Infectious Subviral Particle to Membrane Penetration Active Particle (ISVP-to-ISVP*) Conversion Assay for Mammalian Orthoreovirus

Materials and Reagents

1. Pipette tips
   1. 0.1-10 µl capacity (USA Scientific, catalog number: 1111-3700 )
      
   2. 1-200 µl capacity (VWR, catalog number: 89079-474 )
      
   3. 100-1,250 µl capacity (VWR, catalog number: 53508-924 )
2. PCR 8-well tube strips (VWR, catalog number: 20170-004 )
   
3. 50 ml centrifuge tube (VWR, catalog number: 89039-660 )
   
4. 1.7 ml microcentrifuge tubes (MIDSCI, catalog number: AVSS1700 )
   
5. Purified reovirus stocks (see [Berard and Coombs, 2009; Kobayashi et al., 2010] for propagation and purification procedure)
   
6. Crushed ice
   
7. Standard SDS-PAGE materials and reagents (e.g., 10% SDS-polyacrylamide mini gels)
   
8. Coomassie Brilliant Blue stain and destain solutions (Bio-Rad Laboratories, catalog number: 1610435 )
   
9. Bleach (Biz4USA, Janitorial Supplies, catalog number: CLO30966CT )
   
10. 2-Amino-2-(hydroxymethyl)-1,3-propanediol (Tris) (MP Biomedicals, catalog number: 02103133 )
    
11. Magnesium chloride hexahydrate (MgCl₂·6H₂O) (Sigma-Aldrich, catalog number: M9272 )
    
12. Sodium chloride (NaCl) (Merck, catalog number: SX0420-3 )
    
13. 0.1 N hydrochloric acid (Sigma-Aldrich, catalog number: 2104 )
    
14. N sodium hydroxide (Sigma-Aldrich, catalog number: 2105 )
    
15. 4x Laemmli sample buffer (Bio-Rad Laboratories, catalog number: 1610747 )
    
16. Nα-p-tosyl-L-lysine chloromethyl ketone (TLCK)-treated chymotrypsin (Worthington Biochemical, catalog number: LS001432 )
    
17. Trypsin (Sigma-Aldrich, catalog number: T6567 )
    
18. Phenylmethylsulfonyl fluoride (PMSF) (Sigma-Aldrich, catalog number: P7626 )
    
19. Ultrapure DNase/RNase-free distilled H₂O (Thermo Fisher Scientific, Invitrogen^TM, catalog number: 10977015 )
    
20. Isopropyl alcohol (Avantor Performance Materials, Macron, catalog number: 3032-02 )
    
21. 50% bleach (see Recipes)
    
22. Virus storage buffer (VB) (see Recipes)
    
23. 2 mg/ml Nα-p-tosyl-L-lysine chloromethyl ketone (TLCK)-treated chymotrypsin (see Recipes)
    
24. 100 mM phenylmethylsulfonyl fluoride (PMSF) (see Recipes)
    
25. 1 mg/ml trypsin (see Recipes)
    

Equipment

1. Personal protective equipment (PPE)
   
   1. Laboratory coat
      
   2. Gloves
      
   3. Eye protection
      
2. Biosafety level 2 (BSL-2) laboratory facility
   
3. BSL-2 certified tissue culture hood
   
4. Solid and liquid waste containers
   
5. Autoclave
   
6. Ice bucket
   
7. -20 °C freezer
   
8. Micropipettes
   
   1. 0.1-2.5 µl capacity (Eppendorf, catalog number: 3123000012 )
      
   2. 2-20 µl capacity (Eppendorf, catalog number: 3123000039 )
      
   3. 20-200 µl capacity (Eppendorf, catalog number: 3123000055 )
      
   4. 100-1,000 µl capacity (Eppendorf, catalog number: 3123000063 )
      
9. Digital pH meter (VWR, model: SB70P )
   
10. Digital laboratory balance (Mettler Toledo, model: PB1502-S )
    
11. NanoDrop spectrophotometer (Thermo Fisher Scientific, Thermo Scientific^TM, model: ND-1000 )
    
12. Hot plate stirrer (VWR, catalog number: 12365-382 )
    
13. Magnetic stir bar (VWR, catalog number: 58948-273 )
    
14. Thermal cycler (Bio-Rad Laboratories, model: S1000^TM )
    
15. Analog dry block heater (VWR, catalog number: 12621-110 )
    
16. Thermometer (VWR, catalog number: 89095-566 )
    
17. Gel imaging system (LI-COR, model: Odyssey^® Classic )
    
18. 1,000 ml glass beaker (VWR, catalog number: 89000-212 )
    
19. 1,000 ml graduated cylinder (VWR, catalog number: 65000-012 )
    
20. 1,000 ml storage bottle (VWR, catalog number: 89000-240 )
    Note: This product has been discontinued.
    

Software

1. Image Studio Lite (LI-COR)

Procedure

1. Generation of infectious subviral particles (ISVPs)
   1. Propagate and purify reovirus virions as previously described (Berard and Coombs, 2009; Kobayashi et al., 2010). Using a NanoDrop spectrophotometer, determine particle concentration by measuring the optical density of the purified virus stocks at 260 nm (OD₂₆₀; 1 unit at OD₂₆₀ = 2.1 x 10¹² particles/ml) (Smith et al., 1969).
      
   2. In 1 tube of an 8-well tube strip, dilute 2 x 10¹¹ virions into 90 µl of ice cold VB (see Recipes).
      
   3. Add 10 µl of ice cold 2 mg/ml TLCK-treated chymotrypsin (see Recipes) to the diluted virus. Mix by pipetting up and down 3-4 times.
      Note: For an undigested control, substitute 10 µl of ice cold VB for 10 µl of TLCK-treated chymotrypsin.
      
   4. Incubate the reaction for 20 min at 32 °C in a thermal cycler.
      Note: Under these conditions, σ3 is degraded (Joklik, 1972; Borsa et al., 1973a) and µ1 is cleaved (Nibert and Fields, 1992; Chandran et al., 1999).
      
   5. Following digestion, quench chymotrypsin activity by the addition of 1 µl of 100 mM PMSF (see Recipes). Mix by pipetting up and down 3-4 times.
      
   6. Incubate the reaction for 20 min on ice.
      
   7. To confirm that ISVPs are generated, run 2 x 10¹⁰ particles per lane on a 10% SDS-polyacrylamide mini gel. Run the gel for 40-45 min at 200 V constant.
      
   8. Visualize the protein bands by Coomassie Brilliant Blue staining (see Data analysis, Figure 1).
      
   9. Store ISVPs on ice, and use within 2-3 h for ISVP-to-ISVP* conversion experiments.
      
      
2. ISVP-to-ISVP* conversion assay
   1. Add 10 µl of ISVPs to each tube of an 8-well tube strip on ice.
      
   2. Incubate the ISVPs for 1 h on a temperature gradient in a thermal cycler (i.e., each tube is incubated at a different temperature).
      Note: Appropriate temperature ranges will vary based on the incubation time (5 min to 1 h) and the reovirus strain under investigation. Each step in the temperature gradient should be no less than 1 °C. ISVP-to-ISVP* conversion typically occurs between 30 °C and 50 °C. 
      
   3. Incubate the heat treated ISVPs for 5 min on ice.
      
   4. Add 0.9 µl of 1 mg/ml trypsin (see Recipes) to each tube of heat treated ISVPs. Mix by pipetting up and down 3-4 times.
      
   5. Incubate the reactions for 30 min on ice.
      
   Note: As a consequence of ISVP-to-ISVP* conversion, the central δ fragment of µ1 adopts a protease sensitive conformation (Chandran et al., 2002).
   
   6. Following digestion, add 3.3 µl of 4x Laemmli sample buffer to each reaction. Mix by pipetting up and down 3-4 times.
      
   7. Boil each reaction for 5 min at 95 °C.
      
   8. To determine the temperature at which ISVP-to-ISVP* conversion occurs, run 2 x 10¹⁰ particles (i.e., the entire ISVP-to-ISVP* conversion reaction at a given temperature) per lane on a 10% SDS-polyacrylamide mini gel. Run the gel for 40-45 min at 200 V constant.
      
   9. Visualize the protein bands by Coomassie Brilliant Blue staining (see Data analysis, Figure 2).
      

Data analysis

1. Generation of infectious subviral particles (ISVPs)
   1. Record and analyze the results using a gel imaging system and Image Studio Lite software (Figure 1).
      
      1. Virions contain λ1,2,3, µ1C, σ2, and σ3.
         
      2. ISVPs contain λ1,2,3, µ1C, δ, and σ2.
         
      Note: The appearance of δ, the loss of µ1C, and the loss of σ3 indicate the transition from virions to ISVPs. λ1,2,3 and σ2 should remain unchanged.
      
      
      Figure 1. SDS-PAGE gel of reovirus virions and ISVPs 
      
      
   2. ISVP-to-ISVP* conversion assay
      1. All ISVP-to-ISVP* experiments should be repeated for at least three independent replicates.
         
      2. Record and analyze the results using a gel imaging system and Image Studio Lite software (Figure 2).
         1. Trypsin treated ISVPs contain λ1,2,3, µ1C, δ, and σ2.
            
         2. Trypsin treated ISVP*s contain λ1,2,3 and σ2.
            
      Note: The loss of µ1C and δ indicate the transition from ISVPs to ISVP*s. λ1,2,3 and σ2 should remain unchanged.
      
      
      Figure 2. SDS-PAGE gel of heat and trypsin treated reovirus ISVPs. In the experiment shown here, ISVP-to-ISVP* conversion occurred at 44, 45, and 46 °C.

Notes

1. When possible, all procedures are performed in a BSL-2 certified tissue culture hood.
   
2. Laboratory personnel should use appropriate PPE.
   
3. All solid waste is autoclaved prior to disposal.
   
4. All liquid waste is inactivated with 50% bleach prior to disposal.
   

Recipes

1. 50% bleach
   In a storage bottle, dilute 50 ml of 100% bleach into 50 ml of ultrapure H₂O
   
2. Virus storage buffer (VB) (10 mM Tris, pH 7.4, 15 mM MgCl₂, and 150 mM NaCl)
   
   1. In a glass beaker, dissolve the following into 900 ml of ultrapure H₂O:
      1.21 g Tris
      3.05 g MgCl₂·6H₂O
      8.77 g NaCl
      
   2. Mix at room temperature using a magnetic stir bar on a stir plate
      
   3. Adjust to pH 7.4 with 0.1 N hydrochloric acid
      
   4. In a graduated cylinder, bring the final volume up to 1,000 ml with ultrapure water
      
   5. Transfer the solution to a storage bottle
      
   6. Sterilize by autoclaving
      
   7. Store at room temperature
      
3. 2 mg/ml Nα-p-tosyl-L-lysine chloromethyl ketone (TLCK)-treated chymotrypsin
   
   1. In a centrifuge tube, dissolve 100 mg of TLCK-treated chymotrypsin into 50 ml of ultrapure H₂O
      
   2. Mix at room temperature by gently inverting the tube until the solution becomes clear
      
   3. Transfer 1 ml aliquots to microcentrifuge tubes
      
   4. Store at -20 °C
      
4. 100 mM phenylmethylsulfonyl fluoride (PMSF)
   
   1. In a microcentrifuge tube, dissolve 17.4 mg of PMSF into 1 ml of isopropyl alcohol
      
   2. Mix at room temperature by gently inverting the tube until the solution becomes clear
      
   3. Store at -20 °C
      
5. 1 mg/ml trypsin
   
1. In a microcentrifuge tube, dissolve 1 mg of trypsin into 1 ml of ultrapure H₂O
   
2. Mix at room temperature by gently inverting the tube until the solution becomes clear
   
3. Store at -20 °C

Acknowledgments

This protocol was adapted from a previously published study (Chandran et al., 2002). Research reported in this publication was supported by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health under award numbers 1R01AI110637 (to P.D.) and F32AI126643 (to A.J.S.) and by Indiana University Bloomington. The content is solely the responsibility of the authors and does not necessarily represent the official views of the funders. The authors declare no conflict of interest.

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