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Generation of High‐Titer Defective HSV‐1 Vectors

Filip Lim¹, Rachael L. Neve²

¹Universidad Autonoma de Madrid, Madrid, Spain
²Harvard Medical School & McLean Hospital, Belmont, Massachusetts

Publication Name:

Current Protocols in Neuroscience

Unit Number:

Unit 4.13

DOI:

10.1002/0471142301.ns0413s06

Online Posting Date:

May, 2001

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Abstract

There are two types of replication-deficient herpes simplex virus type 1 (HSV-1) vectors: those in which the foreign DNA of interest is cloned into the viral genome itself, and those that are comprised of a plasmid (amplicon) carrying minimal HSV-1 sequences that allow it to be packaged into virus particles with the aid of a helper virus. This unit 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 also delineated. Thorough characterization of the viral vector stock involves measuring (1) the helper virus plaque-forming units per ml (pfu/ml) on 2-2 cells, (2) the wild-type HSV-1 pfu/ml on Vero cells, and (3) the amplicon stock infectious vector units per ml (ivu/ml) on PC12 cells. Support protocols detail methods for determining titers of helper virus and wild-type virus by plaque assay, and of amplicon stocks by vector assay.

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Unit Introduction
Basic Protocol 1: Preparation of Helper Virus Stocks
Support Protocol 1: Titration of Helper and Wild-Type Virus by Plaque Assay
Basic Protocol 2: Packaging Amplicon into Virus Particles
Support Protocol 2: Titration of Amplicon Virus by Vector Assay
Reagents and Solutions
Commentary
Bibliography
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
CMF-DPBS (see recipe for DPBS)
0.05% (w/v) trypsin/0.02% (w/v) EDTA (e.g., Sigma)
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
Screw-cap vials for virus storage

Additional reagents and equipment for counting cells with a hemacytometer (appendix 3B) and for measuring virus titer (see Support Protocol 1)

Table 4.13.1 Properties 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. (1989)
2-2 cell line	Vero cells containing IE 2 (ICP27) gene and promoter	Smith et al. (1992)
Vero cells	African green monkey kidney epithelium cell line	ATCC #CCL81
PC12 cell line	Derived from rat pheochromocytoma (adrenal cells with NGF reponsiveness)	ATCC #CRL 172; Greene and Tischler (1976)
d120 virus	HSV-1 (KOS strain) with deletion in IE 3 (ICP4)	DeLuca et al. (1985)
D30EBA virus	HSV-1 (strain 17⁺) with deletion in IE 3 (ICP4)	Paterson and Everett (1990)
E5 cells	Vero cells containing IE 3 (ICP4) gene	DeLuca and Schaffer (1987)
RR1 cells	BHK cells containing IE 3 (ICP4) gene	Johnson et al. (1992)

^a Abbreviations: BHK, baby hamster kidney; NGF, nerve growth factor.

Support Protocol 1: Titration of Helper and Wild-Type Virus by Plaque Assay

Additional Materials (also see Basic Protocol 1)

Vero cells (for titrating wild-type HSV-1 virus; Table 4.13.1)
Virus stock: helper virus seed stock (see Basic Protocol 1) or wild-type HSV-1 virus at desired dilutions
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 2% or 10% (v/v) FBS (appendix 2A), room temperature and prewarmed to 37°C
CMF-DPBS (see recipe for DPBS)
0.05% (w/v) trypsin/0.02% (w/v) EDTA (e.g., Sigma)
³50 ng/µl DNA for transfection, purified with a Qiagen column (per manufacturer's instructions) and resuspended in water
Opti-MEM (Life Technologies), prewarmed to 37°C
LipofectAMINE (Life Technologies)
DPBS (see recipe), prewarmed to 37°C
5dl1.2 helper virus seed stock (see Basic Protocol 1)
BSA
10%, 30%, and 60% (w/v) sucrose solutions (see recipe)
Laminar flow hood
60-mm, 100-mm, and 150-mm tissue culture plates
Cell lifters (optional)
15- and 50-ml polypropylene conical tubes with plug seals
Dry ice/ethanol bath
15- and 50-ml polystyrene conical tubes
Cup-type sonicator
Beckman Ultra-Clear 25 × 89–mm (SW28) and 14 × 89–mm (SW41) tubes
Beckman SW28 and SW41 (or SW40) ultracentrifuge rotors
18-G needles
5-ml syringes
Screw-cap vials for virus storage
Additional reagents and equipment for counting cells with a hemacytometer (appendix 3B) and for measuring virus titer (see Support Protocol 2)

Support Protocol 2: Titration of Amplicon Virus by 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 Basic Protocol 2)
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--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)

Looking for materials? Suppliers Guide

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Figures

Figure 4.13.1

Maps of several currently available HSV-1 amplicon vectors. (A) pHSVPrpUC (Geller et al., 1993) is the basic cloning vector. (B) pHSVlac (Geller and Breakefield, 1988) and (C) pHSVgfp (Aboody-Guterman et al., 1997) express E. coli -galactosidase and A.victoria green fluorescent protein, respectively. (D) pHSVflag (Geller et al., 1993) carries the Flag (Prickett et al., 1989) epitope for N-terminal fusions of this tag onto expressed proteins. MCS, multiple cloning site.

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Figure 4.13.2

Diagram illustrating helper virus–dependent packaging of amplicons.

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Figure 4.13.3

Overview of the packaging procedure as described for collection of a P2 stock (see Basic Protocol 2, steps to and to ). An alternate procedure allows the addition of a P3 passage, followed by sucrose gradient purification of virus (not shown; see Basic Protocol 2, steps to ).

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Figure 4.13.4

Illustration of the titration procedure used to determine the optimal amount of a given helper virus preparation for superinfection.

View Image

Literature Cited

Literature Cited
	Aboody-Guterman, K.S., Pechan, P.A., Rainov, N.G., Sena-Esteves, M., Jacobs, A., Snyder, E.Y., Wild, P., Schraner, E., Tobler, K., Breakefield, X.O., and Fraefel, C. 1997. Green fluorescent protein as a reporter for retrovirus and helper virus-free HSV-1 amplicon vector-mediated gene transfer into neural cells in culture and in vivo. Neuroreport 8:3801-3808.
	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.
	Geller, A.I. and Breakefield, X.O. 1988. A defective HSV-1 vector expresses Escherichia coli beta-galactosidase in cultured peripheral neurons. Science 241:1667-1669.
	Geller, A.I., Keyomarsi, K., Bryan, J., and Pardee, A.B. 1990. An efficient deletion mutant packaging system for defective herpes simplex virus vectors: Potential applications to human gene therapy and neuronal physiology. Proc. Natl. Acad. Sci. U.S.A. 90:7603-7607.
	Geller, A.I., During, M.J., Haycock, J.W., Freese, A., and Neve, R. 1993. Long-term increases in neurotransmitter release from neuronal cells expressing a constitutively active adenylate cyclase from a herpes simplex virus type 1 vector. Proc. Natl. Acad. Sci.U.S.A. 90:7603-7607.
	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. 66:2952-2965.
	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.
	Prickett, K.S., Amberg, D.C., and Hopp, T.P. 1989. A calcium-dependent antibody for identification and purification of recombinant proteins. BioTechniques 7:580-589.
	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 eucaryotic defective-virus cloning-amplifying vector. Cell 30:305-310.
Key Reference
	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 laboratories that currently use the HSV-1 amplicon vector system.