Detection of Helper Virus in Retrovirus Stocks

Constance Cepko1, Warren Pear2

1 Harvard Medical School, Boston, Massachusetts, 2 University of Pennsylvania, Philadelphia, Pennsylvania
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 9.13
DOI:  10.1002/0471142727.mb0913s36
Online Posting Date:  May, 2001
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Abstract

Helper virus is a replication‐competent virus that is sometimes present in stocks of replication‐incompetent virus. There are several types of applications in which the presence of helper virus can be problematic. If animal infections are being done, helper virus can lead to leukemia, particularly if the infection is carried out pre‐ or neonatally. If retroviruses are being used for lineage analysis, helper virus may cause horizontal spread of the marker virus, creating false lineage relationships. This unit describes protocols for the detection of helper virus by a selectable marker assay, by rescue of an integrated provirus, or by measuring reverse transcriptase activity.

     
 
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Table of Contents

  • Basic Protocol 1: Detection of Helper Virus Through Horizontal Spread of Drug Resistance
  • Alternate Protocol 1: Proviral Rescue to Detect Replication‐Competent Retrovirus
  • Alternate Protocol 2: Reverse Transcriptase Assay to Detect Helper Virus
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Tables
     
 
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Materials

Basic Protocol 1: Detection of Helper Virus Through Horizontal Spread of Drug Resistance

  Materials
  • NIH 3T3 cells (used only with murine viruses)
  • Three titered virus stocks: test virus containing neo marker, helper virus–free control containing neo marker (negative control), and wild‐type helper virus
  • 800 µg/ml polybrene, filter sterilized (stored at −20°C)
  • Complete DMEM containing 10% calf serum (DMEM‐10, appendix 3F; prepared with calf serum instead of FBS)
  • 6‐cm dishes
  • 0.45‐µm filter
  • Additional reagents and equipment for determination of viral titer (unit 9.10)
NOTE: All solutions and equipment coming into contact with cells must be sterile, and proper sterile technique should be used accordingly.NOTE: All incubations involving tissue culture cells should be performed in a humidified 37°C, 5% CO 2 incubator unless otherwise noted.

Alternate Protocol 1: Proviral Rescue to Detect Replication‐Competent Retrovirus

  • BAG‐3T3 indicator cells: NIH 3T3 cells containing a single copy per cell of an integrated BAG provirus (Fig. )
NOTE: All solutions and equipment coming into contact with cells must be sterile, and proper sterile technique should be used accordingly.NOTE: All incubations involving tissue culture cells should be performed in a humidified 37°C, 5% CO 2 incubator unless otherwise noted.

Alternate Protocol 2: Reverse Transcriptase Assay to Detect Helper Virus

  • recipeReverse transcriptase (RT) reaction cocktail (see recipe)
  • Virus supernatants: test and control samples (see protocol 1, step )
  • 2× SSC ( appendix 22)
  • 95% ethanol
  • 96‐well microtiter dish
  • Whatman DE52 or DE81 paper, precut to 2.5‐cm circles or sheets cut to accommodate the number of samples
NOTE: All solutions and equipment coming into contact with cells must be sterile, and proper sterile technique should be used accordingly.NOTE: All incubations involving tissue culture cells should be performed in a humidified 37°C, 5% CO 2 incubator unless otherwise noted.
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Figures

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Literature Cited

Literature Cited
   Danos, O. and Mulligan, R.C. 1988. Safe and efficient generation of recombinant retroviruses with amphotropic and ecotropic host ranges. Proc. Natl. Acad. Sci. U.S.A. 85:6460‐6464.
   Eckner, R.J. and Hettrick, K.L. 1977. Defective Friend spleen focus‐forming virus: Interfering properties and isolation free from standard leukemia‐inducing helper virus. J. Virol. 24:383‐396.
   Goff, S., Trakman, P., and Baltimore, D. 1981. Isolation and properties of murine leukemia virus mutants: Use of a rapid assay for release of virion reverse transcriptase. J. Virol. 38:239‐248.
   Miller, A.D. and Buttimore, C. 1986. Redesign of retrovirus packaging cell lines to avoid recombination leading to helper virus production. Mol. Cell. Biol. 6:2895‐2902.
   Morgan, B.A. and Fekete, D.M. 1996. Manipulating gene expression with replication competent retroviruses. Methods Cell Biol. 51:185‐218.
   Omer, C.A. and Faras, A.J., 1982. Mechanism of release of the avian retrovirus tRNATrp primer molecule from viral DNA by ribonuclease H during reverse transcription. Cell 30:797‐805.
   Rowe, W.P., Pugh, W.E., and Hartley, J.W. 1970. Plaque assay techniques for murine leukemia viruses. Virology 42:1136‐1139.
   Stoker, A.W. and Bissell, M.J. 1987. Quantitative immunocytochemical assay for infectious avian retroviruses. J. Gen. Virol. 68:2481‐2485.
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