Purification of HCV-remodeled and Control ER Membranes

Materials and Reagents

1. Huh7 human hepatoma cells, stably replicating a wt JFH1 subgenomic replicon (NS4B^wt) and a recombinant replicon cell line harboring the HA epitope and the adaptive mutation Q31R in NS4B (NS4B^HA) (Paul et al., 2013) and Huh7 hepatoma cells stably overexpressing C-terminally HA-tagged calnexin (CANX^HA) (Paul et al., 2013)
   
2. 100 U/ml Pencillin and Streptomycin (Life Technologies, Gibco^®, catalog number: 15140122 )
   
3. 10% (v/v) Fetal Calf Serum (Life Technologies, Gibco^®, catalog number: 10270-106 )
   
4. 1x nonessential amino acids (Life Technologies, catalog number: 11140-035 )
   
5. Geneticin (Life Technologies, Gibco^®, catalog number: 11811-098 )
   
6. Blasticidin (Life Technologies, Gibco^®, catalog number: R210-01 )
   
7. Bradford reagent
   
8. NaN₃ (Sigma-Aldrich, catalog number: S2002 )
   
9. Hemagglutinin peptide (Sigma-Aldrich, catalog number: I2149 )
   
10. Anti-HA agarose beads (Sigma-Aldrich, catalog number: A2095 )
    
11. 1x protease inhibitor cocktail (Roche Diagnostics, catalog number: 04693116001 )
    
12. Dulbecco’s Modified Eagle’s Medium (DMEM) (Life Technologies, Gibco^®, catalog number: 41965-039 ) (see Recipes)
    
13. Bovine serum albumin (BSA) standard (see Recipes)
    
14. PBS (sterile) (see Recipes)
    
15. Hypotonic buffer (see Recipes)
    
16. Sucrose (see Recipes)
    
17. 5 M NaCl solution (see Recipes)
    
18. IP washing buffer I (see Recipes)
    
19. IP washing buffer II (see Recipes)
    
20. IP elution buffer (see Recipes)
    

Equipment

1. 15 cm cell culture dishes
   
2. 37 °C, 5% CO₂ cell culture incubator
   
3. Cell scraper (e.g. SARSTEDT AG, catalog number: 83.1830 )
   
4. Dounce homogenizer (1 ml) (e.g. WHEATON, catalog number: 357538 )
   
5. Table top cooling centrifuge with swinging buckets for 15 ml and 50 ml falcon tubes (e.g. Heraeus Multifuge 3-SR)
   
6. Open top polyclear ultracentrifuge tubes for SW40 rotor (Seton Identification Products, catalog number: 7031 )
   
7. Ultracentrifuge and SW40 rotor and buckets (e.g. Beckman Optima LE80K Ultracentrifuge and Beckman SW40Ti rotor and buckets)
   
8. Lab stand and clamp
   
9. 19 G needle (e.g. BD, catalog number: 301500 )
   
10. Spectrophotometer (e.g. Amersham Ultrospec 2100 pro) and cuvettes (e.g. SARSTEDT AG, catalog number: 67.742 )
    
11. Rotator wheel for Eppendorf tubes (e.g. Labinco, model: L28, catalog number: 2800 )
    
12. Table top cooling centrifuge for Eppendorf reaction tubes (e.g. Eppendorf, model: 5417R )
    
13. Eppendorf Thermomixer compact
    

Procedure

1. Cells are grown in DMEM_cplt in the incubator in a 5% CO₂ environment at 37 °C, in presence of 0.5 mg/ml geneticin in case of replicon cell lines and 10 µg/ml blasticidin for CANX^HA overexpressing cells. Around 5-7.5 x 10⁷ cells are needed for the experiment, which equals six 15 cm cell culture dishes.
   Note: Cell confluence should not exceed 90% at the day of harvest.
   
2. Cells are washed twice in PBS and subsequently scraped in 5 ml PBS per dish and transferred into a 50 ml falcon tube.
   
3. The cells are pelleted at 700 x g for 10 min at 4 °C.
   
4. The supernatant is removed and cells are resuspended in 2 ml hypotonic buffer containing 1x protease inhibitor cocktail.
   
5. Cells are incubated on ice for 30 min.
   
6. Cells are broken by 40 strokes with the dounce homogenizer.
   Note: Turn pestle on the bottom of mortar chamber by 90° during each stroke.
   
7. Cell debris and nuclei are pelleted by centrifugation at 700 x g for 10 min at 4 °C.
   
8. Preparation of discontinuous sucrose gradient in ultra-centrifugation SW40 tubes:
   Pipette 4 ml 70% sucrose on the bottom and consecutively overlay carefully with 3 ml 60% sucrose, 2 ml 40% sucrose and 2 ml 30% sucrose.
   
9. 2 ml post-nuclear supernatants (see step 7) are carefully layered on top of the sucrose gradient.
   
10. Membrane fractions are separated in the sucrose gradient by ultra-centrifugation for 4 h at 130,000 x g using a SW40 rotor.
    
11. Place tubes in the clamp of a lab stand, and carefully make a hole at the bottom using a 19 G needle. Collect 13 1 ml fractions, with fraction number 1 being the lowest one and number 13 representing the top of the gradient.
    Note: The elution speed of the gradient should be around 1 drop per second, which can be adjusted by the hole size on the bottom.
    
12. Gradient fractions can be analyzed for their protein and HCV RNA content. Typically the majority of HCV RNA and proteins are found in fractions 7-11 (Paul et al., 2013), which thus are subsequently pooled.
    
13. Equilibrate pooled fractions 7-11 to 150 mM NaCl by adding an appropriate volume of 5 M NaCl solution (e.g. 150 µl 5 M NaCl for 5 ml fractions 7-11), and store a small aliquot (~50 µl as input samples).
    
14. Determine protein concentration in pooled fractions by using the Bradford assay and a BSA standard curve.
    
15. Wash 25 µl anti-HA agarose beads with 500 µl IP washing buffer I in 2 ml Eppendorf tubes (for each NS4B^wt, NS4B^HA and CANX^HA). Pellet beads by centrifugation at 3,800 x g for 3 min at 4 °C and remove supernatant.
    
16. Incubate beads with membrane fraction containing a total of around 1 mg protein (approximately 1-1.5 ml) for 4 h on a rotator wheel at 4 °C.
    Note: The total protein input should be the same for the three IP samples.
    
17. Pellet beads and bound material by centrifugation at 3,800 x g for 3 min at 4 °C and remove supernatant.
    
18. Wash beads three times with 1 ml IP washing buffer I and in between each washing step pellet beads as described in step 17.
    
19. Add 1ml of IP washing buffer I and transfer beads and bound material into a new eppendorf tube and pellet beads as described in step 17.
    
20. Wash beads with 1 ml IP washing buffer II and pellet beads as described in step 17.
    
21. Elution of bound material:
    1. Add 100 µl IP elution buffer to the beads and shake the samples for 15 min at 1,100 rpm at 25 °C.
       
    2. Pellet beads by centrifugation at 3,800 x g for 3 min at 25 °C.
       
    3. Collect and keep supernatant.
       
    4. Add 100 µl IP elution buffer to the beads and shake the samples for 15 min at 1,100 rpm at 25 °C.
       
    5. Pellet beads by centrifugation at 3,800 x g for 3 min at 25 °C.
       
    6. Collect and pool with supernatant from step 21c.
22. IP elution fractions can be analyzed in a variety of subsequent assays, including western blot, RNA quantification as well as electron microscopy analyses (Paul et al., 2013).
    

Recipes

1. Complete Dulbecco’s Modified Eagle’s Medium (DMEM_cplt)
   2 mM L-glutamine
   1x nonessential amino acids
   Supplemented with 100 U/ml Pencillin and Streptomycin
   10% (v/v) Fetal Calf Serum (heat inactivated at 56 °C for 30 min)
   
2. BSA standard
   Dissolve 0.1 g of BSA in 10 ml IP washing buffer I (final concentration 10 µg/µl)
   Use 1, 2, 3 and 4 µl for the BSA standard in the Bradford assay
   
3. PBS
   8 mM Na₂HPO₄
   2 mM NaH₂PO₄
   140 mM NaCl
   0.27 mM KCl
   0.176 mM KH₂PO₄
   
4. Hypotonic buffer
   10 mM Tris (pH 8)
   1.5mM MgCl₂
   10 mM NaAc
   
5. Sucrose
   70% dissolve 70 g sucrose in 100 ml hypotonic buffer
   60% dissolve 60 g sucrose in 100 ml hypotonic buffer
   40% dissolve 40 g sucrose in 100 ml hypotonic buffer
   30% dissolve 30 g sucrose in 100 ml hypotonic buffer
   
6. 5 M NaCl solution
   Dissolve 1,461 g NaCl in 500 ml water
   
7. IP washing buffer I
   Hypotonic buffer containing 40% Sucrose and 150 mM NaCl
   
8. IP washing buffer II
   Hypotonic buffer containing 10% Sucrose and 150 mM NaCl
   
9. IP elution buffer
   IP washing buffer II containing 250 µg/ml hemagglutinin peptide and 0.01 % NaN₃
   

Acknowledgments

The protocol described here has been published originally in a short form in Paul et al. (2013). Research was supported by the DFG TRR83 TP13.

References

1. Paul, D., Hoppe, S., Saher, G., Krijnse-Locker, J. and Bartenschlager, R. (2013). Morphological and biochemical characterization of the membranous hepatitis C virus replication compartment. J Virol 87(19): 10612-10627.