Purification and Proteomic Analysis of Alphavirus Particles from Sindbis Virus Grown in Mammalian and Insect Cells
哺乳动物和昆虫细胞中的辛德比斯病毒的α病毒颗粒的纯化和蛋白质组分析   

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Journal of Virology
Jun 2018

 

Abstract

Current mass spectrometry (MS) methods and new instrumentation now allow for more accurate identification of proteins in low abundance than previous protein fractionation and identification methods. It was of interest if this method could serve to define the virus proteome of a membrane-containing virus. To evaluate the efficacy of mass spec to determine the proteome of medically important viruses, Sindbis virus (SINV), the prototypical alphavirus was chosen for evaluation. This model system was chosen specifically because the alphaviruses contain members which are human pathogens, this virus is well defined biochemically and structurally, and grows to high titers in both vertebrate and non-vertebrate host cells. The SINV proteome was investigated using this method to determine if host proteins are specifically packaged into infectious virions. It was also of interest if the SINV proteome, when grown in multiple host cells representing vertebrate and mosquito hosts, incorporated specific host proteins from all hosts. Observation of recurrent or distinctive proteins in the virus proteome aided in the determination of proteins incorporated into the virion as opposed to those bound to the particle exterior. Mass spectrometry analysis identified the total protein content of purified virions within limits of detection. The most significant finding was that in addition to the host proteins, SINV non-structural protein 2 (nsP2) was detected within virions grown in all host cells examined. This analysis identified host factors not previously associated with alphavirus entry, replication, or egress, identifying at least one host factor integrally involved in alphavirus replication. Key to the success of this analysis is the method of virus purification which must deliver measurably infectious virus free of high levels of contaminants. For SINV and other members of the alphavirus family, this is accomplished by isopycnic centrifugation through potassium tartrate, followed by a high salt wash.

Keywords: Sindbis virus (辛德比斯病毒), Mass spectrometry (质谱), Virus proteome (病毒蛋白质组), Virus purification (病毒纯化), Alphavirus (α病毒), Proteomics (蛋白质组学)

Background

The terms proteome and proteomics were coined by Marc Wilkins to describe the systematic evaluation of proteins in a model system using a detailed study of structure, function and regulation of its biology including aberrations which lead to disease (Wilkins et al., 1996 and 2009). However, virus proteomes have been under investigation long before the field of proteomics evolved in an attempt to understand the mechanisms of virus-host interactions in vitro and to evaluate virus pathogenesis of the animal host. To this end it was of interest to evaluate the virus proteome of a model system in a family of viruses which contained medically significant pathogens. Sindbis virus was chosen for this investigation because it is a member of the Alphavirus genus, Family Togaviridae, which contains a significant number of human pathogens of medical importance, it is structurally stable, grows to high titers, is well described in the literature and can be grown in vertebrate and non-vertebrate host cells. Sindbis virus has been the subject of many studies because it is a relatively simple membrane-containing +RNA icosahedral virus (Figure 1). The viral 42S genome is infectious and serves as the template for a 26S subgenomic RNA which encodes the structural genes, organized in the sequence C-PE2(E3/E2)-6K(TF)-E1 3’UTR-polyA (Figure 2). The genomic strand encodes the non-structural proteins in the order 5’UTR-nsP1-nsP2-nsP3-nsP4 (Strauss, J. H. and Strauss, E. G., 1994). The process of virus assembly has marked differences when occurring in vertebrate or invertebrate cells (Brown, 1986). In vertebrate cells the nucleocapsid is pre-assembled in the cytoplasm with the genomic RNA prior to association with the viral protein modified plasma membrane and virus budding from the plasma membrane. In invertebrate cells the virus nucleocapsid and fully matured virus are assembled in endosomes termed “virus factories” prior to fusion of the virus-containing endosomes with the plasma membrane, releasing infectious particles. Depending on the virus’ host cells origin, the glycosylation patterns of the glycoproteins will be that specified by the cellular biochemistry and the specific lipid bilayer will be that of the host cell. The virus particles released from either host can be equally infectious and if the SVHR strain of Sindbis virus is used close to 100% viability is achieved (Vancini et al., 2013). This is an important detail because virus infectivity requires all of its components to be in their metastable conformation and native physical state to be functional that is, infectious (Hernandez et al., 2014). Thus, not all virus particles will be amenable to this method of analysis because the particle structure must be 1) of regular stoichiometry 2) capable of rigorous purification without the loss of infectivity and 3) express a very low particle to PFU ratio. These factors are important to be able to discern any protein contaminants from proteins that are carried within the virus particle. Because of the symmetry of the virus particle and the stoichiometry, the protein concentration can be used to calculate the number of physical particles from the number of infectious particles using the plaque assay to determine infectious particle titer. This specific analysis cannot be applied to non-symmetrical viruses.


Figure 1. A cartoon of an alphavirus. The cartoon depicts an enveloped, spherical, icosahedral, 65-70 nm in diameter, capsid with a T = 4 icosahedral symmetry made of 240 monomers 1:1:1 stoichiometric ratio of E1:E2:C. The envelope contains 80 spikes, each spike is a trimer of E1/E2 protein dimers. Reprinted with permission from ViralZone, SBI Swiss Institute of Bioinformatics. Figures from ViralZone, with permission.


Figure 2. Alphavirus genomic organization. This genome is monopartite, linear, ssRNA(+) genome of 11-12 kb. The genome is capped (c) and polyadenylated. P123, precursor to nsP1, 2 and 3. Virus proteins are matured by both host and viral proteases. RdRp, RNA dependent RNA polymerase, TF, transframe protein (made from ribosomal frame shift in 6k protein) and DLP (downstream loop), stable RNA structure in the coding sequence of the 5’ region of the subgenomic RNA. Figures from ViralZone, with permission.

Stock Virus growth in BHK cells or C7-10 cells
The heat resistant SINV (SVHR) strain was used in this study. This strain was isolated by Burge and Pfefferkorn in 1966 by collecting virus that was resistant to heating to 54 °C. The choice of virus strain is important because this strain produces high titers (1010 PFU/ml) and low particle/PFU (~1 particle/PFU), ratios of highly infectious and physically stable virus. BHK and C7-10 (Aedes albopictus) cells were obtained from internal collections and are the favored cells to grow SVHR. Virus was harvested from 10 T-75 flasks (Corning) which produces enough virus to form a large visible band in a 30 ml potassium tartrate gradient and sufficient material for the mass spec analysis. Cells were infected at an MOI of 10 PFU/ml, for Sindbis virus infections and allowed to replicate for a single cycle and harvested at 18 h post infection to ensure that no cell lysis took place. A single cycle of SINV growth from mammalian cells is ~12 h and from C7-10 cells is about 24 h. The supernatants were clarified by low speed centrifugation (Sorvall RC-5B Super speed centrifuge at 2,000 rpm, 700 x g). Twenty microliter of the resulting virus supernatant was loaded onto a 15-35% linear potassium tartrate gradient and twice purified by isopycnic ultracentrifugation (Beckman SW-28 rotor, 18 h at 10,000 x g). The resulting band of purified virus was collected and washed twice by pelleting the virus in 5 ml 1x PBS in an SW-40 Beckman-40 rotor at 45,000 rpm (12,000 x g) for 30 min and collecting the pellet.

Virus titration by Plaque assay
The assay of virus titer by plaque formation, “plaque assay” is the most accurate method for measuring of the amount of infectious virus. This assay is used to determine the titer, in plaque-forming units (PFU) per ml, of virus by infecting a standardized monolayer of cells with a known volume of a known dilution of a virus-containing solution. The infection is contained under agarose which only allows diffusion of the virus to adjacent cells. Virus from a single initially infected cell infects adjacent cells producing a “plaque” or clearing (formed by lysed cells) localized to the original site of infection by an overlay of 1% agarose in 1x EMEM. Plaques of SVHR are visible to the naked eye after neutral red staining within 2-3 days of incubation at 37 °C. Begin with an estimate of what the titer could be, if the titer is estimated to be around 108 PFU/ml, a flask infected with a dilution of 10-6 would show 20-200 plaques; in this case, infecting flasks with dilutions of 10-5, 10-6, and 10-7 PFU/ml should give adequate data to make a relatively accurate calculation. If the titer of the virus is completely unknown, it may be necessary to infect flasks or plates with a wide range of dilutions (10-1 to 10-8). The number and quality of the plaques seen in a given assay can be influenced by a number of factors, including the pH and/or temperature of media, dilution buffer, agarose overlay, or the condition of the cell monolayer. Due to the sensitivity of this assay, it is important to include both positive and negative controls within each assay. The negative control is a flask that is inoculated with diluent only, and the positive control consists of one dilution of SVHR stock virus of known titer sufficient to give ~20 plaques. This number of plaques is significant statistically.

It is important that when going from a high concentration to a low concentration the pipet tip is changed to avoid “carry over” contamination. However, when pipetting from a low concentration to a higher concentration, as is done when the wells are inoculated, a single pipet tip can be used.

Materials and Reagents

Note: These can be from any supplier that offers Cell Culture grade materials or reagents except where specified. All culture flasks and plates are standard and can be from any supplier except where noted.
All fetal bovine serum (FBS) should be heat inactivated at 56 °C for 30 min.

  1. Sub-culture of vertebrate and invertebrate cells
    Sub-culture of BHK-21 cells
    1. 6-well plates
    2. Sterile tissue culture supplies (pipets, vented or plug cap flasks and 15 ml conical tubes)
    3. Heat-inactivated fetal bovine serum (FBS)
    4. Tryptose phosphate broth
    5. L-glutamine
    6. Gentamicin sulfate
    7. KCl
    8. KH2PO4
    9. NaCl 
    10. Na2HPO4 or Na2HPO4·7H2O
    11. CaCl2 or CaCl2·2H2O
    12. MgCl2·6H2O
    13. Complete E-MEM, warmed to 37 °C (Earl’s salts minimal essential medium, completed with 10% heat inactivated fetal bovine serum, FBS) 
    14. 1x PBS-D (see Recipes)
    15. Trypsin stock, 0.25%, warmed to 37 °C (see Recipes)

    Sub-culture of C7-10 cells
    1. Sterile tissue culture supplies (pipets, vented or plug cap flasks and 15 ml conical tubes)
    2. 70% ethanol
    3. 1x MEM

  2. Virus growth and titration
    Stock virus preparation
    1. Serological pipettes (5 ml, 10 ml and 25 ml)
    2. FBS (fetal bovine serum)
    3. Sterile Tissue culture grade water: for a detailed discussion of purified water and specifications for cell culture and other grades of water see Laboratory Water (The national institutes of health, March, 2013)
    4. Gentamicin sulfate
    5. L-glutamine
    6. KCl
    7. KH2PO4
    8. NaCl 
    9. Na2HPO4 or Na2HPO4·7H2O
    10. CaCl2 or CaCl2·2H2O
    11. MgCl2·6H2O
    12. Neutral red
    13. Complete E-MEM, 1x and 2x (see Recipes)
    14. Gentamicin sulfate, 100x (mammalian cells only) (see Recipes)
    15. L-glutamine 100x (see Recipes)
    16. HEPES (pH 7.2-7.4), 1 M (see Recipes)
    17. 2% Neutral red stock solution (see Recipes)
    18. PBS-D (10x) (see Recipes)
    19. SVHR diluent (see Recipes)
    20. Tryptose phosphate broth (TPB) (optional for ATCC cells) (see Recipes)

    Virus titration by plaque formation on BHK cells
    BHK cells preparation
    1. Sterile tissue culture supplies (pipets, vented or plug cap flasks and 15 ml conical tubes)
    2. Heat-inactivated fetal bovine serum (FBS)
    3. Tryptose phosphate broth
    4. L-glutamine
    5. Gentamicin sulfate
    6. Trypsin
    7. KCl
    8. KH2PO4
    9. NaCl 
    10. Na2HPO4 or Na2HPO4·7H2O
    11. CaCl2
    12. CaCl2·2H2O
    13. KH2PO4
    14. MgCl2·6H2O
    15. Complete E-MEM, warmed to 37 °C (see Recipes)
    16. 1x PBS-D (see Recipes)
    17. Trypsin stock, 0.25%, warmed to 37 °C (see Recipes)

    Virus titration by Plaque assay
    1. 2% Agarose
    2. Heat inactivated Fetal Bovine Serum (FBS)
    3. 100% glycerol
    4. Heat-inactivated fetal bovine serum (FBS)
    5. Tryptose phosphate broth
    6. L-glutamine
    7. Gentamicin sulfate
    8. Trypsin
    9. KCl
    10. KH2PO4
    11. NaCl 
    12. Na2HPO4 or Na2HPO4·7H2O
    13. CaCl2 or CaCl2·2H2O
    14. KH2PO4
    15. Neutral Red
    16. Complete E-MEM 2x (see Recipes)
    17. 2% Neutral Red (see Recipes)
    18. SVHR Diluent (see Recipes)
    19. 1x PBS-D (see Recipes)
    20. 1 M HEPES (pH 7.4) (see Recipes)

  3. Sindbis virus infection of vertebrate and invertebrate cells
    Sindbis virus infection of BHK cells
    1. Centrifuge tubes (Corning, 15 ml Fisherbrand, catalog number: 05-539-5)
    2. Sindbis virus infection of C7-10 cells
    3. 100% glycerol (sterile)
    4. Tissue culture grade water
    5. Heat-inactivated fetal bovine serum (FBS)
    6. Tryptose phosphate broth
    7. L-glutamine
    8. Gentamicin sulfate
    9. Trypsin
    10. KCl
    11. KH2PO4
    12. NaCl 
    13. Na2HPO4 or Na2HPO4·7H2O
    14. CaCl2 or CaCl2·2H2O
    15. KH2PO4
    16. MgCl2·6H2O
    17. Tryptose phosphate broth (TPB; Difco)
    18. Complete E-MEM (see Recipes)
    19. Gentamicin sulfate 100x (see Recipes)
    20. L-glutamine 100x (see Recipes)
    21. 1 M HEPES (see Recipes)
    22. TPB-tryptose phosphate broth (see Recipes)
    23. PBS-D (10x) (see Recipes)

    Sindbis virus infection of C7-10 cells
    1. Glycerol
    2. Liquid N2
    3. Heat-inactivated fetal bovine serum (FBS)
    4. Tryptose phosphate broth
    5. L-glutamine
    6. Gentamicin sulfate
    7. Trypsin
    8. KCl
    9. KH2PO4
    10. NaCl 
    11. Na2HPO4 or Na2HPO4·7H2O
    12. CaCl2 or CaCl2·2H2O
    13. KH2PO4
    14. MgCl2·6H2O
    15. Completed E-MEM medium (see Recipes)
    16. 1x PBS-D (see Recipes)

  4. Purification of Sindbis virus
    Purification and Concentration by isopycnic centrifugation
    1. Centrifuge tubes (ultra-clear) 
    2. Small tube clamp
    3. Waste beaker
    4. 2 ml cryotube
    5. 15% potassium tartrate (dibasic, hemihydrate) in PBS-D (filter sterilize to store and store at 4 °C)
    6. 35% potassium tartrate in PBS-D (filter sterilize to store and store at 4 °C)
    7. BCA assay (PierceTM Rapid Gold BCA Protein Assay Kit) (Thermo Fisher Scientific, catalog number: A53225)
    8. KCl
    9. KH2PO4
    10. NaCl 
    11. Na2HPO4 or Na2HPO4·7H2O
    12. CaCl2 or CaCl2·2H2O
    13. KH2PO4
    14. MgCl2·6H2O
    15. PBS-D pH 7.4 (see Recipes)

    Concentration of virus by Polyethylene glycol (PEG) precipitation
    1. Kimax high strength glass centrifuge tube (Kimax, catalog number: #Z2514888) (plain 30 ml)
    2. Polyallomer centrifuge tubes
    3. Polyethylene glycol-8000 (PEG-8000)
    4. 15% potassium tartrate (dibasic, hemihydrate) in PBS-D 
    5. 35% potassium tartrate in PBS-D
    6. BCA assay (PierceTM Rapid Gold BCA Protein Assay Kit) (Thermo Fisher Scientific, catalog number: A53225)
    7. KCl
    8. KH2PO4
    9. NaCl 
    10. Na2HPO4 or Na2HPO4·7H2O
    11. CaCl2 or CaCl2·2H2O
    12. KH2PO4
    13. MgCl2·6H2O
    14. Tris Buffer, pH 7.0
    15. EDTA
    16. PBS-D, pH 7.4 (see Recipes)
    17. PEG Buffer (see Recipes)
    18. 2 M NaCl (see Recipes)

  5. Calculation of particle to PFU ratio
    None

  6. Mass spectrometry and proteomic analysis
    Note: All reagents should be LCV-MS grade.
    Protein Extraction and Digestion
    1. Lo-bind Centrifuge tubes (Eppendorf) 
    2. Spin filter (Millipore-Ultracell YM-30)
    3. Pierce C18 spin columns (Thermo Fisher Scientific)
    4. Parafilm
    5. Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, catalog number: A53225)
    6. Mammalian Protein Extraction Reagent (M-PER) supplemented with 50 mM dithiothreitol (Thermo Fisher Scientific, catalog number: 78501) 
    7. 0.05 M iodoacetamide in UA buffer
    8. Trypsin/Lys-C prepared in 100 mM TEAB to 10 µg/ml 
    9. DTT 
    10. CaCl2
    11. CaCl2·2H2O
    12. KH2PO4
    13. MgCl2·6H2O
    14. NaCl 
    15. Urea
    16. Tris-HCl pH 8.5
    17. TEAB (Sigma, catalog number: T7408-100ML)
    18. 100% trifluoroacetic acid 
    19. IAA powder 
    20. Sterile 1x PBS (see Recipes)
    21. UA buffer (see Recipes)
    22. 100 mM triethylammonium bicarbonate (TEAB) (see Recipes)
    23. 0.5 M NaCl (see Recipes)
    24. 10% trifluoroacetic acid (TFA) (see Recipes)
    25. M-PER supplemented with 50 mM dithiothreitol (DTT)
    26. Iodoacetamide (IAA) solution (0.05 M IAA in UA buffer) (see Recipes)
    27. Trypsin digestion solution (see Recipes)

    LC-MS/MS Data Acquisition
    1. Picofrit 15 cm x 75 µm ID HPLC column packed with 5 µm BioBasic C18 particles 300Å (New Objective)
    2. 3% acetonitrile/0.1% formic acid
    3. 100% formic acid (Thermo Scientific, catalog number: 28905)
    4. 100% acetonitrile
    5. A Buffer (see Recipes)
    6. B Buffer (see Recipes)

Equipment

  1. Sub-culture of vertebrate and invertebrate cells
    1. 25 cm2 flask
    2. 75 cm2 flask
    3. CO2 Cell culture incubator (Any supplier is adequate)
      Note: Any supplier is adequate; the choices are price/quality and capacity. All that is necessary is a water-jacketed incubator which can be regulated to 5% CO2 in a water-saturated atmosphere. This will require a medical grade CO2 tank (liquid gas under pressure) and a pressure regulator specific for that tank. Two incubators will be required if the cells are to be grown simultaneously because the vertebrate cells are incubated at 38 °C and invertebrate cells at 28 °C.
    4. Biological safety cell culture hood
    5. Inverted microscope
    6. Hemacytometer

  2. Virus growth and titration
    Stock virus preparation
    1. CO2 Cell culture incubator

    Virus titration by Plaque assay
    1. Rocker platform (Bellco Biotechnology)
    2. 37 °C water bath
    3. Dilution tube rack
    4. Ice bucket

  3. Sindbis virus infection of vertebrate and invertebrate cells
    Sindbis virus infection of BHK cells
    1. Platform rocker (Bellco Biotechnology)
    2. Hemocytometer

    Sindbis virus infection of C7-10 cells
    1. 75 cm2 flasks
    2. Centrifuge
    3. Hemocytometer

  4. Purification of Sindbis virus
    Purification and Concentration by isopycnic centrifugation
    1. Inverted microscope
    2. Platform rocker (Bellco Biotechnology)
    3. Hemocytometer or cell counting device
    4. CorningTM polypropylene tubes (micro centrifuge tubes, self-standing and conical) 
    5. Sorvall RC-5B Super speed centrifuge
    6. Beckman ultracentrifuge 
    7. Beckman SW-28 rotor
    8. Polyallomer 38 ml tubes
    9. Beckman SW-55Ti
    10. Polyallomer 5 ml tubes
    11. Ring stand 
    12. Small tube clamp 
    13. Hand held low-intensity lamp 
    14. 30-100 ml gradient former (Bio-Rad, model 385)
    15. Medium stir plate

    Concentration of virus by Polyethylene glycol (PEG) precipitation
    1. Sorvall RC-5B Super speed centrifuge
    2. Beckman ultracentrifuge and appropriate rotor and tubes for the volume used
    3. 30-100 ml gradient former (Bio-Rad, model 385)

  5. Calculation of particle to PFU ratio
    None

  6. Mass spectrometry and proteomic analysis
    Protein Extraction and Digestion
    1. Tabletop centrifuge capable of reaching up to 14,000 x g
    2. Shaker heat block
    3. Vortex
    4. SpeedVac Concentrator

    LC-MS/MS Data Acquisition
    1. Orbitrap ELITE mass spectrometer (or equivalent; e.g., Orbitrap QE+, Orbitrap Fusion, Orbitrap Fusion Lumos)
    2. Easy-nLC II liquid chromatography system (or equivalent HPLC/UHPLC nanoflow pump system)

Software

Data Processing:

  1. High performance computer meeting the minimum specifications to run Proteome Discoverer Software (Currently available version: 2.2; Thermo Fisher Scientific)
  2. PANTHER classification system (http://www.pantherdb.org/)

Procedure

Protocol outline:

  1. Sub-culture of vertebrate and invertebrate cells
    1. Sub-culture of BHK cells
    2. Sub-culture of C7-10 cells
  2. Virus growth and titration
    1. Preparation of stock virus
    2. Virus titration by plaque assay
  3. Sindbis virus infection of vertebrate and invertebrate cells
    1. Sindbis virus infection of BHK cells
    2. Sindbis virus infection of C7-10 cells
  4. Purification of Sindbis virus
    1. Concentration by isopycnic centrifugation
    2. Concentration by polyethylene glycol (PEG) precipitation
  5. Calculation of particle/PFU ratio
  6. Mass Spectrometry and Proteomic Analysis
    1. Protein Extraction and Digestion
    2. LC-MS/MS Data Acquisition
    3. Data Processing

  1. Sub-culture of vertebrate and invertebrate cells
    Sub-culture of BHK-21 cells
    Individual stocks of BHK cells may require different passage schedules and may have different levels of viable passages. It is good practice to keep track of the number of passages that an individual culture can be sub-cultured so that a schedule of cell thawing and storage can be established.
      Fresh newly thawed cells can be passaged up to 30 times. If you are not going to split them immediately, cells can be incubated at 28 °C for 2 to 3 days but must be split at least once to recover normal growth before use in experiments.
    1. Wash a confluent BHK cell monolayer once with 1x PBS-D, using 5 ml for a 25 cm2 flask or 15 ml for a 75 cm2 flask.
    2. Decant PBS-D and add trypsin to the monolayer. Add 2 ml to a 25 cm2 flasks, or 5 ml to a 75 cm2 flask. Incubate at room temperature until the cells begin to detach from the flask. Disrupt cell clumps by pipetting up and down using a 5 to 10 ml serological pipet. 
    3. Add BHK cell culture medium to a volume of 1:1 to stop trypsin. Resuspend cells in the trypsin solution and check for cell clumps by under the microscope. If there are significant remaining clumps, pipet up and down using a 5 to 10 ml serological pipet. 
    4. Pipet cell solution into a conical tube of the appropriate size.
    5. Spin tube(s) at 500 x g (medium speed on a tabletop centrifuge) for 3 to 5 min to form a firm pellet. Do not overfill tube. Do not invert tube to mix.
    6. Decant supernatant and replace with the appropriate amount of medium. 
      1. For preparation of a single 25-cm2 flask: resuspend in 3 ml E-MEM. Add 1 ml of cells to 10 ml of medium in the flask. Up to 3 flasks can be prepared from one initial 25 cm2 flask, at a 1:3 split ratio (area of the flask). 
      2. For preparation of a large number of 25-cm2 flasks (plaque flasks or plates): To a sterile bottle, add 10 ml E-MEM per each flask to be prepared. If you are using 6-well plates use 2 ml/well. Remove some medium from the bottle and resuspend cells. Add back to the rest of the medium and mix well. To prepare multiple flasks or plates aliquot 10 ml of the cell mixture into each flask 25-cm2 flasks, swirling continuously. One 75 cm2 flask will make 9-25 cm2 flasks or 4- 6-well plates are (9.5 cm2/well x 6 wells = 57 cm2 total) using 2 ml /well.
        Note: This protocol ensures that the cell monolayers will be uniform and contain approximately the same number of cells. Care should be taken when placing the flasks into the incubator so as not to disturb the cell suspension.
      3. For preparation of a single 75-cm2 flask: Aliquot 1 ml of cells from a single 25 cm2 flask into 30 ml complete E-MEM.
    7. Incubate cells in flasks at 37 °C for 24 h or until confluent.
    8. If medium does not hold pH of < 8, adjust pH with 1 M HEPES to a final concentration of 8 mM HEPES. This is normally not necessary however if the pH needs adjusting then it should be between pH 7 and 8.

    Sub-culture of C7-10 cells
    1. Maintain mosquito cell lines in semi-suspension in tissue culture flasks at 28 °C in a 5% CO2 humidified environment. Subculture up to every other day as described below. The cells need to be split when they begin to clump or float. These cells like to be concentrated; do not split more than 1/3x the area of the monolayer.
      Notes:
      1. Semi-suspension cells adhere loosely to the substrate initially, and then begin to float as they age. Some cells may stick tightly to the substrate.
      2. Do not attempt to completely disrupt the clumps or scrape the cells, this damages the cells.
    2. Clean the area under the hood with 70% ethanol. Clean all materials going under the hood with 70% ethanol and remove unneeded materials.
    3. Place media at room temperature to warm.
    4. Remove cell flasks from the incubator. Observe cell media for changes in color (red) and clarity.
    5. Visualize cells under a microscope at 20x magnification to check monolayer for irregularities, and confluency. Cells need to be 95%-100% confluent prior to passage. Cells will be at a density of ~ 2 x 108 cells/75 cm2 flask in 25 ml of medium.
    6. Hit the side of the flask sharply on any soft surface several times to loosen any cells that are stuck to the surface of the flask.
      Notes:
      1. If the cells are aggregated into clumps, try to break up the clumps by pipetting up and down several times using a 5-10 ml pipet, before transferring the cells to a new flask. 
      2. Small clumps are acceptable.
    7. Transfer one-third of the 25 ml of the medium of the cell suspension to each of three flasks. To each of these flasks, add fresh media to increase the volume to the original 25 ml. Add 25 ml of fresh 1x MEM to the original flask if there are many adherent cells remaining if the cells are needed. If not, omit this step.
    8. Cap flasks tightly if the caps are vented, leave loose for non-vented caps.
    9. Return flasks to the incubator set no higher than 26 °C. Mosquito cells will go into heat-shock at temperatures higher than 34°C.

  2. Virus growth and titration
    Stock virus preparation
    It is standard practice to grow a stock of virus from which additional virus stocks will be grown prior to any additional work with the virus. This practice avoids the production of defective interfering particles which will accumulate upon successive serial passage of high concentrations of virus. Generally, an MOI (multiplicity of infection) of 0.01 PFU (plaque forming units) per cell is required for production of stock virus. To calculate the correct MOI, the number of cells to be infected must be known. Then multiply the number of cells by the required MOI, e.g., 106 total cells to be infected x MOI of 0.01 = 104 PFU of virus inoculum is needed.
    If your stock virus is 1 x 109 PFU/ml and you need a 104 PFU inoculum, do the following:
    1. Make a serial dilution of the stock virus, begin by making a 1:10 dilution of the stock virus to a total of 1 ml. This will be 100 µl of virus supernatant in 900 µl of PBS-D + 3% FBS. This is a 10-1 virus dilution. 
    2. Make a serial dilution of this sample so that you get -1 through -4 dilutions in 5 separate tubes (see Figure 3). The amount of virus that you need is in the -4 dilution tube, 1 ml of virus of 10-4 PFU/ml.


      Figure 3. Serial passage scheme. The figure is modified from http://www.virology.ws/2009/07/06/detecting-viruses-the-plaque-assay/. This work is licensed under a <a rel="license" href="http://creativecommons.org/licenses/by/3.0/">Creative Commons Attribution 3.0 Unported License</a>.

    3. Make as much of this dilution as you need to infect all of your flasks with 1 ml/75 cm2 flask. Do not be tempted to use an inoculum of less than a 50 µl measurement of virus because the only correct way to dilute virus is to make a serial dilution. Virus particles do not go into true solution and care must be taken to suspend them correctly, otherwise the counts will be artificially high or low. Alternatively, you can use 100 µl of the -5 dilution up to a volume of 1 ml or use the entire -5 dilution to make 10 ml of the virus and arrive at the correct MOI.
    4. Refreeze your initial stock virus; this is your primary stock from which you will grow subsequent stocks. Virus is quantitated by plaque assay on the cells from which the virus produces CPE which is usually the cell line from which it is produced (in this case BHK). However Sindbis virus does not produce CPE in many insect cells (e.g., U4.4 of C7-10 cells), which cannot be used for the plaque assay.
      Notes:
      1. Stock virus can be grown from vertebrate or invertebrate cells.
      2. Sindbis virus is very sticky and will bind to glass and plastic surfaces. We have found that CorningTM polypropylene tubes (microcentrifuge tubes, self-standing and conical) bind fewer virus particles than other brands of plastic. Disposable glass tubes are used to make the virus dilutions required for the plaque assay. 
      3. Tissue culture grade reagents from all major suppliers have been found suitable for the cell culture and virus production portions of this protocol.

    Virus titration by plaque formation on BHK cells
    Preparation of BHK cells
    Note: Individual stocks of BHK cells, e.g., from ATCC, may require different passage schedules and may have different numbers of viable passages.
    1. Wash a confluent BHK cell monolayer once with 1x PBS-D, using 5 ml for a 25 cm2 flask or 15 ml for a 75 cm2 flask.
    2. Decant PBS-D and add trypsin to the monolayer. Add 2 ml to a 25 cm2 flasks, or 5 ml to a 75 cm2 flask. Incubate at room temperature until the cells begin to detach from the flask. Disrupt cell clumps by pipetting up and down using a 5 to 10 ml serological pipet. 
    3. Add BHK medium 1:1 to stop trypsin. Resuspend cells in the trypsin solution and check for cell clumps by under the microscope. If there are significant remaining clumps, pipet up and down using a 5 to 10 ml serological pipet. 
    4. Pipet cell solution into a conical tube of the appropriate size, e.g., 10 or 50 ml tubes.
    5. Spin tube(s) at 500 x g (medium speed on a tabletop centrifuge) for 3 to 5 min to form a firm pellet. Do not overfill tube. Do not invert tube to mix.
    6. Decant supernatant and replace with the appropriate amount of medium 3 ml for 25-cm2 flask and 9 ml for a 75 cm2 flask.
      1. For preparation of a single 25-cm2 flask: Resuspend in 3 ml E-MEM. Add 1 ml of cells to 10 ml of medium in the flask. Up to 3 flasks can be prepared from one initial 25 cm2 flask, at a 1:3 split ratio (area of the flask). Cap flasks tightly (vented caps, leave loose for non-vented caps).
      2. 9-25 cm2 flasks can be prepared from 1-75 cm2 flask.

    The materials for plaque assay
    Many of the materials used for the plaque assays are used for both mammalian and mosquito cell assays. It is important to make the serial dilutions of virus immediately prior to infection of the plaque assay flasks/plates to limit the amount of virus binding to the tubes.
    1. Determine the number of plates/flasks required, with two wells per dilution of virus (plated in duplicate), plus a few control plates or wells. The day before the plaque assay, split BHK cells into 6-well plates containing ~6 x 106 cell/plate = 1 x 106 cells/well. In general, the use of 6-well plates has replaced the use of individual flasks; however when learning the technique the use of flasks in much simpler.
      Note: The number of cells required is determined by the confluency required at the time of the assay. Because confluency is related to the area the cells cover, this number is manipulated by knowing the area of the vessel the cells will grow on. Thus, the area of the flasks (rectangular) or wells (circular) will vary but the number of cells/cm2 is constant. (e.g., 1 x 106 cells. For a 6- well plate, each well is 9.6 cm2 seeded with 1 x 106 cells/well x 6 wells for a total of ~58 cm2 onto which a total of ~6 x 106 cells is seeded. These numbers are relative to every specific culture of any cell line and may need to be adjusted up or down. The important thing is the % confluency on the day of the assay during which the cells should still be in log phase. 
    2. Allow cell monolayers to become ~80%-90% confluent by the time of the plaque assay.
      Note: If the cells are ready in the morning and the assay is to be done in the afternoon, the cells can be stored at room temperature with the caps closed tightly, or moved to a 28 °C incubator to slow growth.
    3. Remove the virus to be titered from the freezer and thaw on ice. 
    4. Fill the required number of dilution tubes with 900 μl diluent (SVHR diluent; 1x PBS-D/3% FBS (see Recipes). You need one tube/dilution. One tube for the control (not infected) and one for the positive infected control.
    5. Prepare serial dilutions of the virus by adding 100 μl virus to the first tube containing 900 µl of diluent (10-1), vortex that dilution at full speed, removing 100 μl, and adding it to the next dilution tube (10-2). Continue this process until the desired dilutions have been made. Change pipet tips each time to avoid “carry-over” effect of contaminating virus.
      Note: Dilutions should be made immediately prior to use. Virus and dilutions should be kept on ice at all times. 
    6. Once the dilutions have been made, pour growth media out of flasks (into a sterile waste beaker) and pipet out of plates with a serological pipet (leave enough liquid to cover the monolayer) and infect each flask or well of a 6-well plate with 200 μl of the appropriate dilution. In this case, the area of the vessel no longer matters because the calculation will convert plaques at each dilution to PFU/ml.
    7. Do not let monolayers dry out while adding virus–i.e., do not pour/pipette media off too many flasks/plates at one time or try to drain every drop. DRY MONOLAYERS ARE DEAD MONOLAYERS.
    8. Place flasks on a rocking platform at room temperature for one hour. Do not rock plates; the liquid will only swirl around the edge of the well. Place the plates at the appropriate temperature, 38 °C for BHK cells and hand rock every 15 min.
      Note: The caps on the flasks should be tightened while they are rocking since they are not in the appropriate CO2 environment unless they are vented, then place these in the incubator.
    9. After an hour of rocking/incubating, remove the inoculum (pipette) and overlay the monolayer with 7 ml of a 1:1 mixture of 2% agarose in dH2O and complete 2x EMEM with 7 ml for T-25 cm2 flasks and 2 ml for each well of a 6-well plate.
      Notes:
      1. The agarose should be hot enough that it will not solidify too quickly, but cool enough to allow one to touch the bottle before mixing with the medium (~60-70 °C). Bottles of media at the correct temperature should not feel excessively hot to the touch. Do not try to move flasks/plates before the overlay has solidified, because the cell monolayer will tear. Solidified agar will appear cloudy. 
      2. Not all agarose is tolerated by cells in culture. In general, agarose formulated for gel electrophoresis or chromatography is not suitable for tissue culture. Use Sigma agarose () (Sigma-Aldrich, St Louis, MO, catalog number: A6013).
    10. Incubate flasks for 2 days at 37 °C, 5% CO2.
      Note: Before staining monolayers, hold the flasks up to the light to see if plaques are visible. The plaques will appear more opaque than the rest of the monolayer. Visible plaques are an indication that the assay has worked up to this step. 
    11. To stain the monolayer, add 5 ml of 1:1 mixture of 2% agarose in dH2O and 2x PBS-D with 3% neutral red stain (3% of the total amount of agarose required). Cover the flasks to protect cells from light.
      Note: Cells become light sensitive after they take up neutral red and should be protected from light.
    12. If necessary, when the plaques are faint, return flasks (plates) to 37 °C for 4 h or 28 °C overnight prior to counting plaques.
      Note: You should expect to see clear plaques surrounded by red, living cells. The number of plaques per flask should roughly follow the dilutions made (e.g., 1 plaque on 10-6 flask, 10 plaques on 10-5 flask, and 100 plaques on 10-4 flask). Plates can be used to produce duplicates (6-well plates) or triplicates (24-well plates). If there are no plaques, check the monolayers under the microscope, the cells may be lysed.
    13. Count plaques and calculate the titer.
    14. To calculate the virus titer: e.g., with 2 plaques at -7 dilution and an inoculum of 200 µl, convert this to plaques/ml or 2 plaques x 1/200 µl x 103 µl/1 ml = 10 plaques/ml from a 1/10-7 dilution = 10 x 107 or 1 x 108 PFU/ml. Thus the equation is #plaques/dilution factor (ml) x volume of inoculum (ml) = PFU/ml. Unless the number of virus particles = number of plaques/ml then this unit is a plaque forming unit. This will be explained in the section calculating the particle to PFU ratio.
      Note: If there are no plaques but the positive control worked then the sample has no virus or the incorrect dilutions were plated. If there are no plaques in the control sample check the cells under the microscope for lysis, lysed cells will not take up stain or produce a plaque. Cell lysis will occur if the osmolarity and tonicity of the cultures are not correct. Crystals of stain mean that the agarose was too hot when it was added. Agarose that is too hot will also kill the cells.

  3. Sindbis virus infection of vertebrate and invertebrate cells
    Sindbis virus infection of BHK cells
    1. Subculture BHK cells the day prior to infection such that the monolayer is ~90% confluent at the time of infection. 
    2. Calculate the amount of virus needed for the desired multiplicity of infection (MOI).
      No. cells x MOI = PFU needed.
      For a single cycle MOI = 10, stock virus MOI = 0.01
      For 75 cm2 flask, the number of cells is ~ 2 x 107cells
      For 25 cm2 flask, the number of cells is ~6 x 106 cells
      For a 6-well plate, the number of cells is about 1 x 106 cells/plate.
      Plates are also used if a small amount of inoculum is used, e.g., stock virus volume titer is too low to infect too many cells and the virus must be amplified before use.
    3. Remove the virus from -80 °C freezer and thaw the vial on ice. Dilute inoculum to the desired concentration in 1x PBS-D/3% FBS. Refreeze unused stock virus.
      Notes:
      1. A total volume of at least 1 ml is required for infection of 75 cm2 monolayer, and a minimum of 200 μl is required for a 25 cm2 monolayer. A minimum of 200 μl is required for a 6-well plate. Close the cap tightly and secure to rocker platform. Do not rock plates.
      2. SVHR is heat stable however, many other strains and mutants of Sindbis are not, and thus must be thawed on ice to retain infectivity.
    4. Place flask on a rocking platform for 1 h at room temperature, with caps tightened. Alternatively, infect in the incubator at 37 °C with intermittent hand rocking. 
    5. Remove inoculum and add fresh, complete 1x E-MEM media. Add 5-7 ml media to a 75-cm2 monolayer or 3 ml to a 25-cm2 monolayer. Leave the caps loosened during incubation to allow for CO2 exchange. If more concentrated virus is desired, add 3 ml medium to a 75-cm2 monolayer. Do not allow the monolayer to dry out.
    6. Virus should be harvested once CPE becomes evident, which is generally between 18 (BHK) and 24 h (C7-10) post-infection.
      Note: Cell lines will differ in the time period in which CPE is demonstrated. Some indicators of CPE in BHK cells include the cells becoming long and thin, cytoplasm with a “foamy” appearance, aggregations of cell nuclei, and, in advanced stages, cell lysis. Do not allow the cells to lyse for any type of virus analysis work.
    7. Remove medium from flasks to conical centrifuge tubes (Corning, 15 ml Fisherbrand Cat # 05-539-5 or 50 ml centrifuge tubes, Fisherbrand Cat # 05-539-6). Combine like samples if desired. Spin samples for 10 min in a tabletop centrifuge at ~700 x g to remove cell debris. Decant virus to a new tube and bring the solution to 10% glycerol which acts as a cryoprotectant. Aliquot the virus supernatant into tubes appropriate for freezing and flash freeze virus using liquid N2
    8. Store virus at -80 °C.
      Note: Membrane containing viruses will undergo a freeze-thaw at -65 °C due to phase transition in ice. Thus a freezer failure resulting in warming to -65 °C should be considered a thaw. Sindbis virus will lose some titer (~ 0.5 logs) upon freeze-thaw but is still infectious.

    Sindbis virus infection of C7-10 cells
    Mosquito cells are used when a comparative analysis of virus titers from the invertebrate host are required. To infect mosquito cells, a confluent monolayer of single cells (no clumps) must be formed. Mosquito cells do not adhere well to the substrate, and tend to lift from the flasks regardless of the medium used. To temporarily circumvent this problem, mosquito cultures can be starved for serum for one hour, which causes the cells to adhere to the substrate more tightly. Mosquito cultures in our lab have been adapted to use in E-MEM, however, all other treatments of the cells are consistent with cells which are in other insect media.
    1. Spin down for 5 min at 200-500 x g approximately 1½ -75 cm2 flasks of cells per 1-75 cm2 flask needed for infection.
      Note: Mosquito cells grow as semi-suspension cultures and are easily removed from the flask by tapping the flask against a hard surface, or alternatively by pipetting up and down with a 5-10 ml pipet. Do not spin excessively as the cells will die during pelleting or resuspension. Some cells will be lost during the process.
    2. Wash the cell pellet once with 1x PBS-D to remove any remaining medium. 
    3. Resuspend the final cell pellet in 1 ml/flask needed for infection, of E-MEM medium deficient in FBS.
    4. Count the cells using a hemocytometer. 
    5. Aliquot approximately 6 x 107 cells per 75-cm2 flask with enough medium (without FBS) to cover the monolayer. 
    6. Incubate the flask of cells at 28 °C for one hour, or until cells are well attached to the surface of the flask. 
    7. Once cells are attached, remove medium and add the desired amount of virus to the monolayer. Close caps tightly. 
    8. Rock the flask slowly at room temperature for one hour.
      Note: Rocking slowly is critical to keep a minimum number of cells from lifting from the flask.
    9. Most of the cells should remain attached to the flask after rocking. If cells have lifted from the monolayer they should be removed with the inoculum. 
    10. Fresh, complete E-MEM medium should be added to each flask.
      Note: To increase the concentration of virus, minimize the volume of fresh medium added to the flasks. A minimum of 3 ml medium/75 cm2 is required to cover the cells and support metabolism. Take care that the flasks are level to ensure complete coverage of the monolayers.
    11. Virus may be harvested from 24 to 72 hpi depending on the multiplicity (MOI) used. Cytopathic effect (CPE) will not be evident in mosquito cells, in which case virus will be harvested based on time post-infection rather than CPE. 
    12. For normal storage: Harvest virus and store in sterile 10% glycerol. For best preservation of the virus titer quick freeze tubes in liquid N2.
      Note: Virus frozen and thawed will lose 1/3 to 1/5 log of titer. This is not a significant loss for use to infect cells. For preservation of the highest infectivity level, virus can be stored at 4 °C for up to 5 days in neutral pH buffer. This method preserves the most infectivity and is used for virus to be analyzed in structural studies and for the calculation of particle to PFU ratios.

  4. Purification of Sindbis virus
    Purification and concentration by isopycnic centrifugation
    The virus supernatant is harvested into conical tubes and cell debris is removed from the supernatant by low-speed centrifugation. This is a critical step and if it is omitted, too much debris will interfere with the following gradient purification steps. Clarify virus supernatant by centrifugation in the appropriate size conical tube for 10 min at 1,800 x g.
    1. Virus supernatant is combined into an appropriate size container omitting addition of the glycerol.
      Note: From this step forward, you should not consider your samples sterile. This is not usually a problem and antibiotic is added to the 2x E-MEM media. 
    2. Determine the number of gradients which will be needed.
      Typically, 20 ml of virus is layered onto the initial potassium tartrate step gradient in the 38 ml tubes. This gradient is formed by carefully layering 12 ml of 15% potassium tartrate onto a layer of 6 ml of 35% potassium tartrate, or making a 10% to 35% continuous potassium tartrate gradient. Read the manufacturer’s manual for instructions on use of the gradient former. The tubes are placed into the rotor buckets and weighed upright (we use a small beaker cushioned with tissue). PBS-D is added drop- wise to balance the tubes. Take special care to weigh the buckets and tubes, and load the centrifuge rotor according to the manufacturer’s tolerances and specifications found in the manual. Failure to do so can result in severe damage to the rotor and the instrument.
      Note: The rotor, buckets and solutions should be pre-chilled to 4 °C. If an odd number of gradients are required a “blank” gradient is made and overlaid with buffer to balance the rotor. 
    3. The gradients are centrifuged overnight (this is purely for convenience) at 24,000 rpm in an SW-28 Beckman rotor (or 100,000 x g) and 4 °C. 
    4. After the run is completed, remove a tube carefully from the bucket and attach to a ring stand using a small tube clamp. A virus band of iridescent blue should be visible at the potassium tartrate 15%-35% step interface when the gradient is illuminated from the side with a hand held light of 20 watts. See Figure 4 Density gradient purified Sindbis virus band. Repeat for each of the gradients. 
    5. Collect the band by puncturing the bottom of the tube and letting the tartrate solution flow into a waste beaker, while collecting the virus band into a 2 ml cryotube. Discard the remaining solution. Alternatively, the band can be collected from the side of the tube using a needle and syringe.
    6. Pool the virus-containing samples and dilute with 1.5 volumes of ice-cold PBS-D. This solution should be less dense than 15% potassium tartrate. To check that your concentration is correct, your virus solution should not sink into an aliquot of 15% potassium tartrate. 
    7. This diluted virus is then layered over a continuous 15%-35% potassium tartrate linear gradient of a volume not less than ¾ of the added virus sample. The first gradient may be a step or linear gradient but the second should be a linear one. Do not overfill. 
    8. The continuous gradients are run for 2 h at 26,000 rpm in an SW 40ti Beckman rotor (120,000 x g) and the virus band collected as described in Step D6 above. The virus band should appear about 1/3 down the length of the tube. The virus band may be stored at 4 °C in the tartrate solution. The refractive index of the final virus-tartrate solution is 1.3665 which is ~28% tartrate (Rumble, 2005). 
    9. Collect a sample of the virus to titrate by plaque assay and to determine protein concentration by BCA assay (PierceTM Rapid Gold BCA Protein Assay Kit, Thermo Fisher).
      Note: Properly grown and purified SVHR should give a particle/PFU value of ~1.


      Figure 4. Shown is the opalescent blue band of purified Sindbis virus in a 15-35% potassium tartrate gradient. Note the flocculent pellet at the bottom of the tube. This will be removed after the second K-tartrate gradient.

    Concentration of virus by Polyethylene glycol (PEG) precipitation
    Note: This is an alternative purification step used to concentrate the virus particles but infectivity will be lost.
    1. Infect BHK cells with Sindbis virus as previously described for virus purification. The number of flasks infected will vary depending on how much virus you need. 
    2. Pour culture medium into a centrifuge tube. 
    3. Spin supernatant for clarification, removing cells and cell debris (from this step proceed as in Steps D2-D9 in virus purification protocol.
    4. Transfer supernatant to a fresh tube.
    5. Add 0.25 volumes of 40% polyethylene glycol-8000 in 2 M NaCl and mix well.
    6. Incubate about 24 h at 4 °C for virus precipitation.
    7. Centrifuge at 10,000 rpm (12,000 x g) for 10 min.
    8. Discard the supernatant into a waste beaker and drain well (invert the tube and use a kimwipe to absorb any traces of PEG). 
    9. Re-dissolve the pellet in 5 ml of 1x PEG buffer.
    10. Transfer to a Kimax glass centrifuge tube.
    11. Centrifuge at 12,000 x g for 10 min (Sorval)–this is a further clarification step.
    12. Use the supernatant for gradient purification as described (from this step proceed as in Steps D2-D9 in virus purification protocol.
    Notes:
    1. We did not use PEG concentration of Sindbis virus in our proteomic protocol but include the results in this protocol to demonstrate the importance of purification of infectious virus particles compared to non-infectious particles to extrapolate conclusions on functions of the virus system in question. 
    2. We find that using the SVHR strain of Sindbis virus, PEG concentration followed by isopycnic purification increases the particle/PFU ratio by 10 fold. This increase in the number of non-infectious particles in the interpretation of the experimental outcomes should be seriously evaluated and noted in the results.

  5. Calculation of particle to PFU ratio
    1. A precise protein concentration of the purified virus is required for this calculated value to be accurate. The tartrate solution or PEG will interfere with this assay; the sample must be diluted 1:10.
    2. SVHR virus proteins MW in Daltons = total combined weight of structural proteins (C, E2 and E1) = 130 KDa (by direct BCA measurement, use this measured number and not the calculated value). 
    3. This value is multiplied by 240 copies/virus particle (T = 4 particle:60 T = 240 copies). See Johnson and Speir (1997) for T calculation.
      130 KDa (total virus protein, see Step E2) x 240 (copies/particle see Step E3 above) = 3.12 x 107 Da of protein/virus particle
      Convert from Da to grams [1 Dalton = 1.66 x 10-24 g (CRC tables)]
      3.12 x 107 Da/virus particle x 1.66 x 10-24 g/Da = 5.18 x 10-17 g/particle
      e.g., if you have 1011 PFU/ml SVHR you will have 208 µg/ml of protein (measured by BCA assay)
      = 2.08 x 10-4 g/ml.
      Note: PFU and particles are not the same unit. PFU or plaque forming units is a measure of infectivity of the virus because all virus particles are not infectious. The number of particles refers to the number of physical particles in a solution, which is normally greater than the number of PFU.
      Calculate particles/ml:
      2.08 x 10-4 g/ml/5.18 x 10-17 g/particle = 0.4 x 1013 particles/ml
      0.4 x 1013 particles/ml/1 x 1011 PFU/ml = 0.4 x 102 particles/PFU = 40 particles/PFU
      Note: For Sindbis SVHR the particle/PFU ratio from BHK or C7-10 cells should be ~1 part/PFU.

  6. Mass spectrometry and proteomic analysis
    1. To visually determine if the sample contains contamination a sample of the purified population was then visualized by transmission electron microscopy to check for contaminating cellular organelles or membrane fragments (Coombs and Brown, 1987). This method is beyond the scope of this protocol but detailed in the reference provided above.
    2. Additionally, to check for co-purified protein contaminants, check protein content by running ~10 μl of virus on a 4-12% Bis-Tris SDS-PAGE gradient gel (Invitrogen, Novex) as described by the manufacturer.
    3. Stain the gel with silver in the method of Wray or with coomassie blue (Wray et al., 1981). Excise the visualized bands (silver stain only) and perform an in-gel digestion prior to LC-MS/MS analysis for protein identification (Glaros et al., 2015).

    Protein extraction and digestion
    Viral preparations and their respective negative controls, medium from the cell monolayers harvested prior to infection were processed for LC-MS/MS analysis using the filter-aided sample preparation (FASP) method (Wiśniewski et al., 2009).
    1. Following purification, determine the total protein concentration using a Pierce BCA protein assay kit, following the manufacturer’s instructions.
    2. Normalize all preparations using sterile PBS to 0.5 µg/µl; aliquot out 10 µg of each sample into Lo-bind Eppendorf tubes.
    3. For each sample, mix 10 µg of total protein (20 µl) 1:1 with M-PER supplemented with 50 mM dithiothreitol (DTT) and heat at 95 °C in a heat block at 400 rpm for 10 min to reduce and denature proteins.
    4. Cool samples to room temperature, and mix with 200 µl of UA buffer (8 M urea, 100 mM Tris-HCl, pH 8.5).
    5. Apply each preparation to a 30 kDa filter spin column (Millipore-Ultracell YM-30), and centrifuge at 14,000 x g for 30 min at room temperature to collect all proteins onto the filter membrane.
    6. Alkylate proteins by adding 100 µl of iodoacetamide (IAA) solution (0.05 M IAA in UA buffer) to each filter membrane and incubate at room temperature in the dark for 20 min. Following alkylation, centrifuge the samples at 14,000 x g for 20 min to remove the alkylation solution.
    7. Wash each sample by applying 100 µl of UA buffer to the filter and centrifuging at room temperature at 14,000 x g for 30 min. Repeat this washing step two more times for a total of three washes. Then wash three more times each with 100 µl of 100 mM triethylammonium bicarbonate (TEAB).
    8. To digest the captured protein, apply 100 µl of a trypsin digestion solution (Trypsin/Lys-C prepared in 100 mM TEAB to 10 µg/ml) on each membrane and incubate in sealed tubes overnight (wrap the closed tube caps with parafilm for a better seal) in a heat block at 37 °C, 400 rpm.
    9. After incubation, collect the peptides for LC-MS/MS analysis. Place filter units in a new, clean tube and centrifuge each tube for 30 min at 14,000 x g at room temperature. Wash the membrane by centrifugation one time with 50 µl of 100 mM TEAB and once with 50 µl of 0.5 M NaCl. Collect both of these washes and pool with the final peptide eluate.
    10. Acidify each sample with 10% trifluoroacetic acid until the final pH is ~2-3.
    11. Prior to mass spectrometric analysis, desalt each sample using Pierce C18 spin columns according to the manufacturer’s directions. Eluate from the desalting process should be dried to completeness in a clean Lo-bind Eppendorf microcentrifuge tube using a SpeedVac at 30 °C and stored at -80 °C until LC-MS/MS analysis.

    LC-MS/MS data acquisition
    1. Reconstitute dried peptides in “A Buffer” (3% acetonitrile/0.1% formic acid) and resolve on VIRGIN Picofrit 15 cm x 75 µm ID HPLC column packed with 5 µm BioBasic C18 particles 300Å using a multistep gradient [e.g., 130 min; 0-5 min: 5-10% B, 6-110 min: 10-35% B, and 111-130 min: 35-95% B]. A virgin or unused column is important to ensure that all peptides identified are from the sample and not from a previous LC-MS/MS run which could result from ‘carry over’ if using a column that already had a sample applied. The liquid chromatography gradient should be chosen to allow for adequate separation without excessive time. These parameters depend upon the column and instrument used for analysis. For the gradient, the A buffer is 3% acetonitrile/0.1% formic acid and the B buffer is 95% acetonitrile/0.1% formic acid. Tryptic peptides should be analyzed in at least technical triplicates.
    2. For the Orbitrap ELITE the following configurations were used. This should serve as a guide for similar instrument parameters if using other mass spectrometers. These parameters are instrument specific and you should consult the instrument’s manufacture or the literature for recommendations. Generic settings established from the literature used for bottom up (tryptic peptide analysis) proteomics are likely adequate. A variety of instrument methods have been curated and peer-reviewed which are available at www.massspectrometrymethods.org.
      Example instrument settings used for an orbitrap ELITE
      1. Orbitrap MS1 scans are performed at a resolution of 120,000 at 400 m/z, with a scan range of 110-2,000 m/z.
      2. The top 20 precursors are selected for MS2 data-dependent fragmentation. An MS2 spectrum is acquired using the iontrap scanning in normal mode (Top 20 Method).
      3. The minimum signal required to trigger a data-dependent scan is 5000.
      4. Collision-induced dissociation (CID) is used to generate MS2 spectra with the following settings: normalized collision energy 35%, default charge state 2, isolation width 2 m/z, and activation time 10 ms.
      5. AGC target is set to 1 x 106 for MS and 5 x 104 for MS/MS with a maximum accumulation time of 200 ms.
      6. Dynamic exclusion is set for 60 s for up to 500 targets with a 5 ppm mass window.
      7. A lock mass of 445.120025 is used for internal calibration to improve mass accuracy.

    Data Processing
    1. Process spectra data using Proteome Discoverer with the embedded SEQUEST search algorithm against a Sindbis virus polyprotein database (Uniprot ID: P03317) merged with either Homo sapiens (RefSeq Tax ID: 9606) or Cricetulus griseus (RefSeq Tax ID: 10029). Download the Aedes albopictus-deducted proteome (Chen et al., 2015) data from VectorBase (Giraldo-Calderón et al., 2015) and additional peptides from the NCBI TSA database.
    2. Organize the non-redundant proteomes on excel spreadsheets (Ribeiro et al., 2004) and annotate as described previously (Karim et al., 2011). Merge the resulting FASTA file with the SINV polyprotein and use it to search against the SINV mosquito preparations.
    3. Set dynamic modifications for carbamidomethylation of cysteine [+57.02 Da], oxidation of methionine [+15.99 Da], and N-terminal acetylation [+42.011].
    4. MS/MS spectra are searched with a precursor mass tolerance of 10 ppm and a fragment mass tolerance of 0.6 Da.
    5. Trypsin is specified as the protease with a maximum number of missed cleavages set to 2.
    6. False discovery rate using PERCOLATOR (Kall et al., 2007) is set to < 1% to score high confidence peptide identifications.
    7. Perform grouping and functional analysis using the PANTHER classification system for the human background only with each protein id’s accession number (Mi et al., 2016).

Recipes

  1. Complete E-MEM, 1x and 2x
    Note: Do not warm 2x MEM until the supplements have been added, it will precipitate.
    10% heat-inactivated fetal bovine serum (FBS; any suitable FBS, we test sample lots of FBS prior to purchase)
    5% tryptose phosphate broth (TPB; see Recipe 11; This is only added to the BHK cells)
    0.02% L-glutamine (see Recipe 3 for 100x)
    1x gentamicin sulfate (see Recipe 2 for 100x)

    For 1x medium
    Mix the following:
    400 ml 2x E-MEM (see Recipe 1)
    400 ml TC H2O
    80 ml FBS
    8 ml L-glutamine
    *8 ml gentamicin sulfate
    Store up to 2 weeks at 4 °C
    *Note: Gentamicin is added to medium only for virus growth and not for general maintenance of any of the cell cultures.
    For 2x medium (used to titer viruses by plaque assay)
    Prepare 536 ml medium by mixing the following:
    400 ml 2x E-MEM (incomplete, serum-free; see Recipe 1)
    80 ml FBS
    40 ml TPB
    8 ml 100x L-glutamine
    8 ml 100x gentamicin sulfate
    Store up to 6 months
    IMPORTANT NOTE: Complete medium with supplements are not true percent solutions. The percentage indicated represents the percentage of the original volume of medium. This also holds for any other supplements added to the medium. For example, if the initial volume of the medium is 400 ml, 10% FBS is 40 ml. Addition of other supplements, such as 5% TPB would be 20 ml TPB, bringing the total volume to 460 ml. If problems arise with the cell cultures, growth of the virus, or plaque formation, discard old solutions and make fresh solutions. 
  2. Gentamicin sulfate, 100x
    Dilute 0.5 g gentamicin sulfate up to 100 ml in tissue-culture-grade water
    Store up to 6 months at 4 °C
    Dilution to 1x will give a final concentration of 50 μg/ml. If necessary, 100 μg/ml may be used
    CAUTION: Antibiotics can become contaminated. If persistent problems are experienced with contaminated cultures, test all solutions in the absence of antibiotics. 
  3. L-glutamine, 100x
    Weigh out 2.92 g L-glutamine and dissolve in a total volume of 100 ml 1x PBS-D (see Recipe 7 for 10x)
    Filter sterilize and divide into 50-ml aliquots in 100-ml bottles
    Store frozen up to 6 months; once thawed, discard after 2 weeks
    Note: Glutamine is essential and very labile. It is thus added to already complete medium as a precaution.
  4. HEPES (pH 7.2-7.4), 1 M
    Weigh out 238.3 g HEPES and dissolve in a total volume of 1 L tissue-culture grade water
    Adjust pH to 7.2-7.4 with NaOH
    Autoclave for 30 min to sterilize
    Store up to 1 year at room temperature
  5. PEG Buffer
    0.4 M NaCl
    0.01 M Tris Buffer pH 7.0
    0.001 M EDTA
  6. Neutral red stock solution, 2%
    Dissolve 2 g neutral red and adjust the volume to 100 ml with tissue-culture-grade water
    Stir overnight at room temperature and filter sterilize
    Store up to 1 year at room temperature
    Notes:
    1. Some stain will be lost in the filtration process and each batch may differ. This stain is quite viscous and may require more than one filter to sterilize the entire quantity. 
    2. Alternatively, neutral red stock solution may be purchased ready made from many suppliers.
  7. PBS-D, 10x
    2.0 g KCl
    2.0 g KH2PO4
    80.0 g NaCl
    11.3 g Na2HPO4 or 21.6 g Na2HPO4·7H2O
    Tissue-culture-grade H2O to 1 L
    Autoclave for 20 min
    Dilute to 1x with sterile tissue-culture-grade water
    Store up to 1 year at room temperature
    Note: PBS-D is PBS (phosphate buffered saline) deficient in calcium and magnesium. This can be made as a 1x solution. Also see annotations to the recipe for PBS with calcium and magnesium. It is critical that the pH of the 1x solution is ~7.4 because low pH will inactivate all Alphaviruses.
  8. PBS with calcium and magnesium, 1x
    Prepare solutions 1 and 2 (which are 10x stock solutions) and autoclave separately for 20 min
    Note: PBS cannot be made as a concentrated solution, it will precipitate.
    Solution 1:
    1.0 g CaCl2
    1.3 g CaCl2·2H2O
    2.0 g KH2PO4 (dissolve separately, then add to mixture)
    1.0 g MgCl2·6H2O
    80.0 g NaCl
    Adjust volume to 1 L with tissue-culture-grade H2O
    Solution 2:
    21.6 g Na2HPO4·7H2O or 11.3 g anhydrous Na2HPO4
    Adjust volume to 1 L with tissue-culture-grade H2O
    For 1x solution:
    Add 50 ml solution 1 to 400 ml tissue-culture-grade water
    Mix, then add 50 ml solution 2
    Do not directly mix solutions 1 and 2 together: salts will precipitate
    Store solutions at room temperature up to 1 year or until salts begin to precipitate
    Check that the final pH of the 1x solution is ~7.2 to 7.4
    IMPORTANT NOTE: Do not adjust the pH of the 10x solutions 1 and 2, because this will result in the wrong pH for the 1x solution. It is critical that all solutions that Sindbis virus comes in contact with are neutral pH. Acidic pH quickly inactivates the virus. 
  9. Phenol red, 0.5%
    Note: Do not confuse with neutral red.
    Dissolve 1 g phenol red in 200 ml tissue-culture-grade water
    Filter sterilize and store up to 1 year at room temperature
  10. SVHR diluent (used to dilute virus to be titrated by plaque assay)
    Supplement 1x PBS-D (see Recipe 7 for 10x) to 3% FBS with heat-inactivated fetal bovine serum (FBS)
    Store up to 2 months at 4 °C
  11. Trypsin stock, 0.25%
    0.5 g trypsin
    0.2 g disodium EDTA
    0.6 g phenol red
    Adjust volume to 200 ml with 1x PBS-D (see Recipe 7 for 10x)
    Adjust pH with 1 N NaOH until a cherry red color is achieved
    Divide into 25-ml aliquots
    Store frozen up to 6 months at -20 °C
    Note: Prior to use, thaw in a 37 °C water bath and dilute to 100 ml with versene solution (see Recipe 13). Do not heat trypsin at 37 °C for > 20 min, as auto-digestion of the enzyme will occur. 
  12. Tryptose phosphate broth (TPB)
    Weigh out 29.5 g tryptose phosphate broth (TPB; Difco)
    Dissolve in a total volume of 1 L tissue culture grade water
    Autoclave TPB for 20 min in two separate 500-ml volumes to sterilize
    Do not leave in the autoclave for longer periods since this solution will caramelize
    Cool and store up to 6 months at 4 °C
  13. Versene solution
    500 ml 1x PBS-D (see Recipe 7 for 10x)
    5 ml 0.1 M EDTA (see Recipe 14)
    1.5 ml 0.5% phenol red (see Recipe 9)
    Adjust pH to 7.4 with NaOH
    Filter sterilize (if sterile stock solutions were not used)
    Store up to 6 months at room temperature
  14. 0.1 M EDTA solution
    Use the disodium salt form 3.36 g/100 ml
    The pH will drop below 5.3 and solution will remain cloudy until its pH is adjusted to 7.0. Adjust the pH to 8.0 with NaOH
    Autoclave to sterilize
  15. M-PER Supplemented with 50 mM dithiothreitol (DTT)
    Notes: 
    1. You will need a minimum of 20 μl of DTT-supplemented M-PER for every sample you process. 
    2. Leftover 1 M DTT stock can be stored at -20 °C for up to 30 days (it is best make 50-100 μl aliquots to prevent any freeze/thaw cycles).
  16. UA buffer (8 M urea, 100 mM Tris-HCl pH 8.5) (10 ml)
    Note: You will need around 500 μl of UA buffer per sample.
    Add 4.8048 g urea (Thermo Scientific) to 6 ml mass spec grade water and dissolve (the solution will become cold, so it may be necessary to heat the solution gently in a water bath to get the urea to dissolve completely). Add 1 ml 1 M Tris-HCl pH 8.5 and add mass spec grade water to 10 ml
  17. IAA solution (0.05 M iodoacetamide in UA buffer) (1 ml)
  18. 100 mM triethylammonium bicarbonate (TEAB) (3 ml)
    Add 300 μl 1 M TEAB (Sigma) to 2.7 ml mass spec grade water and vortex
    Recommended to keep solution on ice
  19. Trypsin digestion solution (Trypsin/Lys-C prepared in 100 mM TEAB to 10 µg/ml)
    Add 2,000 μl 100 mM TEAB to a single 20 μg vial of Trypsin/Lys-C (Promega) for a final concentration of 10 μg/ml, and gently pipet-mix until completely dissolved. DO NOT VORTEX. Recommended to keep solution on ice
  20. 0.5 M NaCl (5 ml)
    Note: this will be an excess of solution, but it is a more manageable to weigh out NaCl for this volume).
    Weigh out 146.1 mg NaCl (Sigma) and add mass spec grade water to 5 ml
    Vortex until dissolved
  21. 10% trifluoroacetic acid
    Add 1 ml of 100% trifluoroacetic acid (Thermo Scientific) to 9 ml mass spec grade water and vortex
  22. A Buffer (3% acetonitrile/0.1% formic acid)
    Add 1 ml 100% formic acid (Thermo Scientific) to 30 ml 100% acetonitrile, then add mass spec grade water to 1 L
  23. B buffer (95% acetonitrile/0.1% formic acid)
    Add 1 ml 100% formic acid to 950 ml 100% acetonitrile, then add mass spec grade water to 1 L

Acknowledgments

RH and DF were supported by the Clayton Foundation for Research, Carson City NV and the College of Agriculture and Natural Sciences, North Carolina State University. TG and GR were supported by the U.S. Army Combat Capabilities Development Command (CCDC) Chemical Biological Center through an internal basic research grant. The opinions presented here are those of the authors and are not the official policy of the U.S. Army, CCDC, or the U.S. Government. Information in this report is cleared for public release and distribution is unlimited. This protocol is adapted from the publication. Schuchman R, Kilianski A, Piper A, Vancini R, Ribeiro JMC, Sprague TR, Nasar F, Boyd G, Hernandez R, Glaros T. 2018. Comparative Characterization of the Sindbis Virus Proteome from Mammalian and Invertebrate Hosts Identifies nsP2 as a Component of the Virion and Sorting Nexin 5 as a Significant Host Factor for Alphavirus Replication. J Virol 92.

Competing interests

The Authors declare no competing interests.

References

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  2. Chen, X. G., Jiang, X., Gu, J., Xu, M., Wu, Y., Deng, Y., Zhang, C., Bonizzoni, M., Dermauw, W., Vontas, J., Armbruster, P., Huang, X., Yang, Y., Zhang, H., He, W., Peng, H., Liu, Y., Wu, K., Chen, J., Lirakis, M., Topalis, P., Van Leeuwen, T., Hall, A. B., Jiang, X., Thorpe, C., Mueller, R. L., Sun, C., Waterhouse, R. M., Yan, G., Tu, Z. J., Fang, X. and James, A. A. (2015). Genome sequence of the Asian Tiger mosquito, Aedes albopictus, reveals insights into its biology, genetics, and evolution. Proc Natl Acad Sci U S A 112(44): E5907-5915.
  3. Coombs, K. and Brown, D. T. (1987). Topological organization of Sindbis virus capsid protein in isolated nucleocapsids. Virus Res 7(2): 131-149.
  4. Giraldo-Calderón, G. I., Emrich, S. J., MacCallum, R. M., Maslen, G., Dialynas, E., Topalis, P., Ho, N., Gesing, S., VectorBase, C., Madey, G., Collins, F. H. and Lawson, D. (2015). VectorBase: an updated bioinformatics resource for invertebrate vectors and other organisms related with human diseases. Nucleic Acids Res 43 (Database issue): D707-713.
  5. Glaros, T. G., Blancett, C. D., Bell, T. M., Natesan, M. and Ulrich, R. G. (2015). Serum biomarkers of Burkholderia mallei infection elucidated by proteomic imaging of skin and lung abscesses. Clin Proteomics 12(1): 7.
  6. Hernandez, R., Brown, D. T. and Paredes, A. (2014). Structural differences observed in arboviruses of the alphavirus and flavivirus genera. Adv Virol 2014: 259382.
  7. Johnson, J. E. and Speir, J. A. (1997). Quasi-equivalent viruses: a paradigm for protein assemblies. J Mol Biol 269(5): 665-675.
  8. Kall, L., Canterbury, J. D., Weston, J., Noble, W. S. and MacCoss, M. J. (2007). Semi-supervised learning for peptide identification from shotgun proteomics datasets. Nat Methods 4(11): 923-925.
  9. Karim, S., Singh, P. and Ribeiro, J. M. (2011). A deep insight into the sialotranscriptome of the gulf coast tick, Amblyomma maculatum. PLOS One 6: e28525.
  10. Mi, H., Poudel, S., Muruganujan, A., Casagrande, J. T. and Thomas, P. D. (2016). PANTHER version 10: expanded protein families and functions, and analysis tools. Nucleic Acids Res 44(D1): D336-342.
  11. Ribeiro, J. M., Topalis, P. and Louis, C. (2004). AnoXcel: an Anopheles gambiae protein database. Insect Mol Biol 13(5): 449-457.
  12. Rumble, J. R. (2005). CRC Handbook of Chemistry and Physics. Internet Version 2005, CRC Press, Boca Raton, FL.
  13. Strauss, J. H. and Strauss, E. G. (1994). The alphaviruses: gene expression, replication, and evolution. Microbiol Rev 58(3): 491-562.
  14. Vancini, R., Wang, G., Ferreira, D., Hernandez, R. and Brown, D. T. (2013). Alphavirus genome delivery occurs directly at the plasma membrane in a time- and temperature-dependent process. Journal of Virology 87(8): 4352-4359.
  15. Wilkins, M. (2009). Proteomics data mining. Expert Rev Proteomics 6(6): 599-603.
  16. Wilkins, M. R., Pasquali, C., Appel, R. D., Ou, K., Golaz, O., Sanchez, J. C., Yan, J. X., Gooley, A. A., Hughes, G., Humphery-Smith, I., Williams, K. L. and Hochstrasser, D. F. (1996). From proteins to proteomes: large scale protein identification by two-dimensional electrophoresis and amino acid analysis. Biotechnology (N Y) 14(1): 61-65.
  17. Wiśniewski, J. R., Zougman, A., Nagaraj, N. and Mann, M. (2009). Universal sample preparation method for proteome analysis. Nat Methods 6(5): 359-362.
  18. Wray, W., Boulikas, T., Wray, V. P. and Hancock, R. (1981). Silver staining of proteins in polyacrylamide gels. Anal Biochem 118(1): 197-203.

简介

摘要:目前的质谱(MS)方法和新仪器现在允许比以前的蛋白质分级分离和鉴定方法更精确地鉴定低丰度的蛋白质。如果该方法可用于定义含有膜的病毒的病毒蛋白质组,则是有意义的。为了评估质谱确定医学上重要的病毒,辛德毕斯病毒(SINV)的蛋白质组的功效,选择原型甲病毒进行评估。选择该模型系统是因为甲病毒含有人类病原体的成员,该病毒在生物化学和结构上有明确的定义,并且在脊椎动物和非脊椎动物宿主细胞中都生长至高滴度。使用该方法研究SINV蛋白质组以确定宿主蛋白是否被特异性包装到感染性病毒粒子中。如果SINV蛋白质组在代表脊椎动物和蚊子宿主的多个宿主细胞中生长时掺入来自所有宿主的特异性宿主蛋白,那么也是有意义的。观察病毒蛋白质组中的复发或独特蛋白质有助于测定掺入病毒粒子的蛋白质,而不是与粒子外部结合的蛋白质。质谱分析在检测限内鉴定纯化的病毒粒子的总蛋白质含量。最重要的发现是除了宿主蛋白之外,在检查的所有宿主细胞中生长的病毒粒子中检测到SINV非结构蛋白2(nsP2)。该分析鉴定了先前未与甲病毒进入,复制或出口相关的宿主因子,其鉴定至少一种与甲病毒复制整合的宿主因子。该分析成功的关键是病毒纯化方法,该方法必须提供不含高水平污染物的可测量的感染性病毒。对于SINV和甲病毒属的其他成员,这通过通过酒石酸钾进行等密度离心,然后进行高盐洗涤来实现。


背景:术语蛋白质组和蛋白质组学是由Marc Wilkins创造的,用于描述模型系统中蛋白质的系统评估,使用其生物学的结构,功能和调节的详细研究,包括导致疾病的畸变(Wilkins et al。,1996年和2009年)。然而,病毒蛋白质组早在蛋白质组学领域发展之前就已经开始研究,试图了解体外病毒 - 宿主相互作用的机制并评估动物宿主的病毒发病机制。为此,有意义的是评估含有医学上重要的病原体的病毒家族中的模型系统的病毒蛋白质组。辛德毕斯病毒被选择进行这项调查,因为它是甲病毒属的一个成员,家庭披膜病毒科,其中含有大量具有医学重要性的人类病原体,它结构稳定,长到高滴度,在文献中有很好的描述,可以在脊椎动物和非脊椎动物宿主细胞中生长。辛德毕斯病毒已成为许多研究的主题,因为它是一种相对简单的含有膜的+ RNA二十面体病毒(图1)。病毒42S基因组具有感染性,可作为编码结构基因的26S亚基因组RNA的模板,按序列C-PE2(E3 / E2)-6K(TF)-E1 3'UTR-polyA组织(图2) 。基因组链以5'UTR-nsP1-nsP2-nsP3-nsP4的顺序编码非结构蛋白(Strauss,J.H。和Strauss,E.G.,1994)。病毒组装过程在脊椎动物或无脊椎动物细胞中发生显着差异(Brown,1986)。在脊椎动物细胞中,核壳体在与病毒蛋白质修饰的质膜结合并且从质膜出芽的病毒之前与基因组RNA预组装在细胞质中。在无脊椎动物细胞中,病毒核衣壳和完全成熟的病毒在含有病毒的内体与质膜融合之前被组装在称为“病毒工厂”的内体中,释放出感染性颗粒。取决于病毒的宿主细胞来源,糖蛋白的糖基化模式将是细胞生物化学所指定的,并且特定的脂质双层将是宿主细胞的糖基化模式。从任一宿主释放的病毒颗粒可以具有相同的感染性,并且如果使用辛德毕斯病毒的SVHR菌株,则实现接近100%的生存力(Vancini 等人,,2013)。这是一个重要的细节,因为病毒感染性要求其所有组分处于亚稳态构象和天然物理状态才具有传染性(Hernandez et al。,2014)。因此,并非所有病毒颗粒都适合这种分析方法,因为颗粒结构必须是1)常规化学计量2)能够严格纯化而不损失感染性和3)表现出非常低的颗粒与PFU比率。这些因素对于能够从病毒颗粒中携带的蛋白质中辨别任何蛋白质污染物是重要的。由于病毒颗粒的对称性和化学计量,蛋白质浓度可用于使用噬斑测定法从感染性颗粒的数量计算物理颗粒的数量,以确定感染性颗粒滴度。此特定分析不适用于非对称病毒。


图1.甲病毒的漫画。该漫画描绘了一个有包裹的球形,二十面体,直径65-70纳米,衣壳上有一个 T = 4二十面体对称性由240个单体1:1:1的化学计量比E1:E2:C组成。信封包含80个尖峰,每个尖峰是E1 / E2蛋白二聚体的三聚体。经SBI瑞士生物信息学研究所ViralZone许可转载。来自ViralZone的数据,经许可。


图2.甲病毒基因组组织。该基因组是单分子的,线性的, ssRNA(+)基因组 11-12 kb。基因组被限制(c)和多腺苷酸化。 P123,nsP1,2和3的前体。病毒蛋白由宿主和病毒蛋白酶成熟。 RdRp,RNA依赖性RNA聚合酶,TF,跨框蛋白(由6k蛋白中的核糖体框架移位制成)和DLP(下游环),在亚基因组RNA的5'区域的编码序列中稳定的RNA结构。来自ViralZone的数据,经许可。

BHK细胞或C7-10细胞中的病毒株生长
在该研究中使用耐热SINV(SVHR)菌株。该菌株于1966年由Burge和Pfefferkorn通过收集抗加热至54℃的病毒分离。病毒株的选择很重要,因为该菌株产生高滴度(10 10 PFU / ml)和低粒子/ PFU(~1粒子/ PFU),高感染性和物理稳定性病毒的比例。 BHK和C7-10(白纹伊蚊)细胞从内部收集获得,并且是生长SVHR的优选细胞。从10个T-75烧瓶(Corning)中收获病毒,其产生足够的病毒以在30ml酒石酸钾梯度中形成大的可见条带,并且用于质谱分析。对于辛德毕斯病毒感染,以10PFU / ml的MOI感染细胞,并使其复制一个循环,并在感染后18小时收获,以确保不发生细胞裂解。来自哺乳动物细胞的单个SINV生长周期为~12小时,而来自C7-10细胞的单周期为约24小时。通过低速离心(Sorvall RC-5B超速离心机在2,000rpm,700 xg )澄清上清液。将20微升所得病毒上清液上样到15-35%线性酒石酸钾梯度上,并通过等密度超速离心(Beckman SW-28转子,在10,000 x g 下18小时)纯化两次。收集所得的纯化病毒带并通过将病毒在SW-40 Beckman-40转子中的5ml 1x PBS中以45,000rpm(12,000 xg )沉淀30分钟并收集沉淀物洗涤两次。 。

通过噬菌斑检测进行病毒滴定
通过斑块形成测定病毒滴度,“噬斑测定”是测量感染性病毒量的最准确方法。该测定用于通过用已知体积的已知稀释的含病毒溶液感染标准化的单层细胞来测定每毫升噬斑形成单位(PFU)的滴度。感染包含在琼脂糖中,琼脂糖仅允许病毒扩散到邻近细胞。来自单个最初感染的细胞的病毒感染相邻细胞,产生“斑块”或清除(由裂解细胞形成),通过在1x EMEM中覆盖1%琼脂糖定位于原始感染部位。在37℃温育2-3天后,中性红染色后肉眼可见SVHR斑块。开始估计滴度可能是多少,如果滴度估计大约10 8 PFU / ml,感染稀释度为10 -6 的烧瓶会显示20-200个斑块;在这种情况下,用10 -5 ,10 -6 和10 -7 PFU / ml的稀释液感染烧瓶应该给出足够的数据做一个相对准确的计算。如果病毒滴度完全未知,则可能需要用各种稀释液(10 -1 至10 -8 )感染烧瓶或平板。在给定测定中看到的斑块的数量和质量可受许多因素影响,包括培养基的pH和/或温度,稀释缓冲液,琼脂糖覆盖层或细胞单层的条件。由于该测定的灵敏度,在每个测定中包括阳性和阴性对照是重要的。阴性对照是仅用稀释剂接种的烧瓶,阳性对照由已知滴度的一种稀释的SVHR原种病毒组成,足以产生~20个噬菌斑。这些斑块的数量在统计学上是显着的。

重要的是,当从高浓度到低浓度时,移液管尖端被改变以避免“遗留”污染。然而,当从低浓度移液到较高浓度时,如接种孔时所做的那样,可以使用单个移液管尖端。

关键字:辛德比斯病毒, 质谱, 病毒蛋白质组, 病毒纯化, α病毒, 蛋白质组学

材料和试剂

注意:这些可以来自任何提供细胞培养级材料或试剂的供应商,除非另有说明。所有培养瓶和培养皿都是标准的,除非另有说明,否则可以从任何供应商处获得。
所有胎牛血清(FBS)应在56°C下加热灭活30分钟。

  1. 脊椎动物和无脊椎动物细胞的亚培养
    BHK-21细胞的亚培养
    1. 6孔板
    2. 无菌组织培养用品(移液管,通气或塞帽烧瓶和15毫升锥形管)
    3. 热灭活胎牛血清(FBS)
    4. 胰蛋白胨磷酸盐肉汤
    5. L-谷氨酰胺
    6. 庆大霉素硫酸盐
    7. 氯化钾
    8. KH <子> 2 PO <子> 4
    9. 氯化钠&NBSP;
    10. Na 2 HPO 4 或Na 2 HPO 4 ·7H 2 O
    11. CaCl 2 或CaCl 2 ·2H 2 O
    12. 的MgCl <子> 2 ·6H <子> 2 0
    13. 完成E-MEM,温热至37°C(伯爵盐最低必需培养基,10%热灭活胎牛血清,FBS完成)&nbsp;
    14. 1x PBS-D(见食谱)
    15. 胰蛋白酶原液,0.25%,温热至37°C(见食谱)

    C7-10细胞的亚培养
    1. 无菌组织培养用品(移液管,通气或塞帽烧瓶和15毫升锥形管)
    2. 70%乙醇
    3. 1x MEM

  2. 病毒生长和滴定
    库存病毒准备
    1. 血清移液器(5毫升,10毫升和25毫升)
    2. FBS(胎牛血清)
    3. 无菌组织培养级水:有关纯净水的详细讨论以及细胞培养和其他等级水的规格,请参阅实验室用水(国家卫生研究院,2013年3月)
    4. 庆大霉素硫酸盐
    5. L-谷氨酰胺
    6. 氯化钾
    7. KH <子> 2 PO <子> 4
    8. 氯化钠&NBSP;
    9. Na 2 HPO 4 或Na 2 HPO 4 ·7H 2 O
    10. CaCl 2 或CaCl 2 ·2H 2 O
    11. 的MgCl <子> 2 ·6H <子> 2 0
    12. 中性红
    13. 完整的E-MEM,1x和2x(参见食谱)
    14. 硫酸庆大霉素,100x(仅哺乳动物细胞)(见食谱)
    15. L-谷氨酰胺100x(见食谱)
    16. HEPES(pH 7.2-7.4),1 M(见食谱)
    17. 2%中性红色原液(见食谱)
    18. PBS-D(10x)(见食谱)
    19. SVHR稀释剂(见食谱)
    20. 胰蛋白胨磷酸盐肉汤(TPB)(ATCC细胞可选)(见食谱)

    BHK细胞斑块形成的病毒滴定
    BHK细胞制备
    1. 无菌组织培养用品(移液管,通气或塞帽烧瓶和15毫升锥形管)
    2. 热灭活胎牛血清(FBS)
    3. 胰蛋白胨磷酸盐肉汤
    4. L-谷氨酰胺
    5. 庆大霉素硫酸盐
    6. 胰蛋白酶
    7. 氯化钾
    8. KH <子> 2 PO <子> 4
    9. 氯化钠&NBSP;
    10. Na 2 HPO 4 或Na 2 HPO 4 ·7H 2 O
    11. 氯化钙<子> 2
    12. 氯化钙<子> 2 ·2H <子> 2 0
    13. KH <子> 2 PO <子> 4
    14. 的MgCl <子> 2 ·6H <子> 2 0
    15. 完整的E-MEM,温度升至37°C(参见食谱)
    16. 1x PBS-D(见食谱)
    17. 胰蛋白酶原液,0.25%,温热至37°C(见食谱)

    斑块检测的病毒滴定
    1. 2%琼脂糖
    2. 热灭活的胎牛血清(FBS)
    3. 100%甘油
    4. 热灭活胎牛血清(FBS)
    5. 胰蛋白胨磷酸盐肉汤
    6. L-谷氨酰胺
    7. 庆大霉素硫酸盐
    8. 胰蛋白酶
    9. 氯化钾
    10. KH <子> 2 PO <子> 4
    11. 氯化钠&NBSP;
    12. Na 2 HPO 4 或Na 2 HPO 4 ·7H 2 O
    13. CaCl 2 或CaCl 2 ·2H 2 O
    14. KH <子> 2 PO <子> 4
    15. 中性红
    16. 完整的E-MEM 2x(参见食谱)
    17. 2%中性红(见食谱)
    18. SVHR Diluent(见食谱)
    19. 1x PBS-D(见食谱)
    20. 1 M HEPES(pH 7.4)(见食谱)

  3. 辛德毕斯病毒感染脊椎动物和无脊椎动物细胞 辛德毕斯病毒感染BHK细胞
    1. 离心管(Corning,15 ml Fisherbrand,目录号:05-539-5)
    2. 辛德毕斯病毒感染C7-10细胞
    3. 100%甘油(无菌)
    4. 组织培养级水
    5. 热灭活胎牛血清(FBS)
    6. 胰蛋白胨磷酸盐肉汤
    7. L-谷氨酰胺
    8. 庆大霉素硫酸盐
    9. 胰蛋白酶
    10. 氯化钾
    11. KH <子> 2 PO <子> 4
    12. 氯化钠&NBSP;
    13. Na 2 HPO 4 或Na 2 HPO 4 ·7H 2 O
    14. CaCl 2 或CaCl 2 ·2H 2 O
    15. KH <子> 2 PO <子> 4
    16. 的MgCl <子> 2 ·6H <子> 2 0
    17. 胰蛋白胨磷酸盐肉汤(TPB; Difco)
    18. 完整的E-MEM(参见食谱)
    19. 硫酸庆大霉素100x(见食谱)
    20. L-谷氨酰胺100x(见食谱)
    21. 1 M HEPES(见食谱)
    22. TPB-胰蛋白胨磷酸盐肉汤(见食谱)
    23. PBS-D(10x)(见食谱)

    辛德毕斯病毒感染C7-10细胞
    1. 甘油
    2. 液体N 2
    3. 热灭活胎牛血清(FBS)
    4. 胰蛋白胨磷酸盐肉汤
    5. L-谷氨酰胺
    6. 庆大霉素硫酸盐
    7. 胰蛋白酶
    8. 氯化钾
    9. KH <子> 2 PO <子> 4
    10. 氯化钠&NBSP;
    11. Na 2 HPO 4 或Na 2 HPO 4 ·7H 2 O
    12. CaCl 2 或CaCl 2 ·2H 2 O
    13. KH <子> 2 PO <子> 4
    14. 的MgCl <子> 2 ·6H <子> 2 0
    15. 完成的E-MEM培养基(参见食谱)
    16. 1x PBS-D(见食谱)

  4. 辛德毕斯病毒的纯化
    通过等密度离心纯化和浓缩
    1. 离心管(超清)&nbsp;
    2. 小管夹
    3. 废烧杯
    4. 2毫升冷冻管
    5. PBS-D中15%酒石酸钾(二元,半水合物)(过滤灭菌以储存并储存在4°C)
    6. PBS-D中35%酒石酸钾(过滤除菌以储存并储存在4°C)
    7. BCA测定(Pierce TM Rapid Gold BCA Protein Assay Kit)(Thermo Fisher Scientific,目录号:A53225)
    8. 氯化钾
    9. KH <子> 2 PO <子> 4
    10. 氯化钠&NBSP;
    11. Na 2 HPO 4 或Na 2 HPO 4 ·7H 2 O
    12. CaCl 2 或CaCl 2 ·2H 2 O
    13. KH <子> 2 PO <子> 4
    14. 的MgCl <子> 2 ·6H <子> 2 0
    15. PBS-D pH 7.4(见食谱)

    聚乙二醇(PEG)沉淀病毒浓度
    1. Kimax高强度玻璃离心管(Kimax,目录号:#Z2514888)(普通30毫升)
    2. 聚合物离心管
    3. 聚乙二醇-8000(PEG-8000)
    4. PBS-D中15%酒石酸钾(二元,半水合物)&nbsp;
    5. PBS-D中35%酒石酸钾
    6. BCA测定(Pierce TM Rapid Gold BCA Protein Assay Kit)(Thermo Fisher Scientific,目录号:A53225)
    7. 氯化钾
    8. KH <子> 2 PO <子> 4
    9. 氯化钠&NBSP;
    10. Na 2 HPO 4 或Na 2 HPO 4 ·7H 2 O
    11. CaCl 2 或CaCl 2 ·2H 2 O
    12. KH <子> 2 PO <子> 4
    13. 的MgCl <子> 2 ·6H <子> 2 0
    14. Tris缓冲液,pH 7.0
    15. EDTA
    16. PBS-D,pH 7.4(参见食谱)
    17. PEG缓冲液(见食谱)
    18. 2 M NaCl(见食谱)

  5. 颗粒与PFU比的计算
    没有

  6. 质谱和蛋白质组学分析
    注意:所有试剂应为LCV-MS级。
    蛋白质提取和消化
    1. Lo-bind离心管(Eppendorf)&nbsp;
    2. 旋转过滤器(Millipore-Ultracell YM-30)
    3. Pierce C18离心柱(赛默飞世尔科技)
    4. 封口膜
    5. Pierce BCA蛋白质分析试剂盒(Thermo Fisher Scientific,目录号:A53225)
    6. 哺乳动物蛋白质提取试剂(M-PER)补充有50mM二硫苏糖醇(Thermo Fisher Scientific,目录号:78501)&nbsp;
    7. UA缓冲液中的0.05M碘乙酰胺
    8. 用100mM TEAB至10μg/ ml制备的胰蛋白酶/ Lys-C&nbsp;
    9. DTT&NBSP;
    10. 氯化钙<子> 2
    11. 氯化钙<子> 2 ·2H <子> 2 0
    12. KH <子> 2 PO <子> 4
    13. 的MgCl <子> 2 ·6H <子> 2 0
    14. 氯化钠&NBSP;
    15. 尿素
    16. Tris-HCl pH 8.5
    17. TEAB(Sigma,目录号:T7408-100ML)
    18. 100%三氟乙酸&nbsp;
    19. IAA粉末&nbsp;
    20. 无菌1x PBS(见食谱)
    21. UA缓冲区(参见食谱)
    22. 100 mM三乙基碳酸氢铵(TEAB)(见食谱)
    23. 0.5 M NaCl(参见食谱)
    24. 10%三氟乙酸(TFA)(见食谱)
    25. M-PER补充50 mM二硫苏糖醇(DTT)
    26. 碘乙酰胺(IAA)溶液(UA缓冲液中0.05 M IAA)(见食谱)
    27. 胰蛋白酶消化液(见食谱)

    LC-MS / MS数据采集
    1. Picofrit 15 cm x75μmIDHPLC色谱柱,填充5μmBioBasicC18颗粒300Å(新目标)
    2. 3%乙腈/0.1%甲酸
    3. 100%甲酸(Thermo Scientific,目录号:28905)
    4. 100%乙腈
    5. 缓冲区(参见食谱)
    6. B缓冲液(见食谱)

设备

  1. 脊椎动物和无脊椎动物细胞的亚培养
    1. 25cm 2 烧瓶
    2. 75cm 2 烧瓶
    3. CO 2 细胞培养箱(任何供应商都足够)
      注意:任何供应商都足够;选择的是价格/质量和容量。所有必要的是一个水套式培养箱,可在水饱和的环境中调节至5%CO 2 。这将需要医疗级CO 2 罐(压力下的液态气体)和专用于该罐的压力调节器。如果要同时培养细胞,将需要两个培养箱,因为脊椎动物细胞在38°C温育并在28°C下无脊椎动物细胞。
    4. 生物安全细胞培养罩
    5. 倒置显微镜
    6. 血细胞计数器

  2. 病毒生长和滴定
    库存病毒准备
    1. CO 2 细胞培养箱

    通过噬菌斑检测进行病毒滴定
    1. 摇杆平台(Bellco Biotechnology)
    2. 37°C水浴
    3. 稀释管架
    4. 冰桶

  3. 辛德毕斯病毒感染脊椎动物和无脊椎动物细胞 辛德毕斯病毒感染BHK细胞
    1. 平台摇杆(Bellco Biotechnology)
    2. 血细胞计数器

    辛德毕斯病毒感染C7-10细胞
    1. 75厘米 2 烧瓶
    2. 离心分离机
    3. 血细胞计数器

  4. 辛德毕斯病毒的纯化
    通过等密度离心纯化和浓缩
    1. 倒置显微镜
    2. 平台摇杆(Bellco Biotechnology)
    3. 血细胞计数器或细胞计数装置
    4. 康宁 TM 聚丙烯管(微型离心管,自立式和锥形)&nbsp;
    5. Sorvall RC-5B超高速离心机
    6. 贝克曼超速离心机&nbsp;
    7. Beckman SW-28转子
    8. 聚合物38毫升管
    9. Beckman SW-55Ti
    10. 聚合物5毫升管
    11. 戒指架&nbsp;
    12. 小管夹&nbsp;
    13. 手持低强度灯&nbsp;
    14. 30-100毫升梯度成型剂(Bio-Rad,型号385)
    15. 中型搅拌盘

    聚乙二醇(PEG)沉淀病毒浓度
    1. Sorvall RC-5B超高速离心机
    2. Beckman超速离心机和适当的转子和管用于所用的体积
    3. 30-100毫升梯度形成剂(Bio-Rad,型号385)

  5. 颗粒与PFU比的计算
    没有

  6. 质谱和蛋白质组学分析
    蛋白质提取和消化
    1. 台式离心机能够达到14,000 x g
    2. 振动器加热块
    3. 涡流
    4. SpeedVac集中器

    LC-MS / MS数据采集
    1. Orbitrap ELITE质谱仪(或等效物; 例如,Orbitrap QE +,Orbitrap Fusion,Orbitrap Fusion Lumos)
    2. Easy-nLC II液相色谱系统(或等效的HPLC / UHPLC纳流泵系统)

软件

数据处理:

  1. 符合最低规格的高性能计算机,可运行Proteome Discoverer软件(目前可用版本:2.2; Thermo Fisher Scientific)
  2. PANTHER分类系统( http://www.pantherdb.org/


程序

协议大纲:

  1. 脊椎动物和无脊椎动物细胞的亚培养
    1. BHK细胞的亚培养
    2. C7-10细胞的亚培养
  2. 病毒生长和滴定
    1. 库存病毒的制备
    2. 通过噬斑测定进行病毒滴定
  3. 辛德毕斯病毒感染脊椎动物和无脊椎动物细胞
    1. 辛德毕斯病毒感染BHK细胞
    2. 辛德毕斯病毒感染C7-10细胞
  4. 辛德毕斯病毒的纯化
    1. 通过等密度离心浓缩
    2. 通过聚乙二醇(PEG)沉淀浓缩
  5. 计算颗粒/ PFU比率
  6. 质谱和蛋白质组学分析
    1. 蛋白质提取和消化
    2. LC-MS / MS数据采集
    3. 数据处理

  1. 脊椎动物和无脊椎动物细胞的亚培养
    BHK-21细胞的亚培养
    BHK细胞的个体存量可能需要不同的传代方案,并且可能具有不同水平的存活传代。优良作法是跟踪单个培养物可以传代培养的传代次数,以便建立细胞解冻和储存的时间表。
    &NBSP;新鲜解冻的细胞可以传代多达30次。如果您不打算立即将它们分开,可将细胞在28°C温育2至3天,但必须至少分裂一次以恢复正常生长,然后再进行实验。
    1. 用1x PBS-D洗涤汇合的BHK细胞单层一次,使用5ml用于25cm 2 烧瓶或15ml用于75cm 2 烧瓶。
    2. 倾析PBS-D并将胰蛋白酶加入单层中。将2ml加入25cm 2 烧瓶中,或加入5ml至75cm 2 烧瓶中。在室温下孵育直至细胞开始从烧瓶中分离。使用5至10 ml血清移液管上下移液来破坏细胞团块。&nbsp;
    3. 加入BHK细胞培养基至1:1的体积以停止胰蛋白酶。重悬胰蛋白酶溶液中的细胞,并在显微镜下检查细胞团块。如果有大量残留的团块,使用5至10毫升血清移液管上下移液。&nbsp;
    4. 将细胞溶液吸移到适当大小的锥形管中。
    5. 将旋转管以500 x g (台式离心机中速)旋转3至5分钟以形成坚固的颗粒。不要过度填充管。不要将管倒置混合。
    6. 倒出上清液,并用适量的培养基替换。&nbsp;
      1. 为制备单个25cm 2 烧瓶:重悬于3ml E-MEM中。将1ml细胞加入烧瓶中的10ml培养基中。可以从一个初始的25cm 2 烧瓶中以1:3的分流比(烧瓶面积)制备多达3个烧瓶。&nbsp;
      2. 为了制备大量25cm 2 烧瓶(噬斑烧瓶或平板):在每个待制备的烧瓶中,向无菌瓶中加入10ml E-MEM。如果您使用6孔板,则使用2毫升/孔。从瓶子中取出一些培养基并重悬细胞。加回到培养基的其余部分并充分混合。为了制备多个烧瓶或平板,将10ml细胞混合物等分到每个烧瓶25-cm 2 烧瓶中,连续旋转。一个75 cm 2 烧瓶将制成9-25 cm 2 烧瓶或4-6孔板(9.5 cm 2 / well x 6使用2ml /孔,使孔= 57cm 2 。
        注意:该方案确保细胞单层均匀并含有大致相同数量的细胞。将烧瓶放入培养箱时应小心,以免干扰细胞悬液。
      3. 为制备单个75-cm 2 烧瓶:将1ml来自单个25cm 2 烧瓶的细胞分装到30ml完全E-MEM中。
    7. 将细胞在37℃的烧瓶中孵育24小时或直至融合。
    8. 如果培养基不能保持pH <1。 8,用1M HEPES调节pH至终浓度为8mM HEPES。这通常不是必需的,但是如果需要调节pH,那么它应该在pH 7和8之间。

    C7-10细胞的亚培养
    1. 在28℃,5%CO 2 湿润环境中,在组织培养瓶中保持半悬浮的蚊子细胞系。如下所述,每隔一天进行一次亚文化。细胞在开始结块或漂浮时需要分裂。这些细胞喜欢浓缩;不要超过单层面积的1/3。
      注意:
      1. 半悬浮细胞最初松散地粘附在基质上,然后随着它们的老化而开始漂浮。有些细胞可能会紧紧粘在基质上。
      2. 不要试图完全破坏团块或刮伤细胞,这会破坏细胞。
    2. 用70%乙醇清洁引擎盖下的区域。使用70%乙醇清洁引擎盖下的所有材料,并清除不需要的材料。
    3. 将介质置于室温下加热。
    4. 从培养箱中取出细胞瓶。观察细胞培养基的颜色(红色)和透明度的变化。
    5. 在显微镜下以20x放大率观察细胞以检查单层是否有不规则性和汇合。在传代之前,细胞需要95%-100%汇合。细胞在25ml培养基中的密度为~2×10 8个细胞/ 75cm 2 烧瓶。
    6. 在任何柔软的表面上猛烈地击打烧瓶侧面几次,以松开粘在烧瓶表面的任何细胞。
      注意:
      1. 如果细胞聚集成团块,尝试使用5-10 ml移液管通过上下移液几次来破碎团块,然后将细胞转移到新的烧瓶中。
      2. 可以接受小块。
    7. 将25ml细胞悬浮液培养基的三分之一转移至三个烧瓶中的每一个。在每个烧瓶中加入新鲜培养基,使体积增加到原来的25毫升。如果需要细胞,如果剩下许多粘附细胞,则向原始烧瓶中加入25ml新鲜的1x MEM。如果没有,请省略此步骤。
    8. 如果盖子是通风的,则盖子会紧紧地烧开,对于不通气的盖子而言松开。
    9. 将烧瓶放回不高于26°C的培养箱中。蚊子细胞会在高于34°C的温度下发生热休克。

  2. 病毒生长和滴定
    股票病毒准备
    标准做法是在病毒的任何额外工作之前种植病毒库,从中培育出额外的病毒库存。这种做法避免了有缺陷的干扰颗粒的产生,这些干扰颗粒在连续连续通过高浓度病毒时会累积。通常,生产原种病毒需要每个细胞0.01PFU(噬斑形成单位)的MOI(感染复数)。要计算正确的MOI,必须知道要感染的细胞数量。然后将细胞数乘以所需的MOI,例如,10 6 要感染的总细胞x MOI为0.01 = 10 4 PFU of需要病毒接种物。
    如果您的原种病毒为1 x 10 9 PFU / ml且您需要10 4 PFU接种物,请执行以下操作:
    1. 对原种病毒进行连续稀释,首先将原种病毒1:10稀释至总共1 ml。这将是900μlPBS-D + 3%FBS中的100μl病毒上清液。这是10 -1 病毒稀释液。&nbsp;
    2. 对该样品进行连续稀释,以便在5个单独的管中进行-1至-4稀释(参见图3)。您需要的病毒量在-4稀释管中,1毫升10 -4 PFU / ml的病毒。


      图3.串行通道方案。该图是从 http://www.virology.ws/2009/07/06/detecting-viruses-the-plaque-assay/ 。这项工作是根据&lt; a rel =“license”href =“http://creativecommons.org/licenses/by/3.0/”&gt; Creative Commons Attribution 3.0 Unported License&lt; / a&gt;许可的。

    3. 尽可能多地使用1 ml / 75 cm 2 烧瓶感染所有烧瓶。 不要尝试使用小于50μl病毒测量的接种物,因为稀释病毒的唯一正确方法是进行连续稀释。病毒颗粒不会进入真正的解决方案,必须小心正确地暂停它们,否则计数将被人为地高或低。或者,您可以使用100μl的-5稀释液至1 ml的体积,或使用整个-5稀释液制备10 ml的病毒并达到正确的MOI。
    4. 重新冷冻你的初始病毒;这是您的主要股票,您将从中增加后续股票。通过对病毒产生CPE的细胞进行噬斑测定来定量病毒,CPE通常是产生CPE的细胞系(在这种情况下是BHK)。然而,辛德毕斯病毒在许多昆虫细胞(例如,C4-10细胞的U4.4)中不产生CPE,其不能用于噬斑测定。
      注意:
      1. 股票病毒可以从脊椎动物或无脊椎动物细胞中生长。
      2. 辛德比斯病毒非常粘,会与玻璃和塑料表面结合。我们发现康宁 TM 聚丙烯管(微量离心管,自立式和锥形)比其他品牌的塑料结合更少的病毒颗粒。一次性玻璃管用于制备噬斑测定所需的病毒稀释液。
      3. 已发现来自所有主要供应商的组织培养级试剂适用于本协议的细胞培养和病毒生产部分。

    BHK细胞斑块形成的病毒滴定
    BHK细胞的制备
    注意:来自ATCC的个体BHK细胞库 可能需要不同的传代时间表,并且可能有不同数量的可行传代。
    1. 用1x PBS-D洗涤汇合的BHK细胞单层一次,使用5ml用于25cm 2 烧瓶或15ml用于75cm 2 烧瓶。
    2. 倾析PBS-D并将胰蛋白酶加入单层中。将2ml加入25cm 2 烧瓶中,或加入5ml至75cm 2 烧瓶中。在室温下孵育直至细胞开始从烧瓶中分离。使用5至10 ml血清移液管上下移液来破坏细胞团块。&nbsp;
    3. 加入BHK培养基1:1以停止胰蛋白酶。重悬胰蛋白酶溶液中的细胞,并在显微镜下检查细胞团块。如果有大量残留的团块,使用5至10毫升血清移液管上下移液。&nbsp;
    4. 将细胞溶液吸移到适当大小的锥形管中,例如,10或50ml管。
    5. 将旋转管以500 x g (台式离心机中速)旋转3至5分钟以形成坚固的颗粒。不要过度填充管。不要将管倒置混合。
    6. 倒出上清液,用适量的培养基替换3ml用于25-cm 2 烧瓶,用9ml替换75cm 2 烧瓶。
      1. 制备单个25cm 2 烧瓶:重悬于3ml E-MEM中。将1ml细胞加入烧瓶中的10ml培养基中。可以从1个初始的25cm 2 烧瓶中以1:3的分流比(烧瓶面积)制备多达3个烧瓶。盖帽紧紧(通气盖帽,松散的非通气盖帽)。
      2. 可以从1-75cm 2 烧瓶制备9-25cm 2 烧瓶。

    斑块检测的材料
    用于噬斑测定的许多材料用于哺乳动物和蚊子细胞测定。重要的是在感染噬斑测定试剂瓶/平板之前立即进行病毒的连续稀释,以限制病毒与试管结合的量。
    1. 确定所需的平板/烧瓶的数量,每个稀释的病毒两个孔(一式两份铺板),加上一些对照板或孔。在噬斑测定前一天,将BHK细胞分成含有~6×10 6个细胞/平板= 1×10 6个/孔细胞/孔的6孔板。通常,使用6孔板代替了单个烧瓶的使用;然而,当学习技术时,使用烧瓶要简单得多。
      注意:所需细胞数量由测定时所需的汇合度决定。由于融合与细胞覆盖的区域有关,因此通过了解细胞将在其上生长的血管区域来操纵该数量。因此,烧瓶(矩形)或孔(圆形)的面积将变化,但细胞数/ cm 2 是恒定的。 ( 例如 ,1 x 10 6 细胞。对于6孔板,每口井为9.6厘米 2 播种1 x 10 6 细胞/孔×6孔,总共~58 cm 2 ,总共~6 x 10 6 细胞是种子。这些数字与任何细胞系的每种特定培养物相关,可能需要上调或下调。重要的是是测定当天的百分比汇合,在此期间细胞仍应处于对数期。
    2. 在噬斑测定时,允许细胞单层变为~80%-90%汇合。
      注意:如果早晨准备好细胞并且下午进行测定,可以将细胞保存在室温下,盖子盖紧,或移到28°C培养箱中以减缓生长。
    3. 从冰箱中取出要滴定的病毒并在冰上融化。&nbsp;
    4. 用900μl稀释剂(SVHR稀释剂; 1x PBS-D / 3%FBS(见食谱))填充所需数量的稀释管。需要一管/稀释液。一管用于对照(未感染),一管用于阳性感染控制。
    5. 通过向含有900μl稀释剂(10 -1 )的第一个管中加入100μl病毒制备病毒的连续稀释液,以全速涡旋稀释,去除100μl,并将其加入下一个稀释管(10 -2 )。继续此过程,直到达到所需的稀释度。每次更换移液器吸头以避免污染病毒的“遗留”效应。
      注意:稀释应在使用前立即进行。病毒和稀释液应始终保持在冰上。&nbsp;
    6. 稀释完成后,将生长培养基倒入烧瓶中(放入无菌废物烧杯中),用血清移液管吸出平板(留下足够的液体覆盖单层)并感染每个烧瓶或6孔板的孔。用200μl适当的稀释液。在这种情况下,容器的面积不再重要,因为计算将每个稀释度的噬斑转换为PFU / ml。
    7. 添加病毒时不要让单层干燥 - 即,不要一次倒入/吸取过多烧瓶/盘子的介质或尝试排出每滴水。干燥的单层是单层单层。
    8. 将烧瓶在室温下放置在摇动平台上一小时。不要摇板;液体只会围绕井边旋转。将板放置在38℃的适当温度下,用于BHK细胞,每15分钟手动摇动一次。
      注意:烧瓶上的瓶盖应在摇摆时拧紧,因为它们不在适当的CO 2 环境中,除非它们放空,然后将它们放在培养箱中。
    9. 摇动/孵育一小时后,取出接种物(移液管)并用7ml 1%的2%琼脂糖在dH 2 O中的混合物覆盖单层,并完成2x EMEM和7ml用于T-25cm 2 烧瓶,6孔板的每个孔2ml。
      注意:
      1. 琼脂糖应该足够热,以至于它不会太快凝固,但足够冷却,以便在与培养基(~60-70°C)混合之前让人接触瓶子。在正确温度下的瓶子介质不应该感觉过热。在覆盖层固化之前不要尝试移动烧瓶/板,因为细胞单层会撕裂。凝固的琼脂会出现混浊。
      2. 培养细胞不能耐受所有琼脂糖。通常,配制用于凝胶电泳或色谱法的琼脂糖不适用于组织培养。使用Sigma琼脂糖()(Sigma-Aldrich,St Louis,MO,目录号:A6013)。
    10. 将培养瓶在37℃,5%CO 2 下孵育2天。
      注意:在染色单层膜之前,将烧瓶放在光线下,看是否可见斑块。斑块看起来比单层其他部分更不透明。可见斑块表明该测定已完成此步骤。
    11. 为了染色单层,加入5毫升2%琼脂糖在dH 2 O和1x PBS-D中的1:1混合物和3%中性红染色(需要琼脂糖总量的3%) 。盖住烧瓶以保护细胞免受光照。
      注意:细胞在吸收中性红色后变得对光敏感,应避光。
    12. 如有必要,当斑块微弱时,将烧瓶(平板)放回37°C保持4小时或28°C过夜,然后再计数斑块。
      注意:您应该看到被红色活细胞包围的清晰斑块。每个烧瓶的斑块数量应大致遵循所做的稀释( 例如 ,10斑块 -6 < / sup> 烧瓶,10个 -5 烧瓶中的10个噬菌斑,以及10个 <上的100个噬菌斑 -4 烧瓶)。平板可用于生产重复(6孔板)或一式三份(24孔板)。如果没有斑块,检查显微镜下的单层细胞,可能会裂解细胞。
    13. 计数斑块并计算滴度。
    14. 为了计算病毒滴度:例如,在-7稀释的2个噬菌斑和200μl的接种物,将其转化为噬菌斑/ ml或2个噬菌斑x1 /200μl×10 3 < / sup>μl/ 1 ml =来自1/10 -7 稀释度的10个噬菌斑/ ml = 10 x 10 7 或1 x 10 8 PFU / ml。因此,方程式为#斑块/稀释因子(ml)×接种体积(ml)= PFU / ml。除非病毒颗粒的数量=斑块数/ ml,否则该单位是斑块形成单位。这将在计算颗粒与PFU比率的部分中进行解释。
      注意:如果没有斑块,但阳性对照起作用,那么样品没有病毒或不正确的稀释液被接种。如果对照样品中没有斑块,则在显微镜下检查细胞裂解,裂解的细胞不会吸收污渍或产生斑块。如果培养物的渗透压和张力不正确,将发生细胞裂解。污渍晶体意味着加入时琼脂糖太热。太热的琼脂糖也会杀死细胞。
  3. 辛德毕斯病毒感染脊椎动物和无脊椎动物细胞 辛德毕斯病毒感染BHK细胞
    1. 在感染前一天对BHK细胞进行传代培养,使得单层在感染时约90%融合。&nbsp;
    2. 计算所需多重感染(MOI)所需的病毒量。
      编号为x MOI = PFU。
      对于单个循环MOI = 10,原种病毒MOI = 0.01
      对于75 cm 2 烧瓶,细胞数量为~2 x 10 7 细胞
      对于25 cm 2 烧瓶,细胞数量为~6 x 10 6 细胞
      对于6孔板,细胞数约为1×10 6个细胞/板。
      如果使用少量接种物,也使用平板,例如,储备病毒体积滴度太低而不能感染太多细胞,并且在使用前必须扩增病毒。
    3. 从-80°C冰箱中取出病毒并在冰上解冻小瓶。在1x PBS-D / 3%FBS中将接种物稀释至所需浓度。重新冷冻未使用的病毒。
      注意:
      1. 感染75 cm 2 单层需要至少1 ml的总体积,并且至少需要200μl对于25 cm 2 单层。 6孔板至少需要200μl。盖紧盖子并固定在摇臂平台上。不要摇板。
      2. SVHR是热稳定的,但辛德毕斯的许多其他菌株和突变体都没有,因此必须在冰上解冻以保持感染性。
    4. 将烧瓶置于摇动平台上,在室温下放置1小时,盖上盖子。或者,在37°C的培养箱中感染间歇性手摇。&nbsp;
    5. 取出接种物并加入新鲜,完整的1x E-MEM培养基。将5-7ml培养基加入75-cm 2 单层或3ml加入25-cm 2 单层。在孵育期间使帽松开以允许CO 2 交换。如果需要更浓缩的病毒,则将3ml培养基加入75-cm 2 单层中。不要让单层干燥。
    6. 一旦CPE变得明显,应该收获病毒,通常在感染后18(BHK)和24小时(C7-10)之间。
      注意:细胞系在演示CPE的时间段内会有所不同。 BHK细胞中CPE的一些指标包括细胞变得长而细,具有“泡沫”外观的细胞质,细胞核的聚集,以及在晚期阶段的细胞裂解。不允许细胞裂解任何类型的病毒分析工作。
    7. 将培养基从烧瓶中取出至锥形离心管(Corning,15ml Fisherbrand Cat#05-539-5或50ml离心管,Fisherbrand Cat#05-539-6)。如果需要,可以组合样品。在台式离心机中以~700 x g 旋转样品10分钟以除去细胞碎片。将病毒倒入新管中,将溶液加入10%甘油中,作为冷冻保护剂。使用液体N 2 将病毒上清液等分到适于冷冻和快速冷冻病毒的试管中。&nbsp;
    8. 将病毒储存在-80°C。
      注意:由于冰中的相变,含有膜的病毒将在-65°C进行冻融。因此,应该将导致升温至-65°C的冷冻机故障视为解冻。辛德毕斯病毒在冻融后会失去一些滴度(约0.5个对数),但仍具有传染性。

    辛德毕斯病毒感染C7-10细胞
    当需要对来自无脊椎动物宿主的病毒滴度进行比较分析时,使用蚊子细胞。为了感染蚊子细胞,必须形成单细胞的汇合单层(无团块)。蚊子细胞不能很好地粘附到基质上,并且无论使用何种介质,都倾向于从烧瓶中提起。为了暂时避免这个问题,蚊子培养物可以缺乏血清1小时,这导致细胞更紧密地粘附到基质上。我们实验室中的蚊子培养已经适用于E-MEM,然而,细胞的所有其他处理与其他昆虫培养基中的细胞一致。
    1. 在感染所需的每1-75 cm 2 烧瓶的200-500 xg 约1½-75 cm 2 细胞瓶中旋转5分钟。
      注意:蚊子细胞生长为半悬浮培养物,通过在硬质表面上轻敲烧瓶,或者用5-10ml移液管上下吸移,可以很容易地从烧瓶中取出。不要过度旋转,因为在造粒或重新悬浮期间细胞会死亡。在此过程中,某些单元格将丢失。
    2. 用1x PBS-D洗涤细胞沉淀一次以除去任何剩余的培养基。&nbsp;
    3. 将最终细胞沉淀重悬于感染所需的1ml /瓶中,缺乏FBS的E-MEM培养基。
    4. 使用血细胞计数器计数细胞。&nbsp;
    5. 将每个75-cm 2 烧瓶中的大约6×10 6个细胞等分,用足够的培养基(不含FBS)覆盖单层。&nbsp;
    6. 将细胞培养瓶在28°C孵育1小时,或直至细胞牢固地附着在烧瓶表面上。&nbsp;
    7. 一旦附着细胞,移除培养基并将所需量的病毒添加到单层中。严密关闭帽子。&nbsp;
    8. 在室温下缓慢摇动烧瓶一小时。
      注意:缓慢摇动对于保持最小数量的细胞不会从烧瓶中提升至关重要。
    9. 摇动后,大多数细胞应保持附着在烧瓶上。如果细胞从单层中提起,则应使用接种物将其移除。&nbsp;
    10. 应在每个烧瓶中加入新鲜,完整的E-MEM培养基。
      注意:为了增加病毒浓度,尽量减少添加到烧瓶中的新鲜培养基的体积。需要至少3 ml培养基/ 75 cm 2 来覆盖细胞并支持新陈代谢。注意烧瓶是水平的,以确保完全覆盖单层。
    11. 根据使用的多重性(MOI),可以从24至72hpi收获病毒。细胞病变效应(CPE)在蚊子细胞中不明显,在这种情况下,病毒将根据感染后的时间而不是CPE来收获。&nbsp;
    12. 对于正常储存:收获病毒并储存在无菌的10%甘油中。为了最好地保存液体N 2 中的病毒滴度快速冷冻管。
      注意:病毒冷冻和解冻将失去1/3至1/5 log的滴度。这不是用于感染细胞的重大损失。为了保持最高的感染性水平,病毒可以在中性pH缓冲液中在4°C下储存长达5天。该方法保留了最大的感染性,用于在结构研究中分析病毒以及计算颗粒与PFU的比率。

  4. 辛德毕斯病毒的纯化
    通过等密度离心纯化和浓缩
    将病毒上清液收获到锥形管中,通过低速离心从上清液中除去细胞碎片。这是一个关键步骤,如果省略,过多的碎片会干扰下面的梯度纯化步骤。通过在适当大小的锥形管中以1,800 x g 离心10分钟来澄清病毒上清液。
    1. 将病毒上清液合并到适当大小的容器中,省略添加甘油。
      注意:从这一步开始,您不应该认为您的样品是无菌的。这通常不是问题,并且抗生素被添加到2x E-MEM介质中。&nbsp;
    2. 确定所需的渐变数。
      通常,将20ml病毒分层到38ml管中的初始酒石酸钾步骤梯度上。通过将12ml 15%酒石酸钾小心地层压到6ml 35%酒石酸钾层上,或制成10%至35%连续酒石酸钾梯度,形成该梯度。有关使用梯度成形器的说明,请阅读制造商手册。将管放入转子叶片中并直立称重(我们使用带有纸巾的小烧杯)。逐滴添加PBS-D以平衡管。 特别注意称重铲斗和管子,并根据制造商的公差和规格装载离心机转子。否则可能会导致转子和仪器严重损坏。
      注意:转子,铲斗和溶液应预先冷却至4°C。如果需要奇数个梯度,则制作“空白”梯度并用缓冲液覆盖以平衡转子。&nbsp;
    3. 将梯度在SW-28 Beckman转子(或10,000 x g )和4℃下以24,000rpm离心过夜(这纯粹是为了方便)。&nbsp;
    4. 运行完成后,小心地从桶中取出管,并使用小管夹将其固定到环形支架上。当用20瓦的手持光从侧面照射梯度时,在酒石酸钾15%-35%阶梯界面处应该可以看到虹彩蓝的病毒带。参见图4密度梯度纯化的辛德毕斯病毒带。对每个渐变重复。&nbsp;
    5. 通过刺穿管底部并让酒石酸盐溶液流入废烧杯收集带子,同时将病毒带收集到2ml冷冻管中。丢弃剩余的解决方案。或者,可以使用针和注射器从管的侧面收集带。
    6. 将含有病毒的样品汇集并用1.5体积的冰冷PBS-D稀释。该溶液的密度应小于15%酒石酸钾。要检查您的浓度是否正确,您的病毒溶液不应沉入15%酒石酸钾的等分试样中。&nbsp;
    7. 然后将该稀释的病毒在连续的15%-35%酒石酸钾线性梯度上分层,体积不小于添加的病毒样品的3/4。第一梯度可以是阶梯或线性梯度,但第二梯度应该是线性梯度。不要过满。&nbsp;
    8. 连续梯度在SW 40ti Beckman转子(12,000 x g )中以26,000rpm运行2小时,并如上述步骤D6中所述收集病毒带。病毒带应出现在管长度的1/3左右。病毒带可以在4℃下储存在酒石酸盐溶液中。最终病毒 - 酒石酸盐溶液的折射率为1.3665,其为〜28%酒石酸盐(Rumble,2005)。&nbsp;
    9. 通过噬斑测定收集病毒样品以滴定,并通过BCA测定法测定蛋白质浓度(Pierce TM Rapid Gold BCA Protein Assay Kit,Thermo Fisher)。
      注意:正确生长和纯化的SVHR应该使颗粒/ PFU值为~1。


      图4.显示纯化的辛德毕斯病毒在15-35%酒石酸钾梯度中的乳白色蓝带。注意管底部的絮状颗粒。在第二次K-酒石酸盐梯度后,这将被删除。

    聚乙二醇(PEG)沉淀病毒浓度
    注意:这是用于浓缩病毒颗粒的替代纯化步骤,但传染性将会丧失。
    1. 如前所述用Sindbis病毒感染BHK细胞用于病毒纯化。感染的烧瓶数量取决于您需要多少病毒。&nbsp;
    2. 将培养基倒入离心管中。&nbsp;
    3. 旋转上清液用于澄清,除去细胞和细胞碎片(从该步骤开始,如在病毒纯化方案中的步骤D2-D9中那样进行。
    4. 将上清液转移到新管中。
    5. 在2M NaCl中加入0.25体积的40%聚乙二醇-8000并充分混合。
    6. 在4°C孵育约24小时以进行病毒沉淀。
    7. 以10,000rpm(12,000 xg )离心10分钟。
    8. 将上清液丢弃到废物烧杯中并排干(将管倒置并用kimwipe吸收任何痕量的PEG)。&nbsp;
    9. 将沉淀重新溶解在5ml的1x PEG缓冲液中。
    10. 转移到Kimax玻璃离心管。
    11. 在12,000 x g 离心10分钟(Sorval) - 这是进一步的澄清步骤。
    12. 如上所述使用上清液进行梯度纯化(从该步骤开始,按照病毒纯化方案中的步骤D2-D9进行。
    注意:
    1. 我们没有在我们的蛋白质组学方案中使用辛德毕斯病毒的PEG浓度,但包括该方案中的结果,以证明与非感染性颗粒相比纯化感染性病毒颗粒的重要性,以推断有关病毒系统功能的结论&NBSP;
    2. 我们发现使用辛德毕斯病毒的SVHR菌株,PEG浓度随后进行等密度纯化使颗粒/ PFU比率增加10倍。应该认真评估实验结果解释中非感染性颗粒数量的增加,并在结果中注明。

  5. 计算颗粒与PFU的比率
    1. 为了使该计算值准确,需要精制蛋白质浓度的纯化病毒。酒石酸盐溶液或PEG将干扰该测定;样品必须1:10稀释。
    2. SVHR病毒蛋白MW以道尔顿为单位=结构蛋白的总重量( C,E2和E1 )= 130 KDa(通过直接BCA测量,使用此测量数而不是计算值)。&nbsp;
    3. 该值乘以240拷贝/病毒颗粒(T = 4颗粒:60T = 240拷贝)。参见Johnson和Speir(1997)的T计算。
      130KDa(总病毒蛋白,参见步骤E2)×240(拷贝/颗粒参见上述步骤E3)= 3.12×10 7 Da蛋白/病毒颗粒
      从Da转换为克[1道尔顿= 1.66 x 10 -24 g(CRC表)]
      3.12×10 7 Da /病毒颗粒x 1.66 x 10 -24 g / Da = 5.18 x 10 -17 g /颗粒 例如,如果您有10 11 PFU / ml SVHR,您将获得208μg/ ml蛋白质(通过BCA测定法测量)
      = 2.08×10 -4 g / ml。
      注意:PFU和颗粒不是同一个单位。 PFU或噬斑形成单位是病毒感染性的量度,因为所有病毒颗粒都不具有传染性。颗粒数是指溶液中物理颗粒的数量,通常大于PFU的数量。
      计算颗粒/ ml:
      2.08×10 -4 g / ml /5.18×10 -17 g /颗粒= 0.4×10 13 颗粒/ ml
      0.4×10 13 颗粒/ ml / 1×10 11 PFU / ml = 0.4×10 2 颗粒/ PFU = 40颗粒/ PFU <
      注意:对于Sindbis SVHR,BHK或C7-10细胞的颗粒/ PFU比例应为~1份/ PFU。

  6. 质谱和蛋白质组学分析
    1. 为了目测确定样品是否含有污染物,然后通过透射电子显微镜观察纯化群体的样品以检查污染的细胞器或膜片段(Coombs和Brown,1987)。该方法超出了本协议的范围,但在上面提供的参考文献中有详细说明。
    2. 另外,为了检查共纯化的蛋白质污染物,如制造商所述,通过在4-12%Bis-Tris SDS-PAGE梯度凝胶(Invitrogen,Novex)上运行~10μl病毒来检查蛋白质含量。
    3. 在Wray方法或考马斯蓝中用银染色凝胶(Wray 等,,1981)。在进行蛋白质鉴定的LC-MS / MS分析之前,切除可视化条带(仅银色染色)并进行凝胶内消化(Glaros et al。,2015)。

    蛋白质提取和消化
    使用过滤辅助样品制备(FASP)方法处理病毒制剂及其各自的阴性对照,来自感染前收获的细胞单层的培养基用于LC-MS / MS分析(Wiśniewski等人, 2009)。
    1. 纯化后,按照制造商的说明,使用Pierce BCA蛋白质测定试剂盒测定总蛋白质浓度。
    2. 使用无菌PBS将所有制剂标准化至0.5μg/μl;将10μg每种样品等分到Lo-binding Eppendorf管中。
    3. 对于每个样品,将10μg总蛋白(20μl)1:1与补充有50mM二硫苏糖醇(DTT)的M-PER混合,并在95℃下在加热块中以400rpm加热10分钟以减少和变性蛋白质。
    4. 将样品冷却至室温,并与200μlUA缓冲液(8M尿素,100mM Tris-HCl,pH8.5)混合。
    5. 将每种制剂施加到30kDa过滤器旋转柱(Millipore-Ultracell YM-30)上,并在室温下以14,000 x g 离心30分钟以将所有蛋白质收集到滤膜上。
    6. 通过向每个滤膜加入100μl碘乙酰胺(IAA)溶液(在UA缓冲液中0.05M IAA)烷基化蛋白质,并在室温下在黑暗中孵育20分钟。烷基化后,将样品在14,000 x g 离心20分钟以除去烷基化溶液。
    7. 通过向过滤器施加100μlUA缓冲液并在室温下以14,000 x g 离心30分钟来洗涤每个样品。重复该洗涤步骤两次,总共洗涤三次。然后用100μl100mM三乙基碳酸氢铵(TEAB)洗涤三次。
    8. 为了消化捕获的蛋白质,在每个膜上施加100μl胰蛋白酶消化溶液(在100mM TEAB中制备的胰蛋白酶/ Lys-C至10μg/ ml)并在密封管中孵育过夜(用封口膜包裹封闭的管帽用于更好的密封)在37℃,400转/分钟的加热块中。
    9. 孵育后,收集肽用于LC-MS / MS分析。将过滤器单元放入新的干净管中,并在室温下以14,000 x g 离心每个管30分钟。通过用50μl100mMTEAB离心一次洗涤膜,用50μl0.5MNaCl离心一次。用最终的肽洗脱液收集这些洗涤液和池。
    10. 用10%三氟乙酸酸化每个样品直至最终pH为~2-3。
    11. 在进行质谱分析之前,根据制造商的说明,使用Pierce C18离心柱对每个样品进行脱盐。脱盐过程中的洗脱液应在30℃下使用SpeedVac在干净的Lo-bind Eppendorf微量离心管中干燥至完全,并储存在-80°C直至LC-MS / MS分析。

    LC-MS / MS数据采集
    1. 在“A缓冲液”(3%乙腈/0.1%甲酸)中重构干燥的肽,并使用多步梯度[例如 在VIRGIN Picofrit 15 cmx75μmIDHPLC色谱柱上分离,填充5μmBioBasicC18颗粒300Å ,130分钟; 0-5分钟:5-10%B,6-110分钟:10-35%B,和111-130分钟:35-95%B]。原始或未使用的色谱柱对于确保所有鉴定的肽均来自样品而非先前的LC-MS / MS运行非常重要,如果使用已经应用样品的色谱柱,则可能会导致“残留”。应选择液相色谱梯度,以便在没有过多时间的情况下进行充分分离。这些参数取决于用于分析的色谱柱和仪器。对于梯度,A缓冲液是3%乙腈/0.1%甲酸,B缓冲液是95%乙腈/0.1%甲酸。应至少在技术上一式三份地分析胰蛋白酶肽。
    2. 对于Orbitrap ELITE,使用以下配置。如果使用其他质谱仪,这应作为类似仪器参数的指南。这些参数是仪器特定的,您应该查阅仪器的制造商或文献以获取建议。从用于自下而上(胰蛋白酶肽分析)蛋白质组学的文献建立的通用设置可能是足够的。各种仪器方法已经过策划和同行评审,可在 www.massspectrometrymethods.org 上找到。

      用于轨道阱ELITE的仪器设置示例
      1. Orbitrap MS1扫描以1200 m / z的分辨率120,000进行,扫描范围为110 -2 ,000 m / z。
      2. 选择前20种前体用于MS2数据依赖性片段化。在正常模式下使用离子阱扫描获取MS2光谱(Top 20方法)。
      3. 触发数据相关扫描所需的最小信号为5000。
      4. 碰撞诱导解离(CID)用于产生具有以下设置的MS2光谱:标准化碰撞能量35%,默认电荷状态2,隔离宽度2m / z和激活时间10ms。
      5. 对于MS,AGC目标设定为1×10 6 ,对于MS / MS,AGC目标设定为5×10 4 ,最大累积时间为200ms。
      6. 动态排除设置为60秒,最多500个目标,质量窗口为5 ppm。
      7. 锁定质量为445.120025用于内部校准,以提高质量准确度。

    数据处理
    1. 使用Proteome Discoverer处理光谱数据,其具有针对辛德毕斯病毒多蛋白数据库(Uniprot ID:P03317)的嵌入式SEQUEST搜索算法,其与智人(RefSeq Tax ID:9606)或灰仓鼠(Cricetulus griseus)(RefSeq Tax ID:10029)合并。从VectorBase(Giraldo-Calderón等人,2015)下载白纹伊蚊 - 蛋白质组扣除的蛋白质组(Chen et al。,2015)数据和来自NCBI TSA数据库的其他多肽。
    2. 在excel电子表格上组织非冗余蛋白质组(Ribeiro et al。,2004)并如前所述进行注释(Karim et al。,2011)。将得到的FASTA文件与SINV多蛋白合并,并用它来搜索SINV蚊子制剂。
    3. 设定半胱氨酸[+ 57.02 Da]的氨基甲酰化,甲硫氨酸氧化[+15.99 Da]和N-末端乙酰化[+42.011]的动态修饰。
    4. 搜索MS / MS谱,前体质量公差为10ppm,片段质量公差为0.6Da。
    5. 胰蛋白酶被指定为蛋白酶,其最大错过切割数设置为2。
    6. 使用PERCOLATOR(Kall 等人,2007)的错误发现率设定为&lt; 1%得分高信度肽鉴定。
    7. 仅使用每种蛋白质id的登录号,使用PANTHER分类系统对人类背景进行分组和功能分析(Mi et al。,2016)。

食谱

  1. 完整的E-MEM,1x和2x
    注意:在添加补充剂之前不要加热2x MEM,它会沉淀。
    10%热灭活的胎牛血清(FBS;任何合适的FBS,我们在购买之前测试样品批次的FBS)
    5%胰蛋白胨磷酸盐肉汤(TPB;见配方11;这只添加到BHK细胞中)
    0.02%L-谷氨酰胺(参见配方3为100x)
    1x硫酸庆大霉素(参见配方2 100x)

    适用于1x中等
    混合以下内容:
    400毫升2x E-MEM(见配方1)
    400毫升TC H 2 O
    80毫升FBS
    8毫升L-谷氨酰胺
    * 8毫升庆大霉素硫酸盐
    在4°C下储存长达2周
    *注意:庆大霉素仅添加到培养基中用于病毒生长,而不是用于任何细胞培养物的一般维护。
    对于2x培养基(用于通过噬斑测定滴定病毒)
    通过混合以下物质制备536 ml培养基:
    400毫升2x E-MEM(不完全,无血清;参见配方1)
    80毫升FBS
    40毫升TPB
    8毫升100倍L-谷氨酰胺
    8毫升100倍硫酸庆大霉素
    存放长达6个月
    重要提示:含有补充剂的完整培养基不是真正的百分比溶液。指示的百分比表示原始介质体积的百分比。这也适用于添加到培养基中的任何其他补充剂。例如,如果培养基的初始体积是400ml,则10%FBS是40ml。添加其他补充剂,例如5%TPB,将是20ml TPB,使总体积达到460ml。如果细胞培养物出现问题,病毒生长或斑块形成,则丢弃旧溶液并制作新溶液。&nbsp;
  2. 硫酸庆大霉素,100x
    在组织培养级水中稀释0.5 g庆大霉素硫酸盐至100 ml
    在4°C下储存长达6个月
    稀释至1x将得到50μg/ ml的终浓度。如有必要,可以使用100μg/ ml
    注意:抗生素可能会被污染。如果污染培养物出现持续性问题,请在没有抗生素的情况下测试所有溶液。
  3. L-谷氨酰胺,100x
    称取2.92克L-谷氨酰胺,溶于总体积为100毫升的1×PBS-D(见配方7中10倍)
    过滤灭菌并在100毫升瓶中分成50毫升等分试样
    储存冷冻长达6个月;一旦解冻,2周后丢弃
    注意:谷氨酰胺是必需的,非常不稳定。因此,作为预防措施,它已添加到已经完整的介质中。
  4. HEPES(pH 7.2-7.4),1 M
    称出238.3克HEPES并溶解于总体积为1升的组织培养级水中 用NaOH调节pH至7.2-7.4
    高压灭菌30分钟消毒
    在室温下储存长达1年
  5. PEG缓冲液
    0.4 M NaCl
    0.01 M Tris Buffer pH 7.0
    0.001 M EDTA
  6. 中性红色原液,2%
    溶解2克中性红,用组织培养级水将体积调节至100毫升
    在室温下搅拌过夜并过滤灭菌
    在室温下储存长达1年
    注意:
    1. 过滤过程中会丢失一些污渍,每批可能会有所不同。这种污渍非常粘稠,可能需要一个以上的过滤器来消毒整个数量。
    2. 或者,可以从许多供应商处购买中性红色原液。
  7. PBS-D,10x
    2.0克KCl
    2.0克KH 2 PO 4
    80.0克NaCl
    11.3g Na 2 HPO 4 或21.6 g Na 2 HPO 4 ·7H 2 O
    组织培养级H 2 O至1L
    高压灭菌20分钟
    用无菌组织培养级水稀释至1倍
    在室温下储存长达1年
    注意:PBS-D是缺乏钙和镁的PBS(磷酸盐缓冲盐水)。这可以作为1x解决方案。另请参阅含钙和镁的PBS配方注释。至关重要的是1x溶液的pH值为~7.4,因为低pH会使所有的Alphaviruses失活。
  8. 含钙和镁的PBS,1x
    准备溶液1和2(10倍储备溶液)并分别高压灭菌20分钟
    注意:PBS不能作为浓缩溶液制成,它会沉淀。
    解决方案1:
    1.0克CaCl 2
    1.3g CaCl 2 ·2H 2 O
    2.0克KH 2 PO 4 (分别溶解,然后加入混合物中)
    1.0g MgCl 2 ·6H 2 O
    80.0克NaCl
    用组织培养级H 2 O将体积调节至1L 解决方案2:
    21.6g Na 2 HPO 4 ·7H 2 O或11.3g无水Na 2 HPO 4
    用组织培养级H 2 O将体积调节至1L
    适用于1x解决方案:
    加入50毫升溶液1至400毫升组织培养级水
    混合,然后加入50毫升溶液2
    不要将溶液1和2直接混合在一起:盐会沉淀出来 在室温下储存溶液长达1年或直到盐开始沉淀为止 检查1x溶液的最终pH值是否为7.2到7.4
    重要注意事项:不要调整10x溶液1和2的pH值,因为这会导致1x溶液的pH值错误。至关重要的是,辛德毕斯病毒与之接触的所有溶液都是中性pH值。酸性pH值会迅速使病毒失活。&nbsp;
  9. 酚红,0.5%
    注意:不要与中性红混淆。
    将1克酚红溶于200毫升组织培养级水中 过滤灭菌并在室温下储存长达1年
  10. SVHR稀释剂(用于稀释通过噬斑测定滴定的病毒)
    使用热灭活的胎牛血清(FBS)补充1x PBS-D(参见配方7的10倍)至3%FBS
    在4°C下储存长达2个月
  11. 胰蛋白酶原料,0.25%
    0.5克胰蛋白酶
    0.2克EDTA二钠
    0.6克酚红
    用1x PBS-D将体积调节至200 ml(参见配方7 10倍)
    用1N NaOH调节pH直至达到樱桃红色
    分成25毫升的等分试样
    在-20°C下冷藏最多6个月
    注意:使用前,在37°C水浴中解冻,用versene溶液稀释至100 ml(参见配方13)。不要在37℃下加热胰蛋白酶> 20分钟,因为酶会自动消化。&nbsp;
  12. 胰蛋白胨磷酸盐肉汤(TPB)
    称出29.5克胰蛋白胨磷酸盐肉汤(TPB; Difco)
    溶于总体积为1 L的组织培养级水中 高压灭菌TPB在两个独立的500毫升体积中灭菌20分钟进行灭菌 不要在高压灭菌器中放置更长时间,因为这种解决方案会焦糖化 冷却并在4°C下储存长达6个月
  13. Versene解决方案
    500毫升1x PBS-D(见配方7 10倍)
    5毫升0.1M EDTA(见配方14)
    1.5毫升0.5%酚红(见配方9)
    用NaOH调节pH至7.4 过滤消毒(如果不使用无菌储备溶液)
    在室温下储存长达6个月
  14. 0.1 M EDTA溶液
    使用3.36克/ 100毫升的二钠盐形式
    pH将降至5.3以下并且溶液将保持混浊直至其pH调节至7.0。用NaOH调节pH值至8.0 高压灭菌消毒
  15. M-PER补充50mM二硫苏糖醇(DTT)
    注意:&NBSP;
    1. 您处理的每个样品都需要至少20μl补充DTT的M-PER。&nbsp;
    2. 剩余1 M DTT原液可在-20°C下储存长达30天(最好制作50-100μl等分试样以防止任何冻/融循环)。
  16. UA缓冲液(8M尿素,100mM Tris-HCl pH8.5)(10ml)
    注意:每个样品需要大约500μl的UA缓冲液。
    将4.8048克尿素(Thermo Scientific)加入6毫升质谱级水中并溶解(溶液变冷,因此可能需要在水浴中温和加热溶液以使尿素完全溶解)。加入1 ml 1 M Tris-HCl pH 8.5,加入10 mL质谱级水
  17. IAA溶液(UA缓冲液中0.05M碘乙酰胺)(1 ml)
  18. 100mM三乙基碳酸氢铵(TEAB)(3ml)
    将300μl1M TEAB(Sigma)加入2.7 ml质谱级水中并涡旋振荡 建议将溶液保存在冰上
  19. 胰蛋白酶消化液(在100 mM TEAB至10μg/ ml制备的胰蛋白酶/ Lys-C)
    将2,000μl100mMTEAB加入单个20μg小瓶胰蛋白酶/ Lys-C(Promega)中,终浓度为10μg/ ml,轻轻移液混合直至完全溶解。不要VORTEX。建议将溶液保存在冰上
  20. 0.5 M NaCl(5 ml)
    注意:这将是一个过剩的解决方案,但是对于这个体积来说,更容易控制NaCl。)
    称出146.1 mg NaCl(Sigma)并将质谱级水加入5 ml
    涡旋直至溶解
  21. 10%三氟乙酸
    将1ml 100%三氟乙酸(Thermo Scientific)加入9ml质谱级水中并涡旋
  22. 缓冲液(3%乙腈/0.1%甲酸)
    将1 ml 100%甲酸(Thermo Scientific)加入30 ml 100%乙腈中,然后加入质谱级别的水至1 L
  23. B缓冲液(95%乙腈/0.1%甲酸)
    将1ml 100%甲酸加入950ml 100%乙腈中,然后加入质谱级水至1L

致谢

RH和DF由Clayton Foundation for Research,Carson City NV和北卡罗来纳州立大学农业与自然科学学院提供支持。 TG和GR由美国陆军作战能力发展指挥部(CCDC)化学生物中心通过内部基础研究补助金支持。这里提出的意见是作者的意见,不是美国陆军,CCDC或美国政府的官方政策。本报告中的信息已公开发布,分发无限制。该协议改编自该出版物。 Schuchman R,Kilianski A,Piper A,Vancini R,Ribeiro JMC,Sprague TR,Nasar F,Boyd G,Hernandez R,Glaros T. 2018.来自哺乳动物和无脊椎动物宿主的辛德比斯病毒蛋白质组的比较表征将nsP2鉴定为病毒和排序Nexin 5作为甲病毒复制的重要宿主因子。 J Virol 92。

利益争夺

作者宣称没有竞争利益。

参考

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引用:Hernandez, R., Glaros, T., Rizzo, G. and Ferreira, D. F. (2019). Purification and Proteomic Analysis of Alphavirus Particles from Sindbis Virus Grown in Mammalian and Insect Cells. Bio-protocol 9(10): e3239. DOI: 10.21769/BioProtoc.3239.
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