参见作者原研究论文

本实验方案简略版
Mar 2018

本文章节


 

Random Insertional Mutagenesis of a Serotype 2 Dengue Virus Clone
2型登革病毒克隆的随机插入诱变   

引用 收藏 提问与回复 分享您的反馈 Cited by

Abstract

Protein tagging is a powerful method of investigating protein function. However, modifying positive-strand RNA virus proteins in the context of viral infection can be particularly difficult as their compact genomes and multifunctional proteins mean even small changes can inactivate or attenuate the virus. Although targeted approaches to functionally tag viral proteins have been successful, these approaches are time consuming and inefficient. A strategy that has been successfully applied to several RNA viruses is whole-genome transposon insertional mutagenesis. A library of viral genomes, each containing a single randomly placed small insertion, is selected by passaging in cell culture and the insertion sites can be identified using Next Generation Sequencing (NGS). Here we describe a protocol for transposon mutagenesis of the 16681 strain of dengue virus, serotype 2. Mutant dengue virus libraries containing short randomly placed insertions are passaged through mammalian cells and insertions are mapped by NGS of the viable progeny. The protocol is divided into four stages: transposon mutagenesis of a dengue cDNA clone, viral genome transfection into permissive cells, isolation of viral progeny genomes, and sequencing library preparation.

Keywords: Transposon (转座子), Mutagenesis (诱变), RNA virus (RNA病毒), Dengue (登革热), Flavivirus (黄病毒属)

Background

A key aspect of understanding viral pathogenesis is elucidating the viral proteins’ functions during infection. However, viral proteins, particularly those encoded by compact viral genomes, are often multifunctional and therefore more challenging to study, in part because they are often difficult to tag (epitope tags, fluorescent proteins, etc.) in the context of an infectious virus genome without compromising viral infection. One workaround is to express individually tagged proteins in cells, which may result in an incomplete picture of the viral protein’s function as the other viral proteins are not present. It also does not directly determine if the functional tag interferes with the tagged protein’s function(s) in viral infection. Another approach is the empiric tagging of viral proteins in the context of an infectious virus genome, which ensures viral viability but is an inefficient process that is difficult to scale.

Transposon mutagenesis can help dissect the functions of proteins under various experimental conditions and has been used at a whole genome scale to elucidate the role of various proteins during microbial infections. This approach has been successfully applied to a number of positive-strand RNA viruses (Arumugaswami et al., 2008; Beitzel et al., 2010; Teterina et al., 2011; Thorne et al., 2012; Remenyi et al., 2014; Eyre et al., 2017; Fulton et al., 2017). By coupling the transposon mutagenesis approach to Next Generation Sequence, a map of sites in the viral genome that tolerate insertions can be determined with unprecedented resolution. Once these sites are identified, functional tags can be introduced into these sites through site-directed mutagenesis. This protocol describes whole-genome transposon insertion mapping applied to the 16681 strain of serotype 2 dengue virus.

Materials and Reagents

  1. Pipette tips (Fisher Scientific, catalog numbers: 02-707-80 ; 02-707-167 )
  2. 6-well plate (Corning, catalog number: 3506 )
  3. 0.22 μm sterile filter (Corning, catalog number: 431219 )
  4. Cell scraper (Corning, catalog number: 3010 )
  5. 10-beta chemically competent E. coli (New England Biolabs, catalog number: C3019H ) or equivalent–genotype used Δ(ara-leu) 7697 araD139 fhuA ΔlacX74 galK16 galE15 e14- ϕ80dlacZΔM15 recA1 relA1 endA1 nupG rpsL (StrR) rph spoT1 Δ(mrr-hsdRMS-mcrBC)
  6. Primers
    1. Fragment A (SacI/NarI) forward with T7 sequence
      GAAATTAATACGACTCACTATAAGTTGTTAGTCTACGTG
    2. Fragment A (SacI/NarI) reverse
      GTCATAGTGGCGCCTACCATAACCATCACTCTTCCC
    3. Fragment B (NarI/EcoRV) forward
      GGTTATGGTAGGCGCCACTATGACGGATGAC
    4. Fragment B (NarI/EcoRV) reverse
      CTGCTTCCTGATATCTCTGCCTGGTCTTCCC
    5. Fragment C (EcoRV/XbaI) forward
      GACCAGGCAGAGATATCAGGAAGCAGTCCAATCC
    6. Fragment C (EcoRV/XbaI) reverse
      AGAACCTGTTGATTCAACAGCAC
    7. Ion Torrent P1 adaptor top oligo
      CCACTACGCCTCCGCTTTCCTCTCTATGGGCAGTCGGTGAT
    8. Ion Torrent P1 adaptor bottom oligo
      <phos>ATCACCGACTGCCCATAGAGAGGAAAGCGGAGGCGTAGTGGTT
    9. Modified Ion Torrent A adaptor top oligo
      <phos>GGCCGCCTGAGTCGGAGACACGCAGGGATGAGATGGTT
    10. Modified Ion Torrent A adaptor bottom oligo
      CGGACTCAGCCTCTGTGCGTCCCTACTCTACC
  7. SacI (New England Biolabs, catalog number: R3156 )
  8. NarI (New England Biolabs, catalog number: R0191 )
  9. EcoRV (New England Biolabs, catalog number: R3195 )
  10. XbaI (New England Biolabs, catalog number: R0145 )
  11. NotI (New England Biolabs, catalog number: R0189 )
  12. Mutation Generation System kit (Thermo Fisher Scientific, catalog number: F-701 )
  13. Carbenicillin (Fisher Scientific, catalog number: BP26481 )
  14. Kanamycin (Thermo Fisher Scientific, catalog number: 11815024 )
  15. Promega Wizard Maxiprep kit (Promega, catalog number: A7270 )
  16. Promega Gel Purification kit (Promega, catalog number: A9281 )
  17. m7g(5')ppp(5')A RNA Cap Structure analog (New England Biolabs, catalog number: S1405S )
  18. Agarose (Thermo Fisher Scientific, catalog number: 16500100 )
  19. Q5 polymerase (New England Biolabs, catalog number: M0493 )
  20. T7 MEGAscript in vitro RNA translation kit (Thermo Fisher Scientific, catalog number: AM1334 )
  21. ZYMO Quick-RNA Viral kit (ZYMO RESEARCH, catalog number: R1034 )
  22. Applied Biosciences High Capacity cDNA kit (Thermo Fisher Scientific, catalog number: 4368814 )
  23. Ion Library Taqman Quantitation kit (Thermo Fisher Scientific, catalog number: 4468802 )
  24. 10x TBE (Fisher Scientific, catalog number: BP1333 )
  25. T4 DNA ligase (New England Biolabs, catalog number: M0202 )
  26. T4 PNK (New England Biolabs, catalog number: M0201 )
  27. dNTPs (Thermo Fisher Scientific, catalog number: R1121 )
  28. AMPure XP Beads (Beckman Coulter, catalog number: A63881 )
  29. LB medium (Fisher Scientific, catalog number: BP1425-500 )
  30. PEG 8000 (Thermo Fisher Scientific, catalog number: BP233-100 )
  31. Vero cells (ATCC, catalog number: CCL-81 )
  32. TransMessenger transfection reagent (QIAGEN, catalog number: 301525 )
  33. Full-length dengue virus clone 16681 serotype 2 cDNA (in plasmid pD2/IC-30P-NBX, Huang et al., 2010).
    Note: This cDNA was a generous gift of Dr. Huang from the Centers for Disease Control.

Equipment

Note: No specific equipment is necessary and any model of the following should suffice.

  1. Balance
  2. Pipettes
  3. Heat block
  4. Incubator
  5. Thermal cycler
  6. Water bath
  7. Microcentrifuge
  8. Magnet for Ampure XP bead purification

Procedure

  1. Transposon Mutagenesis
    This section describes the process of generating a library consisting of a randomly inserted transposon in a cDNA clone of the viral genome on a plasmid. By excising the bulk of the 1.2 kb transposon using unique NotI sites, the randomly inserted transposon is reduced to 15 nucleotides containing a NotI site that introduces a 5-amino acid insertion without introducing a frameshift or nonsense mutation. This insertion is TGCGGCCGCAN1N2N3N4N5 where N1N2N3N4N5 denotes a duplication of the five nucleotides directly upstream of the insertion.
    For our studies, the dengue cDNA clone was fragmented to eliminate genetic instability and toxicity in E. coli observed with full-length flavivirus clones. However, viral genome fragmentation is not required if the full-length viral genome is genetically stable and nontoxic in E. coli. Note that the starting plasmid must not contain any NotI restriction sites; if any exist they should be destroyed by site-directed mutagenesis.
    1. Divide the full-length dengue virus clone 16681 serotype 2 cDNA (in plasmid pD2/IC-30P-NBX, Huang et al., 2010) into three fragments flanked by SacI and NarI (Fragment A), NarI and EcoRV (Fragment B), EcoRV and XbaI (Fragment C) (Figure 1, step 1) and subclone back into the original vector with a modified multiple cloning site containing SacI, NarI, EcoRV, and XbaI sites (Figure 1, step 2).
    2. Mutagenize the plasmids using MuA Transposase supplied in the Mutation Generation Kit to introduce an engineered transposon randomly into each plasmid. Under these conditions, only one transposon should be introduced into each DNA plasmid.
      1. Calculate amount of target DNA per reaction by multiplying the plasmid size (in kb) by 40 ng.


      2. Incubate reactions at 30 °C for 60 min, and heat inactivate at 75 °C for 10 min.
    3. Dilute the transposase reaction tenfold in sterile nuclease-free water to a total of 200 μl and transform into high-efficiency recA- competent cells following the manufacturer’s protocol using a maximum of 5 μl per transformation. We used chemically competent cells, but electrocompetent cells may be used, as long as the transformation efficiency is at least 108 cfu/μg. If electroporation is used, the reactions should be diluted by ten-fold to avoid arcing. Twenty reactions should be sufficient to generate an estimated nucleotide coverage of at least 10 (see Step A5).
    4. Select transformed E. coli on LB plates containing carbenicillin (to select for the plasmid) and kanamycin (to select for the transposon). 
    5. Count the number of independent colonies to determine the library coverage. At this stage, the library coverage can be calculated by dividing the number of observed colonies by the total number of nucleotides in the plasmid (viral cDNA fragment plus backbone). We recommend a nucleotide coverage of at least 10-fold. Scrape the colonies from the plates into resuspension buffer from a Promega Wizard Maxiprep kit and prepare plasmid DNA. One Promega Wizard Maxiprep column should be used per 10,000 colonies.


      Figure 1. Protocol Schematic. 1. Fragment the full-length dengue virus genome into three parts using unique restriction enzyme cut sites. 2. Clone the fragments into a clean vector backbone. 3. Mutagenize each of these constructs independently using a mutation generation kit to generate randomly inserted transposon containing antibiotic resistance, and select mutated constructs for antibiotic resistance contained on the transposon. 4. Excise the dengue virus fragment from the vector backbone and ligate back into a clean vector to ensure that the transposon was located only in the dengue virus fragment and not the vector backbone. 5. Next, remove the transposon using a unique enzyme restriction site introduced by the transposon and self-ligate constructs to leave a small 15 nucleotide genetic scar (5aa). 6. Amplify the genetic scar containing fragment by PCR in parallel with corresponding wildtype fragments. 7. Stitch together using PCR the genetic scar containing fragment and wildtype fragments to obtain a full-length dengue virus genome. 8. Transcribe the genome in vitro and transfect RNA into permissive cells. Recover viable progeny virus from the transfection, isolate RNA, and synthesize cDNA. Amplify the fragment containing the genetic scar. 9. Next Generation Sequencing libraries were constructed from the PCR amplified genetic scar containing fragments, and sequenced. Reads and trimmed reads used for alignment are displayed.

    6. The transformants will contain a transposon insertion in either the viral cDNA fragment or in the vector backbone. Steps C6-C8 will isolate only the transposon insertions within the viral cDNA fragments (Figure 1, step 4). Digest 5 μg of each plasmid library with Sac1 and Nar1 (Fragment 1), NarI and EcoRV (Fragment 2), or EcoRV and XbaI (Fragment 3) and separate on a 1% TBE-agarose gel. Each digest should contain four different bands consisting of the vector backbone, the vector with transposon insertion, the viral cDNA fragment, and the viral cDNA fragment with the transposon insertion. Excise the bands corresponding to the viral cDNA fragments with the transposon insertion and purify. 
    7. Ligate the gel purified bands back into the original clean vector backbone digested with the appropriate compatible restriction enzymes using T4 DNA Ligase following the manufacturer’s protocol.
    8. Transform the ligation reaction into high-efficiency competent cells following the manufacturer’s protocol and select on LB plates with carbenicillin and kanamycin. The library coverage can be again calculated in a similar method to Step A5. To maintain library coverage above 10-fold coverage, ensure that at least as many colonies are recovered in this step as were recovered in Step A5. Purify plasmid DNA as in Step A5. 
    9. Digest 5 μg of isolated plasmids with NotI and run on a 1% agarose gel. This digestion removes the bulk of the 1.2 kb transposon, leaving behind the 15-nucleotide insertion (Figure 1, step 5). Excise the larger band and discard the 1.2 kb transposon fragment. 
    10. Recircularize the gel-purified band using T4 DNA ligase following the manufacturer’s protocol.
    11. Transform the ligation reaction into high-efficiency competent cells following the manufacturer’s protocol and select on LB-carbenicillin plates. 
    12. Count E. coli colonies and purify plasmid DNA as in Step A5. To ensure high coverage ensure that at least as many colonies are counted and recovered as in Steps A5 and A8.

  2. Viral genome transfection into permissive cells
    This section describes the process of generating full-length mutant dengue genomes in vitro from the fragments by splice overlap extension (SOE) PCR, in vitro transcribing full-length dengue genomes to RNA, and transfecting viral RNA into permissive cells. To maintain library complexity, reactions are performed in at least three replicates for each step and pooled. Several independent transfections of viral RNA into permissive cells should also be performed to minimize bottleneck effects.
    1. PCR amplify the mutagenized dengue fragment libraries from the previous step (Figure 1, step 6).
      1. Amplify the mutagenized Fragment A, B, and C libraries with the forward and reverse primers listed in “Materials and Reagents.”


      2. In addition, amplify the corresponding wild-type viral fragments A, C, AB (Using Fragment A (SacI/NarI) forward with T7 sequence and Fragment B (NarI/EcoRV) reverse), and BC (Using Fragment B (NarI/EcoRV) forward and Fragment C (EcoRV/XbaI) reverse) fragments from an unmodified cDNA copy of dengue virus clone 16681 pD2/IC-30P-NBX.
        Thermal cycler program: (1) 95 °C for 2 min; (2) 95 °C for 20 sec, 60 °C for 20 sec, and 72 °C for 1 min/kb product x 35 cycles; (3) hold at 4 °C. 
    2. Gel-purify PCR products on a 1% agarose gel.
    3. Construct full-length mutant dengue genomes using splicing by overlap extension (SOE) PCR (Figure 1, step 7) in the following PCR reactions. Splice mutagenized fragment A with wildtype fragment BC, mutagenized fragment B with wildtype fragments A and C, and mutagenized fragment C with wildtype fragment AB. The resulting full-length genomes will contain a random insertion within fragment A, B, or C, respectively. To ensure enough material for subsequent steps, perform three independent reactions.
       

      1. Thermal cycler program: (1) 95 °C for 2 min; (2) 95 °C for 20 sec, 72 °C for 1 min/kb product x 10 cycles.
      2. Add 2.5 μl of 10 μM fragment A forward primer and 2.5 μl of 10 μM fragment C reverse primer to each reaction.
      3. Thermal cycler program: (1) 95 °C for 2 min; (2) 95 °C for 20 sec, 60 °C for 20 sec, and 72 °C for 11 min x 35 cycles; (3) hold at 4 °C.
    4. Gel-purify the full-length dengue virus genomes on a 1% agarose gel.
    5. Use 200 ng of purified full-length genome for in vitro transcription using the T7 MEGAscript kit. Note that an m7G(5’) ppp(5’)A RNA cap analog is used in the in vitro transcription reaction as the dengue virus RNA has a 5’ cap, but is not needed for the synthesis of uncapped viral RNA genomes (e.g., hepatitis C virus).
       

      1. Incubate reactions at 37 °C for 6 h. To increase viral RNA yield, add 1 μl of 10 mM ATP, supplied in the T7 MEGAscript kit, to the reaction 1 h after beginning the incubation.
      2. Add 1 μl of the DNase solution supplied in the T7 MEGAscript kit, 30 min prior to completion of the incubation.
    6. Purify the in vitro transcribed viral RNA using the ZYMO Quick-RNA Viral kit following the manufacturer’s protocol. Elute in 50 μl of nuclease-free water prewarmed to 70 °C.
    7. Quantitate the viral RNA. We observed best results from freshly made RNA, although storing at -80 °C only negligibly decreases transfection efficiency.
    8. One day before viral RNA transfection, plate permissive cells in a 6-well plate such that they will be 80-90% confluent on the day of transfection. For dengue virus, plate 3.3 x 105 Vero cells per well of a 6-well plate.
    9. Transfect 2.5 μg of viral RNA per well using the QIAGEN TransMessenger kit with 4 μl enhancer and 8 μl transfection reagent.
      Note: The ratio of RNA to enhancer and transfection reagent should be optimized if cells other than Vero cells will be used.
    10. Wash off transfection complex 2 h after transfection.
      Note: Duration of transfection will vary based on cell type and should be determined experimentally.
    11. After three days, collect supernatant from wells, pool, and spin at 500 x g for 10 min to clarify. Use half of the clarified supernatant to infect naïve cells and store the other half at -80 °C as passage 1. Infect naïve cells by adding undiluted supernatant to Vero cells plated the day before infection at 1.7 x 105 cells per well of a 6-well plate, and replace with fresh medium the day after infection.
    12. After three days, collect supernatant from wells, pool, and spin at 500 x g for 10 min to clarify. Use half of the clarified supernatant to infect naïve cells and store the other half at -80 °C as passage 2. Infect naïve cells as described in Step B11. After three days, collect supernatant from wells, pool, and spin at 500 x g for 10 min to clarify. Store at -80 °C as passage 3.

  3. Isolation of viral progeny genome and Next Generation Sequencing library preparation
    1. Concentrate virus from passage 1, 2, and 3 supernatants by PEG precipitation.
      1. Make a 40% (w/v) PEG8000 solution in PBS and filter through a 0.22 μm sterile filter.
      2. Add 40% PEG8000 to the supernatant to a final concentration of 8% PEG8000, and incubate overnight at 4 °C.
      3. Centrifuge at 7,500 x g for 4 h at 4 °C.
      4. Remove the supernatant and gently resuspend the pellet in 1 ml of sterile PBS by pipetting.
    2. Split the resuspended virus into three equal volumes and isolate viral RNA from each using the ZYMO Quick-RNA Viral kit following the manufacturer’s procedure. Elute in a volume of 20 μl per column and pool the purified viral RNAs.
    3. Reverse transcribe the purified viral RNA into cDNA using the Applied Biosciences High Capacity cDNA kit following the manufacturer’s procedure. Use all 60 μl of the eluted RNA from Step C2 to make cDNA, and pool after cDNA is made.
    4. PCR amplify the recovered cDNA for the genome fragment that contains the transposon insertion using primers designed to generate smaller ~250 bp amplicons for Next Generation Sequencing.


      Thermal cycler program: (1) 95 °C for 2 min; (2) 95 °C for 20 sec, 60 °C for 20 sec, and 72 °C for 1 min/kb product x 35 cycles; (3) hold at 4 °C.
    5. Clean up the amplicons using the PCR cleanup protocol for the Promega Gel Purification Kit.
    6. At this stage of the protocol, amplicons can be quantitated and submitted for library preparation and next-generation sequencing.

  4. Next Generation Sequencing library preparation
    This section of the protocol generates libraries compatible with Torrent PGM sequencing. Note that the library preparation protocol has been modified to select for NotI containing DNA fragments to enrich the sequencing reads for transposon insertion sites, which contain a NotI site. By blunt ligating one adaptor to each end of the DNA fragment, cutting with NotI, and ligating a NotI containing read adaptor, we created sequencing libraries where each read contained the insertion site. In principle, this strategy could be adapted to generate libraries compatible with Illumina sequencing, but this will have to be developed by the user.
    1. Combine amplicons from step C5 in an equimolar fashion to generate a total of 1 μg of input DNA.
    2. Treat input DNA with T4 PNK to phosphorylate the ends for subsequent adapter ligation.


      Incubate reaction at 37 °C for 30 min, and heat inactivate at 65 °C for 20 min.
    3. Mix Ion Torrent P1 adaptor top and Ion Torrent P1 adaptor bottom together in sterile nuclease-free water to a final concentration of 44 μM for each oligo. Heat the tube containing the mixture in a water-filled beaker to 100 °C for 1 min, remove the beaker from heat, and allow it to cool to room temperature on a benchtop.
    4. Blunt ligate the phosphorylated amplicons to the Ion Torrent P1 adapter.


      Incubate reaction for 60 min at 25 °C.
    5. Clean up reactions using 72 μl of AMPure XP beads with a ratio of 1.8 bead volume to reaction volume, following the manufacturer’s protocol, and elute in 35 μl of sterile nuclease-free water.
    6. Digest reactions with NotI.


      Incubate reactions at 37 °C for 60 min, and heat inactivate NotI at 65 °C for 20 min.
    7. Clean up reactions using AMPure XP beads as in Step D4 and eluting in 34 μl of sterile nuclease-free water.
    8. Anneal Modified Ion Torrent A adaptor top and Modified Ion Torrent A adaptor bottom oligos as described in Step D3.
    9. Ligate reactions to the annealed Modified Ion Torrent A adaptor.


      Incubate reaction for 90 min at 16 °C.
    10. Clean up reactions using AMPure XP beads as in Step D4 and eluting in 35 μl of sterile nuclease-free water.
    11. Quantitate the libraries using the Ion Torrent Library quantitation kit following the manufacturer’s protocol.
    12. Submit the quantitated libraries for sequencing on Ion Torrent PGM.

Data analysis

Mapping sequencing reads to the viral genome, and enumerating these results can be challenging to individuals not proficient in bioinformatics. Collaboration with an experienced bioinformatician should be established to help produce accurate results. The following is a broad overview of the approach to determine sites in the genome that tolerate small insertions.

  1. Obtain the raw reads of the sequencing results (such as a FASTQ file). Next, split the reads into two groups; one containing TGCGGCCGCAN1N2N3N4N5 at the beginning of the read (Figure 1, step 9) and the other that did not contain that exact sequence. 
  2. Trim the first ten nucleotides of each read to remove the transposon inserted sequence (Figure 1, step 9), and leave the Dengue genome sequence for alignment.
  3. Using the trimmed reads only, align reads to the dengue genome using the default settings of BOWTIE2. BOWTIE2 can be obtained from http://www.sourceforge.net.
  4. Obtain the alignment file, and extract the start position of the alignment for each read. This site is 5 bases upstream of the actual insertion due to the duplication of the insert caused by the transposase (see Procedure A), so these alignment sites should be reduced by 5.
  5. Enumerate the number of reads for each site in the viral genome.
  6. Normalize the number of reads by dividing the insertion sites counts by the total number of reads from the sequencing runs, so you can compare multiple sequencing runs.

Notes

Detailed recipes are supplied in the protocol above. Troubleshooting tips are available in each of the manufacturer’s protocols and should be read before performing experiments to highlight potential problems with the execution of the said experiment.

Acknowledgments

This protocol was adapted from several previous transposon insertional mutagenesis studies (Arumugaswami et al., 2008; Beitzel et al., 2010; Teterina et al., 2011; Thorne et al., 2012; Remenyi et al., 2014). We thank Dr. Claire Huang (CDC) for the pD2/IC-30-P-NBX infectious cDNA clone. This work was supported by National Institutes of Health grants R01DK097374 (A.W.T.), and the Michigan Institute for Clinical and Health Research (MICHR) grant UL1TR002240 (J.W.P.), and the University of Michigan Center for Gastrointestinal Research (UMCGR), grant 5P30DK034933.

Competing interests

The authors have no conflicts of interest to disclose.

References

  1. 1Arumugaswami, V., Remenyi, R., Kanagavel, V., Sue, E. Y., Ngoc Ho, T., Liu, C., Fontanes, V., Dasgupta, A. and Sun, R. (2008). High-resolution functional profiling of hepatitis C virus genome. PLoS Pathog 4(10): e1000182.
  2. Beitzel, B. F., Bakken, R. R., Smith, J. M. and Schmaljohn, C. S. (2010). High-resolution functional mapping of the venezuelan equine encephalitis virus genome by insertional mutagenesis and massively parallel sequencing. PLoS Pathog 6(10): e1001146.
  3. Eyre, N. S., Johnson, S. M., Eltahla, A. A., Aloi, M., Aloia, A. L., McDevitt, C. A., Bull, R. A. and Beard, M. R. (2017). Genome-wide mutagenesis of dengue virus reveals plasticity of the NS1 protein and enables generation of infectious tagged reporter viruses. J Virol 91(23). 
  4. Fulton, B. O., Sachs, D., Schwarz, M. C., Palese, P. and Evans, M. J. (2017). Transposon mutagenesis of the zika virus genome highlights regions essential for RNA replication and restricted for immune evasion. J Virol 91(15).
  5. Huang, C. Y., Butrapet, S., Moss, K. J., Childers, T., Erb, S. M., Calvert, A. E., Silengo, S. J., Kinney, R. M., Blair, C. D. and Roehrig, J. T. (2010). The dengue virus type 2 envelope protein fusion peptide is essential for membrane fusion. Virology 396(2): 305-315.
  6. Remenyi, R., Qi, H., Su, S. Y., Chen, Z., Wu, N. C., Arumugaswami, V., Truong, S., Chu, V., Stokelman, T., Lo, H. H., Olson, C. A., Wu, T. T., Chen, S. H., Lin, C. Y. and Sun, R. (2014). A comprehensive functional map of the hepatitis C virus genome provides a resource for probing viral proteins. MBio 5(5): e01469-01414.
  7. Teterina, N. L., Lauber, C., Jensen, K. S., Levenson, E. A., Gorbalenya, A. E. and Ehrenfeld, E. (2011). Identification of tolerated insertion sites in poliovirus non-structural proteins. Virology 409(1): 1-11.
  8. Thorne, L., Bailey, D. and Goodfellow, I. (2012). High-resolution functional profiling of the norovirus genome. J Virol 86(21): 11441-11456.

简介

蛋白质标记是研究蛋白质功能的有效方法。然而,在病毒感染的情况下修饰正链RNA病毒蛋白可能特别困难,因为它们的紧密基因组和多功能蛋白意味着即使很小的变化也可以使病毒失活或减弱。尽管功能性标记病毒蛋白的靶向方法已经成功,但这些方法耗时且效率低。已经成功应用于几种RNA病毒的策略是全基因组转座子插入诱变。通过细胞培养中的传代选择病毒基因组文库,每个文库含有单个随机放置的小插入,并且可以使用下一代测序(NGS)鉴定插入位点。在这里,我们描述了用于登革病毒16681株血清型2的转座子诱变的方案。含有短随机放置插入物的突变登革病毒文库通过哺乳动物细胞传代,插入由有活力后代的NGS定位。该方案分为四个阶段:登革热cDNA克隆的转座子诱变,病毒基因组转染到允许细胞,分离病毒后代基因组和测序文库制备。

【背景】 理解病毒发病机制的一个关键方面是阐明病毒蛋白在感染过程中的功能。然而,病毒蛋白,特别是那些由紧凑病毒基因组编码的蛋白,往往是多功能的,因此研究更具挑战性,部分原因是它们通常难以标记(表位标签,荧光蛋白,等)。传染病毒基因组的背景,不会影响病毒感染。一种解决方法是在细胞中表达单独标记的蛋白质,这可能导致病毒蛋白质功能的不完整图像,因为其他病毒蛋白质不存在。它也不能直接确定功能标签是否会干扰病毒感染中标记蛋白的功能。另一种方法是在感染性病毒基因组的背景下对病毒蛋白进行经验标记,其确保病毒活力,但是是难以扩展的低效过程。

转座子诱变可以帮助在各种实验条件下剖析蛋白质的功能,并且已经在全基因组规模上用于阐明各种蛋白质在微生物感染期间的作用。该方法已成功应用于许多正链RNA病毒(Arumugaswami et al。,2008; Beitzel et al。,2010; Teterina et al 。,2011; Thorne et al。,2012; Remenyi et al。,2014; Eyre et al。,2017; Fulton et al。,2017)。通过将转座子诱变方法与下一代序列偶联,可以以前所未有的分辨率确定病毒基因组中耐受插入的位点图谱。一旦确定了这些位点,就可以通过定点诱变将功能标签引入这些位点。该协议描述了应用于血清型2登革热病毒的16681菌株的全基因组转座子插入作图。

关键字:转座子, 诱变, RNA病毒, 登革热, 黄病毒属

材料和试剂

  1. 移液器吸头(Fisher Scientific,目录号:02-707-80; 02-707-167)
  2. 6孔板(康宁,目录号:3506)
  3. 0.22μm无菌过滤器(Corning,目录号:431219)
  4. 细胞刮刀(Corning,目录号:3010)
  5. 10-beta化学感受态 E.大肠杆菌(New England Biolabs,目录号:C3019H)或等效基因型使用Δ(ara-leu)7697 araD139 fhuA Δ lacX74 galK16 galE15 e14-φ80 d lacZ Δ M15 recA1 relA1 endA1 nupG rpsL (Str R ) rph spoT1 Δ(mrr- hsdRMS-的McrBC)
  6. 引物
    1. 片段A(SacI / NarI)向前与T7序列
      GAAATTAATACGACTCACTATAAGTTGTTAGTCTACGTG
    2. 片段A(SacI / NarI)反转
      GTCATAGTGGCGCCTACCATAACCATCACTCTTCCC
    3. 片段B(NarI / EcoRV)前进
      GGTTATGGTAGGCGCCACTATGACGGATGAC
    4. 片段B(NarI / EcoRV)反向
      CTGCTTCCTGATATCTCTGCCTGGTCTTCCC
    5. 片段C(EcoRV / XbaI)前进
      GACCAGGCAGAGATATCAGGAAGCAGTCCAATCC
    6. 片段C(EcoRV / XbaI)反转
      AGAACCTGTTGATTCAACAGCAC
    7. Ion Torrent P1适配器顶级寡核苷酸
      CCACTACGCCTCCGCTTTCCTCTCTATGGGCAGTCGGTGAT
    8. Ion Torrent P1适配器底部oligo
      &LT; PHOS&GT; ATCACCGACTGCCCATAGAGAGGAAAGCGGAGGCGTAGTGGTT
    9. 改良Ion Torrent适配器顶级寡核苷酸
      &LT; PHOS&GT; GGCCGCCTGAGTCGGAGACACGCAGGGATGAGATGGTT
    10. 改良Ion Torrent适配器底部寡头
      CGGACTCAGCCTCTGTGCGTCCCTACTCTACC
  7. SacI(New England Biolabs,目录号:R3156)
  8. NarI(New England Biolabs,目录号:R0191)
  9. EcoRV(New England Biolabs,目录号:R3195)
  10. XbaI(New England Biolabs,目录号:R0145)
  11. NotI(New England Biolabs,目录号:R0189)
  12. 突变生成系统试剂盒(Thermo Fisher Scientific,目录号:F-701)
  13. 羧苄青霉素(Fisher Scientific,目录号:BP26481)
  14. 卡那霉素(赛默飞世尔科技,目录号:11815024)
  15. Promega Wizard Maxiprep套件(Promega,目录号:A7270)
  16. Promega凝胶纯化试剂盒(Promega,目录号:A9281)
  17. m 7 g(5')ppp(5')RNA帽结构类似物(New England Biolabs,目录号:S1405S)
  18. 琼脂糖(赛默飞世尔科技,目录号:16500100)
  19. Q5聚合酶(New England Biolabs,目录号:M0493)
  20. T7 MEGAscript 体外 RNA翻译试剂盒(Thermo Fisher Scientific,目录号:AM1334)
  21. ZYMO Quick-RNA病毒试剂盒(ZYMO RESEARCH,目录号:R1034)
  22. Applied Biosciences高容量cDNA试剂盒(赛默飞世尔科技,目录号:4368814)
  23. Ion Library Taqman定量试剂盒(Thermo Fisher Scientific,目录号:4468802)
  24. 10x TBE(Fisher Scientific,目录号:BP1333)
  25. T4 DNA连接酶(New England Biolabs,目录号:M0202)
  26. T4 PNK(New England Biolabs,目录号:M0201)
  27. dNTPs(赛默飞世尔科技,目录号:R1121)
  28. AMPure XP Beads(Beckman Coulter,目录号:A63881)
  29. LB培养基(Fisher Scientific,目录号:BP1425-500)
  30. PEG 8000(Thermo Fisher Scientific,目录号:BP233-100)
  31. Vero细胞(ATCC,目录号:CCL-81)
  32. TransMessenger转染试剂(QIAGEN,目录号:301525)
  33. 全长登革病毒克隆16681血清型2 cDNA(质粒pD2 / IC-30P-NBX,Huang 等人,2010)。
    注意:这个cDNA是疾病控制中心黄博士的慷慨礼物。

设备

注意:不需要特定设备,以下任何型号都应该足够。

  1. 平衡
  2. 移液器
  3. 加热块
  4. 恒温箱
  5. 热循环仪
  6. 水浴
  7. 微量
  8. 用于Ampure XP珠子净化的磁铁

程序

  1. 转座子诱变
    该部分描述了在质粒上产生由病毒基因组的cDNA克隆中随机插入的转座子组成的文库的过程。通过使用独特的NotI位点切除大部分1.2kb转座子,随机插入的转座子被还原为含有NotI位点的15个核苷酸,其引入5-氨基酸插入而不引入移码或无义突变。这种插入是TGCGGCCGCAN 1 N 2 N 3 N 4 N 5 其中N 1 N 2 N 3 N 4 N 5 表示五个重复直接插入上游的核苷酸。
    对于我们的研究,登革热cDNA克隆被片段化以消除 E中的遗传不稳定性和毒性。用全长黄病毒克隆观察到大肠杆菌。然而,如果全长病毒基因组在 E中具有遗传稳定性和无毒性,则不需要病毒基因组片段化。大肠杆菌。注意,起始质粒不得含有任何NotI限制性位点;如果存在,它们应该被定点突变破坏。
    1. 将全长登革病毒克隆16681血清型2 cDNA(在质粒pD2 / IC-30P-NBX,Huang et al。,2010中)分成三个侧翼为SacI和NarI的片段(片段A), NarI和EcoRV(片段B),EcoRV和XbaI(片段C)(图1,步骤1)和亚克隆回到原始载体,具有修饰的多克隆位点,含有SacI,NarI,EcoRV和XbaI位点(图1,步骤) 2)。
    2. 使用突变生成试剂盒中提供的MuA转座酶诱变质粒,将工程转座子随机引入每个质粒中。在这些条件下,每个DNA质粒中只应引入一个转座子。
      1. 通过将质粒大小(以kb计)乘以40 ng来计算每个反应的靶DNA量。


      2. 在30°C孵育反应60分钟,并在75°C加热灭活10分钟。
    3. 在无菌无核酸酶水中将转座酶反应稀释10倍至总共200μl,并按照制造商的方案转化为高效recA-感受态细胞,每次转化最多5μl。我们使用化学感受态细胞,但是可以使用电感受态细胞,只要转化效率至少为10 8 cfu /μg。如果使用电穿孔,则应将反应稀释10倍以避免电弧放电。二十个反应应该足以产生至少10的估计核苷酸覆盖度(参见步骤A5)。
    4. 选择已转换的 E.在含有羧苄青霉素(以选择质粒)和卡那霉素(以选择转座子)的LB平板上的大肠杆菌。&nbsp;
    5. 计算独立菌落的数量以确定库覆盖率。在此阶段,可以通过将观察到的菌落数除以质粒(病毒cDNA片段加骨架)中的核苷酸总数来计算文库覆盖率。我们建议核苷酸覆盖率至少为10倍。将来自平板的菌落从Promega Wizard Maxiprep试剂盒中刮入重悬浮缓冲液中,并制备质粒DNA。每10,000个菌落应使用一个Promega Wizard Maxiprep柱。


      图1.方案示意图。 1.使用独特的限制性酶切位点将全长登革热病毒基因组分成三部分。 2.将片段克隆到干净的载体骨架中。 3.使用突变生成试剂盒独立诱变这些构建体中的每一个以产生含有抗生素抗性的随机插入的转座子,并选择转座子上包含的抗生素抗性的突变构建体。 4.从载体骨架中切除登革热病毒片段并连接回干净载体,以确保转座子仅位于登革病毒片段而不是载体骨架中。 5.接下来,使用由转座子和自身连接构建体引入的独特酶限制性位点去除转座子,留下小的15个核苷酸遗传瘢痕(5aa)。 6.通过PCR与相应的野生型片段平行扩增含有遗传瘢痕的片段。 7.使用PCR将含有遗传瘢痕的片段和野生型片段拼接在一起,以获得全长登革热病毒基因组。 8.转录基因体外并将RNA转染到允许细胞中。从转染中回收可存活的子代病毒,分离RNA并合成cDNA。扩增含有遗传瘢痕的片段。 9.从含有片段的PCR扩增的遗传瘢痕构建下一代测序文库,并测序。显示用于对齐的读取和修剪读取。

    6. 转化子将在病毒cDNA片段或载体骨架中含有转座子插入。步骤C6-C8将仅分离病毒cDNA片段内的转座子插入(图1,步骤4)。用Sac1和Nar1(片段1),NarI和EcoRV(片段2),或EcoRV和XbaI(片段3)消化5μg每个质粒文库,并在1%TBE-琼脂糖凝胶上分离。每个消化物应包含四个不同的条带,包括载体骨架,带有转座子插入的载体,病毒cDNA片段和带有转座子插入的病毒cDNA片段。用转座子插入切除对应于病毒cDNA片段的条带并纯化。&nbsp;
    7. 按照制造商的方案,使用T4 DNA连接酶将凝胶纯化的条带重新连接回用适当的相容限制酶消化的原始清洁载体骨架。
    8. 按照制造商的方案将连接反应转化为高效感受态细胞,并在含有羧苄青霉素和卡那霉素的LB平板上进行选择。可以用与步骤A5类似的方法再次计算库覆盖率。为了将库覆盖率保持在10倍以上,确保在该步骤中至少回收与步骤A5中回收的菌落一样多的菌落。按步骤A5纯化质粒DNA。&nbsp;
    9. 用NotI消化5μg分离的质粒,并在1%琼脂糖凝胶上电泳。这种消化去除了1.2kb转座子的大部分,留下了15个核苷酸的插入(图1,步骤5)。切除较大的条带并丢弃1.2 kb的转座子片段。&nbsp;
    10. 按照制造商的方案,使用T4 DNA连接酶重新环化凝胶纯化的条带。
    11. 按照制造商的方案将连接反应转化为高效感受态细胞,并在LB-羧苄青霉素平板上选择。&nbsp;
    12. 算上 E.大肠杆菌菌落并如步骤A5中那样纯化质粒DNA。为确保高覆盖率,确保至少按照步骤A5和A8计算和回收多少菌落。

  2. 病毒基因组转染到允许细胞中 本节描述了通过剪接重叠延伸(SOE)PCR,体外转录全长登革热基因组从片段中体外生成全长突变体登革热基因组的过程。 RNA,并将病毒RNA转染到允许的细胞中。为了保持文库复杂性,对每个步骤进行至少三次重复的反应并合并。还应进行几次独立的病毒RNA转染到允许细胞中,以最大限度地减少瓶颈效应。
    1. PCR扩增来自前一步骤的诱变的登革热片段文库(图1,步骤6)。
      1. 使用“材料和试剂”中列出的正向和反向引物扩增诱变的片段A,B和C文库。

      2. 此外,扩增相应的野生型病毒片段A,C,AB(使用片段A(SacI / NarI)向前与T7序列和片段B(NarI / EcoRV)反向)和BC(使用片段B(NarI / EcoRV) )来自登革病毒克隆16681 pD2 / IC-30P-NBX的未修饰cDNA拷贝的正向和片段C(EcoRV / XbaI)反向片段。
        热循环仪程序:(1)95°C,2分钟; (2)95℃20秒,60℃20秒,72℃1分钟/ kb产物×35个循环; (3)保持在4°C&nbsp;
    2. 在1%琼脂糖凝胶上凝胶纯化PCR产物。
    3. 在以下PCR反应中通过重叠延伸(SOE)PCR(图1,步骤7)使用剪接构建全长突变体登革热基因组。剪接突变片段A与野生型片段BC,诱变片段B与野生型片段A和C,和诱变片段C与野生型片段AB。得到的全长基因组将分别在片段A,B或C内包含随机插入。为确保后续步骤有足够的材料,请执行三个独立的反应。

      1. 热循环仪程序:(1)95°C,2分钟; (2)95℃20秒,72℃1分钟/ kb产物×10个循环。
      2. 向每个反应中加入2.5μl10μM片段A正向引物和2.5μl10μM片段C反向引物。
      3. 热循环仪程序:(1)95°C,2分钟; (2)95℃20秒,60℃20秒,72℃11分钟×35个循环; (3)保持在4°C。
    4. 在1%琼脂糖凝胶上凝胶纯化全长登革热病毒基因组。
    5. 使用T7 MEGAscript试剂盒,使用200 ng纯化的全长基因组进行体外转录。注意,m 7 G(5')ppp(5')A RNA帽类似物用于体外转录反应,因为登革病毒RNA具有5'帽,但不需要合成无上限的病毒RNA基因组(例如,丙型肝炎病毒)。


      1. 在37°C孵育反应6小时。为了增加病毒RNA产量,在开始孵育后1小时将1μl10mMATP(在T7 MEGAscript试剂盒中提供)加入到反应中。
      2. 在孵育完成前30分钟,加入1μlT7MEGAscript试剂盒中提供的DNase溶液。
    6. 按照制造商的方案,使用ZYMO Quick-RNA Viral试剂盒纯化体外转录的病毒RNA。在50μl预热至70°C的无核酸酶水中洗脱。
    7. 定量病毒RNA。我们观察到来自新制RNA的最佳结果,尽管在-80°C下储存仅可忽略不计地降低转染效率。
    8. 在病毒RNA转染前一天,在6孔板中的平板允许细胞使得它们在转染当天将为80-90%汇合。对于登革热病毒,在6孔板的每孔中加入3.3×10 5个 Vero细胞。
    9. 使用带有4μl增强子和8μl转染试剂的QIAGEN TransMessenger试剂盒每孔转染2.5μg病毒RNA。
      注意:如果使用Vero细胞以外的细胞,应优化RNA与增强子和转染试剂的比例。
    10. 转染后2小时洗去转染复合物。
      注意:转染的持续时间将根据细胞类型而有所不同,应通过实验确定。
    11. 三天后,从孔中收集上清液,合并并以500μL离心旋转10分钟以澄清。使用一半澄清的上清液感染幼稚细胞并将另一半储存在-80°C作为第1代。通过在感染前一天(1.7 x 10 5 6孔板的每孔细胞,感染后第2天用新鲜培养基替换。
    12. 三天后,从孔中收集上清液,合并并以500μL离心旋转10分钟以澄清。使用一半澄清的上清液感染幼稚细胞,并将另一半储存在-80°C作为第2代。如步骤B11所述感染幼稚细胞。三天后,从孔中收集上清液,合并并以500μL离心旋转10分钟以澄清。储存在-80°C作为第3道。

  3. 病毒后代基因组的分离和下一代测序文库制备
    1. 通过PEG沉淀浓缩来自第1,2和3代上清液的病毒。
      1. 在PBS中制备40%(w / v)PEG8000溶液,并通过0.22μm无菌过滤器过滤。
      2. 向上清液中加入40%PEG8000至终浓度为8%PEG8000,并在4℃下孵育过夜。
      3. 在4,500℃下以7,500 x g 离心4小时。
      4. 除去上清液,通过移液管将沉淀轻轻重悬于1ml无菌PBS中。
    2. 将重悬的病毒分成三等体积,按照制造商的程序使用ZYMO Quick-RNA Viral试剂盒分离各病毒RNA。以每柱20μl的体积洗脱并汇集纯化的病毒RNA。
    3. 使用Applied Biosciences High Capacity cDNA试剂盒按照制造商的程序将纯化的病毒RNA逆转录成cDNA。使用来自步骤C2的所有60μl洗脱的RNA制备cDNA,并在制备cDNA后汇集。
    4. PCR扩增含有转座子插入的基因组片段的回收cDNA,使用设计用于生成较小~250 bp扩增子的引物用于下一代测序。


      热循环仪程序:(1)95°C,2分钟; (2)95℃20秒,60℃20秒,72℃1分钟/ kb产物×35个循环; (3)保持在4°C。
    5. 使用Promega凝胶纯化试剂盒的PCR纯化方案清洗扩增子。
    6. 在该方案的这个阶段,可以对扩增子进行定量分析并提交用于文库制备和下一代测序。

  4. 新一代测序文库制备
    该协议的这一部分生成与Torrent PGM测序兼容的库。注意,文库制备方案已被修改以选择含有DNA片段的NotI,以富集含有NotI位点的转座子插入位点的测序读数。通过将一个衔接子平端连接到DNA片段的每个末端,用NotI切割,并连接含有NotI的读取适配器,我们创建了测序文库,其中每个读数包含插入位点。原则上,该策略可以适用于生成与Illumina测序兼容的文库,但这必须由用户开发。
    1. 将来自步骤C5的扩增子以等摩尔方式组合以产生总共1μg的输入DNA。
    2. 用T4 PNK处理输入DNA以使末端磷酸化以用于随后的衔接子连接。


      在37°C孵育反应30分钟,并在65°C加热灭活20分钟。
    3. 将Ion Torrent P1适配器顶部和Ion Torrent P1适配器底部混合在无菌无核酸酶水中,每种寡核苷酸的最终浓度为44μM。将含有混合物的管在充满水的烧杯中加热至100℃1分钟,将烧杯从加热中取出,并使其在台面上冷却至室温。
    4. 钝器将磷酸化的扩增子连接到Ion Torrent P1适配器。


      在25℃下孵育反应60分钟。
    5. 根据制造商的方案,使用72μl的AMPure XP珠子以1.8个珠子体积与反应体积的比例清洁反应,并在35μl无菌无核酸酶水中洗脱。
    6. 与NotI的消化反应。


      在37°C孵育反应60分钟,并在65°C加热灭活NotI 20分钟。
    7. 如步骤D4中那样使用AMPure XP珠子清洁反应,并在34μl无菌无核酸酶水中洗脱。
    8. 退火改良离子Torrent适配器顶部和改良Ion Torrent适配器底部寡核苷酸,如步骤D3中所述。
    9. Ligate对退火的改良型Ion Torrent A适配器的反应。


      在16°C孵育反应90分钟。
    10. 如步骤D4中那样使用AMPure XP珠子清洁反应,并在35μl无菌无核酸酶水中洗脱。
    11. 按照制造商的方案,使用Ion Torrent Library定量试剂盒对文库进行定量。
    12. 提交定量文库以在Ion Torrent PGM上测序。

数据分析

将测序序列定位到病毒基因组,并且对不熟悉生物信息学的个体来说,列举这些结果可能具有挑战性。应建立与经验丰富的生物信息学家的合作,以帮助产生准确的结果。以下是确定基因组中耐受小插入位点的方法的广泛概述。

  1. 获取测序结果的原始读数(例如FASTQ文件)。接下来,将读取分成两组;一开始含有TGCGGCCGCAN 1 N 2 N 3 N 4 N 5 读取(图1,步骤9)和另一个不包含该确切序列的读数。&nbsp;
  2. 修剪每个读数的前十个核苷酸以去除转座子插入的序列(图1,步骤9),并留下登革热基因组序列用于比对。
  3. 仅使用修剪的读数,使用BOWTIE2的默认设置将读数对齐到登革热基因组。 BOWTIE2可以从 http://www.sourceforge.net 获得。
  4. 获取对齐文件,并为每次读取提取对齐的起始位置。由于转座酶引起的插入重复,该位点位于实际插入的上游5个碱基处(参见程序A),因此这些比对位点应减少5。
  5. 列举病毒基因组中每个位点的读数。
  6. 通过将插入位点计数除以测序运行的总读数来标准化读数,因此您可以比较多个测序运行。

笔记

上述协议中提供了详细的配方。每个制造商的协议中都提供了故障排除提示,在执行实验之前应该阅读这些提示以突出执行所述实验的潜在问题。

致谢

该方案改编自几个先前的转座子插入诱变研究(Arumugaswami et al。,2008; Beitzel et al。,2010; Teterina et al。,2011; Thorne et al。,2012; Remenyi et al。,2014)。作者没有披露利益冲突。我们感谢Claire Huang博士(CDC)的pD2 / IC-30-P-NBX感染性cDNA克隆。这项工作得到美国国立卫生研究院拨款R01DK097374(A.W.T.)和密歇根临床与健康研究所(MICHR)拨款UL1TR002240(J.W.P.)和密歇根大学胃肠道研究中心(UMCGR)的资助,授予5P30DK034933。

参考

  1. 1Arumugaswami,V.,Remenyi,R.,Kanagavel,V.,Sue,E.Y.,Ngoc Ho,T.,Liu,C.,Fontanes,V.,Dasgupta,A。和Sun,R。(2008)。 丙型肝炎病毒基因组的高分辨率功能分析。 PLoS Pathog < / em> 4(10):e1000182。
  2. Beitzel,BF,Bakken,RR,Smith,JM和Schmaljohn,CS(2010)。 High-通过插入诱变和大规模平行测序解析委内瑞拉马脑炎病毒基因组的功能映射。 PLoS Pathog 6(10):e1001146。
  3. Eyre,N.S。,Johnson,S.M.,Eltahla,A.A.,Aloi,M.,Aloia,A.L.,McDevitt,C.A.,Bull,R.A。和Beard,M。R.(2017)。 登革热病毒的全基因组突变揭示了NS1蛋白的可塑性,并能够产生感染性标记的报告病毒。 J Virol 91(23)。&nbsp;
  4. Fulton,B.O。,Sachs,D.,Schwarz,M.C.,Palese,P。和Evans,M。J.(2017)。 寨卡病毒基因组的转座子突变突出了RNA复制必需的区域,并限制免疫逃避。 J Virol 91(15)。
  5. Huang,C.Y.,Butrapet,S.,Moss,K.J.,Childers,T.,Erb,S.M.,Calvert,A.E.,Silengo,S.J.,Kinney,R.M.,Blair,C.D。和Roehrig,J.T。(2010)。 登革病毒2型包膜蛋白融合肽对膜融合至关重要。 病毒学 396(2):305-315。
  6. Remenyi,R.,Qi,H.,Su,SY,Chen,Z.,Wu,NC,Arumugaswami,V.,Truong,S.,Chu,V.,Stokelman,T.,Lo,HH,Olson,CA ,Wu,TT,Chen,SH,Lin,CY和Sun,R。(2014)。 丙型肝炎病毒基因组的综合功能图提供了探测病毒蛋白的资源。 MBio 5(5):e01469-01414。
  7. Teterina,N.L.,Lauber,C.,Jensen,K.S。,Levenson,E.A.,Gorbalenya,A.E。和Ehrenfeld,E。(2011)。 鉴定脊髓灰质炎病毒非结构蛋白中的耐受插入位点。 病毒学 409(1):1-11。
  8. Thorne,L.,Bailey,D。和Goodfellow,I。(2012)。 诺如病毒基因组的高分辨率功能分析。 J Virol 86(21):11441-11456。
登录/注册账号可免费阅读全文
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
引用:Perry, J. W. and Tai, A. W. (2018). Random Insertional Mutagenesis of a Serotype 2 Dengue Virus Clone. Bio-protocol 8(16): e2975. DOI: 10.21769/BioProtoc.2975.
提问与回复
提交问题/评论即表示您同意遵守我们的服务条款。如果您发现恶意或不符合我们的条款的言论,请联系我们:eb@bio-protocol.org。

如果您对本实验方案有任何疑问/意见, 强烈建议您发布在此处。我们将邀请本文作者以及部分用户回答您的问题/意见。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片的形式来说明遇到的问题。

如果您对本实验方案有任何疑问/意见, 强烈建议您发布在此处。我们将邀请本文作者以及部分用户回答您的问题/意见。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片的形式来说明遇到的问题。