauer,D.A.,Clarke,D.,Duarte,E.and Domingo,E。(1989)。 病毒突变频率可以通过病毒体的单克隆抗体中和而大大低估。 J Virol 63(12):5030-5036。
  3. Novella,I.S.,Presloid,J.B.,Zhou,T.,Smith-Tsurkan,S.D.,Ebendick-Corpus,B.E.,Dutta,R.N.,Lust,K.L.and Wilke,C.O.(2010)。 具有适应性差异的水泡性口炎病毒株的基因组进化。 J Virol 84(10):4960-4968。
  4. Novella,I.S.,Presloid,J.B.,Beech,C.and Wilke,C.O。(2013)。 水泡性口炎病毒的适应性和遗传稳定性的一致进化。 87(9):4923-4928。
  5. Valcarcel,J。和Ortin,J。(1989)。 表型隐藏:RNA病毒中突变的遗留,如流感病毒中mar突变体的检测所示。 J Virol 63(9):4107-4109。
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:Novella, I. S. (2014). Measuring Genetic Robustness in Vesicular Stomatitis Virus. Bio-protocol 4(6): e1073. DOI: 10.21769/BioProtoc.1073.
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