Generation of High‐Titer Defective HSV‐1 Vectors

Rachael L. Neve1, Filip Lim2

1 Massachusetts Institute of Technology, Cambridge, Massachusetts, 2 Universidad Autonoma de Madrid, Madrid, Spain
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 4.13
DOI:  10.1002/0471142301.ns0413s62
Online Posting Date:  January, 2013
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Abstract

Basic Protocol 1 describes the generation of helper virus stocks. Preparation of recombinant amplicon vector particles by transfection of amplicon and superinfection of helper virus into cells, and harvesting of packaged particles, is delineated in Basic Protocol 3. Thorough characterization of each amplicon viral vector stock involves measuring (1) the helper virus plaque‐forming units per ml (pfu/ml) on 2‐2 cells and (2) the amplicon stock infectious units per ml (iu/ml) on PC12 cells. The Support Protocols detail methods for determining titers of helper virus by plaque assay, and of amplicon stocks by vector assay. Curr. Protoc. Neurosci. 62:4.13.1‐4.13.15. © 2013 by John Wiley & Sons, Inc.

Keywords: HSV‐1; amplicon; defective vector

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Preparation of Helper Virus Stocks
  • Support Protocol 1: Titration of Helper Virus by Plaque Assay
  • Basic Protocol 2: Packaging Amplicon into Virus Particles
  • Support Protocol 2: Titration of Amplicon Virus by Amplicon Vector Assay
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Preparation of Helper Virus Stocks

  Materials
  • Adherent 2‐2 cells (Table 4.13.1) maintained in supplemented DMEM/10% FBS
  • DPBS with Ca+2/Mg+2, hereafter referred to as DPBS (Mediatech, cat. no. 21‐030‐CM)
  • Ca+2/Mg+2‐free (CMF) DPBS: prepared by diluting 10× CMF‐DPBS (BioWhittaker, cat. no. 17‐515Q) 1:10 with cell culture grade H 2O (Mediatech, cat. no. 25‐055‐CV)
  • TrypLE Express (Life Technologies)
  • Supplemented DMEM (see recipe) containing 2% or 10% (v/v) FBS ( appendix 2A)
  • 5dl1.2 helper virus stock (Table 4.13.1)
  • Plaque agarose (see recipe)
  • Laminar flow hood
  • 60‐ and 100‐mm tissue culture plates
  • Cell lifters
  • 15‐ml polypropylene conical tube with a plug seal
  • Dry ice/ethanol bath
  • 15‐ml polystyrene conical tubes
  • Cup‐type sonicator
  • Benchtop centrifuge
  • Screw‐cap tubes for virus storage
  • Additional reagents and equipment for counting cells with a hemacytometer ( appendix 3B) and for measuring virus titer (see protocol 1)
    Table 4.3.1   MaterialsProperties and Sources for Virus and Cell Lines

    Virus/cell line Properties a Reference
    5dl1.2 virus HSV‐1 (KOS strain) with partial deletion in IE 2 (ICP27) McCarthy et al. ( )
    2‐2 cell line Vero cells containing IE 2 (ICP27) gene and promoter Smith et al. ( )
    Vero cells African green monkey kidney epithelium cell line ATCC #CCL81
    PC12 cell line Derived from rat pheochromocytoma (adrenal cells with NGF responsiveness) ATCC #CRL 172; Greene and Tischler ( )
    dl120 virus HSV‐1 (KOS strain) with deletion in IE 3 (ICP4) DeLuca et al. ( )
    D30EBA virus HSV‐1 (strain 17+) with deletion in IE 3 (ICP4) Paterson and Everett ( )
    E5 cells Vero cells containing IE 3 (ICP4) gene DeLuca and Schaffer ( )
    RR1 cells BHK cells containing IE 3 (ICP4) gene Johnson et al. ( )

     aAbbreviations: BHK, baby hamster kidney; NGF, nerve growth factor.

Support Protocol 1: Titration of Helper Virus by Plaque Assay

  • Plaque‐fixing solution: 5% (v/v) methanol/10% (v/v) acetic acid (store at room temperature)
  • Crystal violet stain (see recipe)
  • Dissecting microscope

Basic Protocol 2: Packaging Amplicon into Virus Particles

  Materials
  • Adherent 2‐2 cells (Table 4.13.1) maintained in supplemented DMEM/10% FBS
  • Supplemented DMEM (see recipe) containing 5% or 10% (v/v) FBS ( appendix 2A), room temperature and prewarmed to 37°C
  • ≥50 ng/µl DNA for transfection, purified with a Qiagen column or its equivalent and resuspended in TE (10 mM Tris, 1 mM EDTA)
  • Opti‐MEM (Life Technologies), prewarmed to 37°C
  • Plus Reagent (Life Technologies)
  • LipofectAMINE 2000 (Life Technologies)
  • DPBS, prewarmed to 37°C
  • 5dl1.2 helper virus amplified from seed stock (see protocol 1)
  • 10× CMF‐DPBS
  • 1 M solution HEPES, pH 7.3 (USB)
  • 10%, 30%, and 60% (w/v) sucrose solutions (see recipe)
  • Laminar flow hood
  • 60‐mm, 100‐mm, and 150‐mm tissue culture plates
  • 1.5‐ml microcentrifuge tubes
  • 15‐ and 50‐ml polypropylene conical tubes
  • Samco 232‐1S fine‐tipped transfer pipets
  • Benchtop centrifuge
  • Cell lifters
  • Dry ice/ethanol bath
  • 37°C water bath
  • 50‐ml polystyrene tubes
  • Cup‐type sonicator
  • Ultra‐Clear 25 × 89–mm (SW28) and 14 × 89–mm (SW41) tubes
  • SW28 and SW41 (or SW40) ultracentrifuge rotors, or their equivalent
  • Clamp and ring stand
  • 18‐G needles
  • 3‐ml syringes
  • Additional reagents and equipment for counting cells with a hemacytometer ( appendix 3B) and for measuring virus titer (see protocol 2)

Support Protocol 2: Titration of Amplicon Virus by Amplicon Vector Assay

  Materials
  • 20 µg/ml poly‐D‐lysine solution (see recipe)
  • PC12 cells (Table 4.13.1)
  • DMEM/10% HS/5% FBS: Supplemented DMEM (see recipe) without G418, containing 10% (v/v) horse serum and 5% (v/v) FBS ( appendix 2A)
  • Amplicon stock to be measured (see protocol 3)
  • PBS ( appendix 2A) with and without 10 mM EDTA ( appendix 2A)
  • 4% (w/v) paraformaldehyde solution (see recipe)
  • TBS (optional; appendix 2A)
  • Fe solution (optional; see recipe)
  • 50 mg/ml 5‐bromo‐4‐chloro‐3‐indolyl‐β‐D‐galactopyranoside (Xgal; optional) in dimethyl sulfoxide (store in aliquots at −20°C)
  • Rabbit (or mouse) anti‐HSV primary antibody (optional)
  • TST (optional): TBS containing 1% (v/v) goat serum and 0.1% (v/v) Triton X‐100 (store at 4°C)
  • Alkaline phosphatase (AP)–conjugated anti‐rabbit (or anti‐mouse) secondary antibody (optional)
  • AP buffer (optional; see recipe)
  • AP substrate solution (optional; see recipe)
  • Laminar flow hood
  • 24‐well tissue culture plates
  • 21‐G needle
  • Dissecting microscope
  • Additional reagents and equipment for counting cells with a hemacytometer ( appendix 3B)
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Figures

Videos

Literature Cited

   Bursztajn, S., DeSouza, R., McPhie, D.L., Berman, S.A., Shioi, J., Robakis, N.K., and Neve, R.L. 1998. Overexpression in neurons of human presenilin‐1 or a presenilin‐1 familial Alzheimer disease mutant does not enhance apoptosis. J. Neurosci. 18:9790‐9799.
   Carlezon, W.A. Jr., Boundy, V.A., Haile, C.N., Kalb, R.G., Neve, R.L., and Nestler, E.J. 1997. Sensitization to morphine induced by viral‐mediated gene transfer. Science 277:812‐814.
   DeLuca, N.A. and Schaffer, P.A. 1987. Activities of herpes simplex virus type 1 (HSV‐1) ICP4 genes specifying nonsense peptides. Nucl. Acids Res. 15:4491‐4511.
   DeLuca, N.A., McCarthy, A.M., and Schaffer, P.A. 1985. Isolation and characterization of deletion mutants of herpes simplex virus type 1 in the gene encoding immediate‐early regulatory protein ICP4. J. Virol. 56:558‐570.
   Greene, L.A. and Tischler, A.S. 1976. Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor. Proc. Natl. Acad. Sci. U.S.A. 73:2424‐2428.
   Johnson, P.A., Miyanohara, A., Levine, F., Cahill, T., and Friedmann, T. 1992. Cytotoxicity of a replication‐defective mutant of herpes simplex virus type 1. J. Virol. 66:2952‐2956.
   Lim, F., Hartley, D., Starr, P., Lang, P., Song, S., Yu, L., Wang, Y., Geller, A.I. 1996. Generation of high‐titer defective HSV‐1 vectors using an IE 2 deletion mutant and quantitative study of expression in cultured cortical cells. BioTechniques 20:460‐470.
   McCarthy, A.M., McMahan, L., and Schaffer, P.A. 1989. Herpes simplex virus type 1 ICP27 deletion mutants exhibit altered patterns of transcription and are DNA deficient. J. Virol. 63:18‐27.
   Neve, R.L., Howe, J.R., Hong, S., and Kalb, R.G. 1997. Introduction of glutamate receptor subunit 1 into motor neurons in vivo using a recombinant herpes simplex virus alters the functional properties of AMPA receptors. Neuroscience 79:435‐447.
   Paterson, T. and Everett, R.D. 1990. A prominent serine‐rich region in Vmw175, the major transcriptional regulator protein of herpes simplex virus type 1, is not essential for virus growth in tissue culture. J. Gen. Virol. 71:1775‐1783.
   Smith, I.L., Hardwicke, M.A., and Sandri‐Goldin, R.M. 1992. Evidence that the herpes simplex virus immediate early protein ICP27 acts post‐transcriptionally during infection to regulate gene expression. Virology 186:74‐86.
   Spaete, R. and Frenkel, N. 1982. The herpes simplex virus amplicon: A new eukaryotic defective‐virus cloning‐amplifying vector. Cell 30:305‐310.
Key References
   Lim et al., 1996. See above.
  This paper describes the background and rationale for the major modifications that have been incorporated into the packaging procedure outlined in this unit.
Internet Resources
  http://bacterio.cbm.uam.es/flim/Ampweb.html
  This Web site contains HSV‐1 packaging protocols and vector maps and sequences, describes biosafety considerations, and lists some laboratories that currently use the HSV‐1 amplicon vector system.
  http://mcgovern.mit.edu/technology/viral‐vector/types‐of‐viruses
  This Web page (click on “Catalog of HSV Vectors”) describes the many HSV‐1 amplicon vectors available today. It is updated frequently.
  http://mcgovern.mit.edu/technology/viral‐vector/viral‐core‐newsletter#catalog%20of%20vectors
  This newsletter describes in detail each HSV‐1 vector available today, with links to maps.
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