Gene Vector and Virus Core–  

Care and Handling of VSV-G Pseudotyped Lentiviruses

lentivirus

It can be useful to think of lentiviruses as small cells.  They have a membrane and a small amount of cytoplasm.  The capsid, which contains the genome, is somewhat like a nucleus.  As such, they are very unstable and should be treated gently like a cell would be.



DON'T:

  1. Don’t expose to environmental extremes (such as pH, temperature, organic solvents, protein denaturants, strong detergents, or cation chelators such as EDTA).  ALL liquids the virus is in (dilution buffers, cell culture media, etc.) should be pH= ~7.2.  Add 10 mm HEPES if in doubt to buffer the pH.
  2. In particular the VSV-G protein, which is on the surface of the virus and is required for infection, is extremely pH sensitive.  It loses about 10-fold in infectious activity for each 0.3 unit drop in pH below ~pH 7.0.  Although this effect is reversible to some degree the reversibility is time dependent.  Because of this pH sensitivity ALL liquids the virus is in (dilution buffers , cell culture media, etc.) should be pH= ~7.2.  Add 10 mM HEPES if in doubt to buffer the pH.
  3. Don’t expose to any condition that disrupts membranes (such as temperature, organic solvents, and strong detergents).  This will result in near complete inactivation because an intact viral membrane is required for viral infection.
  4. Don’t introduce air into the virus by vortexing, blowing bubbles and similar operations which result in protein denaturation.  Denatured VSV-G, which is required for infection, is inactive.
  5. Don’t dry.  Drying will also result in membrane disruption and near complete inactivation of the virus.
  6. Don’t freeze and thaw multiple times.  The titer may drop 2-3 fold (or more) with each freeze-thaw cycle.   Therefore, it is best to avoid.
  7. Don’t expose to hydrophobic plastics (especially polystyrene) for prolonged periods.  Because lentiviruses are surrounded by a mostly hydrophobic membrane they are very sticky and losses can occur if they are exposed to hydrophobic plastics while not frozen.  It is best to store thawed lentiviruses in siliconized or low protein binding tubes and pipette it with similar pipette tips. 
  8. Don’t filter.  Filtering can reduce titer because viruses can stick to the filter.  If filtering is necessary it is essential that filters with 0.45 μm (or larger) pores are used.  Since the diameter of a lentivirus is about 0.15-0.2 μm, if a filter with 0.2 μm (or smaller) pores is used substantial loss of infectious titer can occur.

DO:

  1. Do aliquot and freeze at -80C for long term storage if the virus is not used within ~1 week.  Besides being intrinsically unstable it is possible for microorganisms to grow in residual cell culture media that is in the virus.  Freezing helps prevent this.
  2. Thaw on ice just before use.
  3. Use virus within ~ 6 months of storage at -80C unless the titer is much higher than needed.  Even when stored at -80C lentiviruses will lose ~10x in titer every 6-12 months.

 

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