Construction of Replication‐Defective Herpes Simplex Virus Vectors

William F. Goins1, Peggy Marconi1, David Krisky1, Darren Wolfe1, Joseph C. Glorioso1, Ramesh Ramakrishnan2, David J. Fink3

1 University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, 2 Virusys Corp., North Berwick, Maine, 3 University of Pittsburgh School of Medicine and VA Medical Center, Pittsburgh, Pennsylvania
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 12.11
DOI:  10.1002/0471142905.hg1211s33
Online Posting Date:  August, 2002
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Advances in identification and characterization of gene products responsible for specific diseases of the nervous system have opened opportunities for novel therapies using gene transfer vectors for gene replacement. Herpes simplex virus (HSV)‐based vectors are particularly well suited for gene delivery to neurons of the central and peripheral nervous systems. The authors have developed methods to delete HSV‐1 IE gene functions and to subsequently introduce foreign genes into the HSV‐1 genome using homologous recombination. This unit describes methods for generating cell lines that complement multiple essential gene deletion mutants as well for generating such replication‐defective virus recombinants and inserting foreign DNA sequences into replication‐defective viral genomes, the last step in preparing a vector. Three support protocols describe methods for preparing virus stocks, titering virus, and preparing viral DNA.

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Basic Protocol 1: Construction of HSV‐1 IE Gene–Complementing Cell Lines
  • Basic Protocol 2: Construction of Replication‐Defective Vectors
  • Basic Protocol 3: Insertion of Foreign Gene Sequence into a Replication‐Defective Genomic HSV Vector
  • Support Protocol 1: Preparation of Herpes Simplex Virus Stock
  • Support Protocol 2: Plaque Assay to Titer Virus
  • Support Protocol 3: Isolation of Viral DNA
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Construction of HSV‐1 IE Gene–Complementing Cell Lines

  Materials
  • Vero cells (African green monkey kidney cells; ATCC #CCC81) or other appropriate cell line
  • Trypsin/EDTA: 0.05% (w/v) trypsin/0.3 mM EDTA (Life Technologies)
  • recipeComplete Eagle modified essential medium with and without 10% (v/v) FBS (complete MEM/10% FBS and complete MEM; see recipe)
  • Gel‐purified plasmid DNA fragment
  • recipe2× HEPES‐buffered saline (HeBS, see recipe), pH 7.05
  • 2 M CaCl 2 (prepared as in unit 12.9 but at lower concentration)
  • 20% (v/v) glycerol in recipe2× HeBS (see recipe)
  • 1 mg/ml neomycin sulfate (G418, Life Technologies) or 10 µg/ml puromycin (Clontech), in complete MEM/10% FBS
  • Freezing medium: complete MEM/10% FBS containing 10% (v/v) dimethylsulfoxide (DMSO) or glycerol
  • recipePBS, pH 7.5 (see recipe)
  • recipeDNA extraction buffer (see recipe)
  • 10× PCR amplification buffer with 15 mM MgCl 2 ( appendix 2D)
  • 10 mM 4dNTP mix ( appendix 2D)
  • 100 ng/µl ICP27 primer pair:
    •  5′ primer: 5′‐GCC GCC GCG ACG ACC TGG AAT‐3′
    •  3′ primer: 5′‐TGT GGG GCG CTG GTT GAG GAT‐3′
  • 10 U/µl Taq DNA polymerase
  • DNA probe specific for ICP27 sequences
  • HSV ICP27 mutant
  • HSV IE gene mutant
  • recipe1% (w/v) methylcellulose overlay (see recipe)
  • recipe1% (w/v) crystal violet staining solution (see recipe)
  • 30‐, 60‐, and 100‐mm tissue culture dishes
  • Beckman GPR refrigerated tabletop centrifuge and GH‐3.7 swinging‐bucket rotor
  • 96‐well round‐bottom tissue culture plates
  • 25‐cm2 tissue culture flasks
  • Cell scrapers
  • Additional reagents and equipment for trypsinization of cells and tissue culture ( appendix 3G), agarose gel electrophoresis and Southern blot hybridization (unit 2.7), preparation of midistocks (see protocol 4), and titration of virus stock (see protocol 5)
NOTE: All solutions and equipment coming into contact with cells must be sterile, and proper sterile technique should be used accordingly.NOTE: All culture incubations are performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.

Basic Protocol 2: Construction of Replication‐Defective Vectors

  Materials
  • IE gene–complementing cell line (see protocol 1)
  • Trypsin/EDTA: 0.05% (w/v) trypsin/5.3 mM EDTA solution (Life Technologies)
  • recipeComplete Eagle modified essential medium with and without 10% (v/v) FBS (complete MEM/10% FBS and complete MEM; see recipe)
  • Plasmid with reporter cassette (e.g., HCMV IEp‐lacZ‐BGH pA; see Fig. ) inserted in place of deleted HSV‐1 genes
  • Restriction endonuclease with 8‐bp recognition sequence, and appropriate buffer
  • Viral DNA (see protocol 6)
  • recipe2× HEPES‐buffered saline (HeBS; see recipe), pH 7.05
  • 2 M CaCl 2 (prepared as in unit 12.9 but at lower concentration)
  • 20% (v/v) glycerol in recipe2× HeBS
  • recipe0.1% Xgal staining solution (see recipe)
  • recipePBS, pH 7.5 (see recipe)
  • recipeDNA extraction buffer (see recipe)
  • 10× PCR amplification buffer with 15 mM MgCl 2 ( appendix 2D)
  • 10 mM 4dNTP mix ( appendix 2D)
  • 100 ng/µl ICP27 primer pair:
    •  5′ primer: 5′‐GCC GCC GCG ACG ACC TGG AAT‐3′
    •  3′ primer: 5‐TGT GGG GCG CTG GTT GAG GAT‐3′
  • 100 ng/µl glycoprotein B (gB) primer pair:
    •  5′ primer: 5′‐ATT CTC CTC CGA CGC CAT ATC CAC CAC CTT‐3′
    •  3′ primer: 5′‐AGA AAG CCC CCA TTG GCC AGG TAG T‐3′
  • 10 U/µl Taq DNA polymerase
  • Sterile H 2O
  • DNA probes specific for ICP27 or gB sequences
  • 30‐ and 60‐mm tissue culture dishes
  • Wide‐bore pipet tips (Bio‐Rad)
  • Cell scrapers
  • Beckman GPR refrigerated tabletop centrifuge and GH‐3.7 swinging‐bucket rotor
  • Sonicator with cup horn (VirTis)
  • Rocker platform (e.g., Nutator, Becton Dickinson Primary Care Diagnostics)
  • Multichannel pipettor and reagent reservoirs (Costar)
  • 96‐well flat‐bottom tissue culture plate
  • Additional reagents and equipment for tissue culture ( appendix 3G), titering virus stock (see protocol 5), and producing virus midistocks (see protocol 4)
NOTE: All solutions and equipment coming into contact with cells must be sterile, and proper sterile technique should be used accordingly.NOTE: All culture incubations are performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.

Basic Protocol 3: Insertion of Foreign Gene Sequence into a Replication‐Defective Genomic HSV Vector

  Materials
  • IE gene–complementing cells (see protocol 1)
  • Replication‐defective HSV vector (see protocol 2) digested with PacI under the conditions recommended by the manufacturer
  • Plasmid containing foreign gene cassette, linearized using the appropriate restriction endonuclease
  • Subconfluent monolayer cultures of IE gene–complementing cell line (see protocol 1) in 24‐well tissue culture plates
  • recipeComplete Eagle modified essential medium with and without 10% (v/v) FBS (complete MEM/10% FBS and complete MEM; see recipe)
  • recipeTris‐buffered saline (TBS), pH 7.5 (see recipe)
  • recipeDNA extraction buffer (see recipe)
  • 25:24:1 (v/v/v) phenol/chloroform/isoamyl alcohol ( appendix 3C)
  • Chloroform
  • Isopropanol, −20°C
  • 70% ethanol
  • Total RNA isolation reagent (TRI reagent, Molecular Research Center)
  • 1‐Bromo‐3‐chloropropane (BCP, Aldrich)
  • RNase‐free DNase I (Worthington)
  • 10 U/µl reverse transcriptase and buffer (e.g., SuperScript II and 5× SuperScript II buffers, Life Technologies)
  • 1.0 mM DTT ( appendix 2D for 1 M)
  • 100 ng/µl lacZ primer pair:
    •  5′ primer: 5′‐ACC CCT TCA TTG ACC TCA AC‐3′
    •  3′ primer: 5′‐ATT GGG GGT AGG AAC ACG‐3′
  • 100 ng/µl GAPDH primer pair:
    •  5′ primer: 5′‐TTG CTG ATT CGA GGG GTT AAC CGT CAC GAG‐3′
    •  3′ primer: 5′‐ACC AGA TGA TCA CAC TGC GGT GAT TAC GAT‐3′
  • 100 ng/µul foreign gene primer pair
  • 10 mM 4dNTP mix ( appendix 2D)
  • Placental RNase inhibitor: 50 to 100 U/µl RNasin (Promega)
  • Sterile H 20
  • 10× PCR amplification buffer with 15 nM CaCl 2 ( appendix 2D)
  • DNA probe for foreign gene sequences
  • 10 U/µl Taq DNA polymerase
  • Rocker platform (e.g., Nutator, Becton Dickinson Primary Care Diagnostics)
  • 30‐mm tissue culture dishes
  • 96‐well flat‐bottom and 24‐well tissue culture plates
  • 1.5‐ml microcentrifuge tubes, sterile
  • Pellet pestles (Kontes Glass)
  • Additional reagents and equipment for tissue culture ( appendix 3G) and dot‐blot or Southern blot hybridization (unit 2.7)
NOTE: All solutions and equipment coming into contact with cells must be sterile, and proper sterile technique should be used accordingly.NOTE: All culture incubations are performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.NOTE: When isolating and handling RNA, wear gloves, keep solutions cold and samples on ice when possible, and use new sterile or DEPC‐treated microcentrifuge tubes and pipettor tips.

Support Protocol 1: Preparation of Herpes Simplex Virus Stock

  Materials
  • Permissive cells appropriate for growth of virus
  • Virus stock of known titer (see protocol 5)
  • recipeComplete Eagle modified essential medium with and without 10% (v/v) FBS (complete MEM/10% FBS and complete MEM; see recipe)
  • 1 M HEPES (N‐2‐hydroxyethylpiperazine‐N′‐2‐ethanesulfonic acid)
  • 25‐cm2 tissue culture flasks
  • 15‐ and 50‐ml conical polypropylene centrifuge tubes
  • Beckman GPR refrigerated tabletop centrifuge and GH‐3.7swinging‐bucket rotor
  • Cup‐horn sonicator (VirTis)
  • 50‐ml polypropylene Oak Ridge tubes
  • Beckman J2‐21M preparative centrifuge and JA‐20 rotor (preparative)
  • 850‐cm2 tissue culture roller bottles
  • Roller bottle apparatus
  • Cell scrapers
  • Additional reagents and equipment for tissue culture ( appendix 3G), titering virus (see protocol 5), and virus purification using gradient centrifugation (CPMB UNIT ; optional)
NOTE: All solutions and equipment coming into contact with cells must be sterile, and proper sterile technique should be used accordingly.NOTE: All culture incubations are performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.

Support Protocol 2: Plaque Assay to Titer Virus

  Materials
  • Permissive cells appropriate for growth of virus
  • Virus stock to be titrated
  • recipeComplete Eagle modified essential medium with and without 10% (v/v) FBS (complete MEM/10% FBS and complete MEM; see recipe)
  • recipe1% (w/v) methylcellulose overlay (see recipe)
  • recipe1% (w/v) crystal violet solution (see recipe)
  • 12‐well tissue culture plates
  • Additional reagents and equipment for tissue culture ( appendix 3G)
NOTE: All solutions and equipment coming into contact with cells must be sterile, and proper sterile technique should be used accordingly.NOTE: All culture incubations are performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.

Support Protocol 3: Isolation of Viral DNA

  Materials
  • Monolayers of permissive cells, in 150‐cm2 tissue culture flasks and 60‐mm tissue culture dishes
  • Stock of HSV‐1 virus of known titer (see protocol 4)
  • recipeComplete Eagle modified essential medium with and without 10% (v/v) FBS (complete MEM/10% FBS and complete MEM; see recipe)
  • recipeTris‐buffered saline (TBS), pH 7.5 (see recipe)
  • recipeDNA extraction buffer (see recipe)
  • 25:24:1 (v/v/v) phenol/chloroform/isoamyl alcohol ( appendix 3C)
  • Chloroform
  • Isopropanol, −20°C
  • 70% ethanol
  • Labeled total HSV‐1 viral DNA
  • recipe1% (w/v) methylcellulose overlay (see recipe)
  • recipe1% (w/v) crystal violet staining solution (see recipe)
  • 150‐cm2 tissue culture flask
  • Cell scrapers (optional)
  • 15‐ml conical polypropylene tubes
  • Beckman GPR refrigerated tabletop centrifuge and GH‐3.7 swinging‐bucket rotor or equivalent
  • Rocker platform (e.g., Nutator, Becton Dickinson Primary Care Diagnostics)
  • Heat‐sealed Pasteur pipet
  • Wide‐bore pipet tips (Bio‐Rad)
  • Additional reagents and equipment for tissue culture ( appendix 3G), spectrophotometric quantitation of DNA ( appendix 3D), agarose gel electrophoresis and Southern blot hydridization (unit 2.7), and calcium phosphate transfection of cells (see protocol 2, steps to )
NOTE: All solutions and equipment coming into contact with cells must be sterile, and proper sterile technique should be used accordingly.NOTE: All culture incubations are performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Akkaraju, G.R., Huard, J., Hoffman, E.P., Goins, W.F., Pruchnic, R., Watkins, S.C., Cohen, J.B., and Glorioso, J.C. 1999. Herpes simplex virus vector‐mediated dystrophin gene transfer and expression in MDX mouse skeletal muscle. J. Gene Med. 1:280‐289.
   Alvira, M.R., Cohen, J.B., Goins, W.F., and Glorioso, J.C. 1999. Genetic studies exposing the splicing events involved in HSV‐1 latency associated transcript (LAT) production during lytic and latent infection. J. Virol. 73:3866‐3876.
   Batchelor, A.H. and O'Hare, P.O. 1990. Regulation and cell‐type‐specific activity of a promoter located upstream of the latency‐associated transcript of herpes simplex virus type 1. J. Virol. 64:3269‐3279.
   Batchelor, A.H. and O'Hare, P.O. 1992. Localization of cis‐acting sequence requirements in the promoter of the latency‐associated transcript of herpes simplex virus type 1 required for cell‐type‐specific activity. J. Virol. 66:3573‐3582.
   Chen, X., Schmidt, M.C., Goins, W.F., and Glorioso, J.C. 1995. Two herpes simplex virus type‐1 latency‐active promoters differ in their contribution to latency‐associated transcript expression during lytic and latent infection. J. Virol. 69:7899‐7908.
   Croen, K.D., Ostrove, J.M., Dragovic, L.J., Smialek, J.E., and Straus, S.E. 1987. Latent herpes simplex virus in human trigeminal ganglia. Detection of an immediate early gene “anti‐sense” transcript by in situ hybridization. New Engl. J. Med. 317:1427‐1432.
   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.
   Devi‐Rao, G.B., Goddart, S.A., Hecht, L.M., Rochford, R., Rice, M.K., and Wagner, E.K. 1991. Relationship between polyadenylated and nonpolyadenylated, HSV type 1 latency‐associated transcripts. J. Gen. Virol. 65:2179‐2190.
   Dobson, A.T., Sederati, F., Devi‐Rao, G., Flanagan, W.M., Farrell, M.J., Stevens, J.G., Wagner, E.K., and Feldman, L.T. 1989. Identification of the latency‐associated transcript promoter by expression of rabbit β‐globin mRNA in mouse sensory nerve ganglia latently infected with a recombinant herpes simplex virus. J. Virol. 63:3844‐3851.
   During, M.J., Naegele, J., O' Malley, K.L., and Geller, A.I. 1994. Long‐term behavioral recovery in Parkinsonian rats by an HSV vector expressing tyrosine hydroxylase. Science 266:1399‐1403.
   Evans, C., Goins, W.F., Schmidt, M.C., Robbins, P.D., Ghivizzani, S.C., Oligino, T., Marconi, P., Krisky, D., and Glorioso, J.C. 1997. Progress in development of herpes simplex virus gene vectors for treatment of rheumatoid arthritis. Adv. Drug Delivery Rev. 27:41‐57.
   Farrell, M.J., Dobson, A.T., and Feldman, L.T. 1991. Herpes simplex virus latency‐associated transcript is a stable intron. Proc. Natl. Acad. Sci. U.S.A. 88:790‐794.
   Fink, D.J., Sternberg, L.R., Weber, P.C., Mata, M., Goins, W.F., and Glorioso, J.C. 1992. In vivo expression of β‐galactosidase in hippocampal neurons by HSV‐mediated gene transfer. Hum.Gene Ther. 3:11‐19.
   Fraser, N.W., Lawrence, W.C., Wroblewska, Z., Gilden, D.H., and Koprowski, H. 1981. Herpes simplex virus type 1 DNA in human brain tissue. Proc. Natl. Acad. Sci. U.S.A. 78:6461‐6465.
   French, S.W., Schmidt, M.C., and Glorioso, J.C. 1996. Involvement of an HMG protein in the transcriptional activity of the herpes simplex virus latency active promoter 2. Mol. Cell. Biol. 16:5393‐5399.
   Frye, R.A., Benz, C.C., and Liu, E. 1989. Detection of amplified oncogenes by differential polymerase chain reaction. Oncogene 4:1153‐1157.
   Gage, P.J., Sauer, B., Levine, M., and Glorioso, J.C. 1992. A cell‐free recombination system for site‐specific integration of multigenic shuttle plasmids into the herpes simplex virus type 1 genome. J. Virol. 66:5509‐5515.
   Geller, A.I. and Breakefield, X.O. 1988. A defective HSV‐1 vector expresses Escherichia coli β‐galactosidase in cultured peripheral neurons. Science 241:1667‐1669.
   Geller, A.I. and Freese, A. 1990. Infection of cultured central nervous system neurons with a defective herpes simplex virus 1 vector results in stable expression of Escherichia coli β‐galactosidase. Proc. Natl. Acad. Sci. U.S.A. 87:1149‐1153.
   Goins, W.F., Sternberg, L.R., Croen, K.D., Krause, P.R., Hendricks, R.L., Fink, D.J., Straus, S.E., Levine, M., and Glorioso, J.C. 1994. A novel latency‐active promoter is contained within the herpes simplex virus type 1 UV4 flanking repeats. J. Virol. 68:2239‐2252.
   Goins, W.F., Lee, K.A., Cavalcoli, J.D., O'Malley, M.E., DeKosky, S.T., Fink, D.J., and Glorioso, J.C. 1999. Herpes simplex virus type 1 vector‐mediated expression of nerve growth factor protects dorsal root ganglia neurons for peroxide toxicity. J. Virol. 73:519‐532.
   Goins, W.F., Yoshimura, N., Ozawa, H., Yokoyama, T., Phelan, M., Bennet, N., de Groat, W.C., Glorioso, J.C., and Chancellor, M.B. 2001. Herpes simplex virus vector‐mediated nerve growth factor expression in bladder and afferent neurons: potential treatment for diabetic bladder dysfunction. J. Urol. 165:1748‐1754.
   Graham, F.L. and van der Eb, A.J. 1973. A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology 52:456‐467.
   Huard, J., Akkaraju, G.R., Watkins, S.C., Pike‐Cavalcoli, M., and Glorioso, J.C. 1997. LacZ gene transfer to skeletal muscle using a replicatio‐defective herpes simplex virus type 1 mutant vector. Hum. Gene Ther. 8:439‐452.
   Kaplitt, M.G., Kwong, A.D., Kleopoulos, S.P., Mobbs, C.V., Rabkin, S.D., and Pfaff, D.W. 1994. Preproenkephalin promoter yields region‐specific and long‐term expression in adult brain after direct in vivo gene transfer via a defective herpes simplex viral vector. Proc. Natl. Acad. Sci. U.S.A. 91:8979‐8983.
   Krause, P.R., Croen, K.D., Straus, S.E., and Ostrove, J.M. 1988. Detection and preliminary characterization of herpes simplex virus type transcripts in latently infected human trigeminal ganglia. J. Virol. 62:4819‐4823.
   Krisky, D.M., Wolfe, D., Goins, W.F., Marconi, P.C., Ramakrishman, R., Mata, M., Rouse, R.J.D., Fink, D.J., and Glorioso, J.C. 1998a. Deletion of multiple immediate‐early genes from herpes simplex virus reduces cytotoxicity and permits long‐term gene expression in neurons. Gene Ther. 5:1593‐1603.
   Krisky, D.M., Marconi, P.C., Oligino, T.J., Rouse, R.J.D., Fink, D.J., Cohen, J.B., Watkins, S.C., and Glorioso, J.C. 1998b. Development of herpes simplex virus replication‐defective multigene vectors for combination gene therapy applications. Gene Ther. 5:1517‐1530.
   Krummenacher, C., Zabolotny, J., and Fraser, N. 1997. Selection of a nonconsensus branch point is influenced by an RNA stem‐loop structure and is important to confer stability to the herpes simplex virus 2‐kilobase latency‐associated transcript. J.Virol. 71:5849‐5860.
   Marconi, P., Krisky, D., Oligino, T., Poliani, L., Ramakrishman, R., Goins, W.F., Fink, D.J., and Glorioso, J.C. 1996. Replication‐defective HSVvectors for gene transfer in vivo. Proc. Natl. Acad. Sci. U.S.A. 93:11319‐11320.
   Markert, J., Medlock, M., Rabkin, S., Gillespie, G., Todo, T., Hunter, W., Palmer, C., Feigenbaum, F., Tornatore, C., Tufaro, F., and Martuza, R. 2000. Conditionally replicating herpes simplex virus mutant, G207 for the treatment of malignant glioma: results of a phase I trial. Gene Ther. 7:867‐874.
   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.
   Nicosia, M., Deshmane, S.L., Zabolotny, J.M., Valyi‐Nagy, T., and Fraser, N.W. 1993. Herpes simplex virus type 1 Latency‐Associated Transcript (LAT) promoter deletion mutants can express a 2‐kilobase transcript mapping to the LAT region. J. Virol. 67:7276‐7283.
   Oligino, T., Ghivizzani, S., Wolfe, D., Lechman, E., Krisky, D., Mi, Z., Evans, C., Robbins, P., and Glorioso, J.C. 1999. Intra‐articular delivery of a herpes simplex virus IL‐1 Ra gene vectors reduces inflammation in a rabbit model of arthritis. Gene Ther. 6:1713‐1720.
   Ramakrishnan, R., Fink, D.J., Jiang, G., Desai, P., Glorioso, J.C., and Levine, M. 1994a. Competitive quantitative polymerase chain reaction (PCR) analysis of herpes simplex virus type 1 DNA and LAT RNA in latently infected cells of brain. J. Virol. 68:1864‐1870.
   Ramakrishnan, R., Levine, M., and Fink, D.J. 1994b. A PCR‐based analysis of herpes simplex virus type 1 latency in the rat trigeminal ganglion established with a ribonucleotide reductase‐deficient mutant. J. Virol. 68:7083‐7091.
   Ramakrishnan, R., Poliani, P.L., Levine, M., Glorioso, J.C., and Fink, D.J. 1996. Detection of herpes simplex virus type 1 latency‐associated transcript expression in trigeminal ganglion by an in situ reverse transcriptase PCR. J. Virol. In press.
   Rampling, R., Cruickshank, G., Papanastassiou, V., Nicoll, J., Hadley, D., Brennan, D., Petty, R., MacLean, A., Harland, J., McKie, E., Mabbs, R., and Brown, M. 2000. Toxicity evaluation of replication‐competent herpes simplex virus (ICP 34.5 null mutant 1716) in patients with recurrent malignant glioma. Gene Ther. 7:859‐866.
   Rasty, S., Goins, W.F., and Glorioso, J.C. 1995. Site‐specific integration of multigenic shuttle plasmids into the herpes simplex virus type 1 genome using a cell‐free Cre‐lox recombination system. Methods Mol.Genet. 7:114‐130.
   Samaniego, L. Webb, A., and DeLuca, N. 1995. Functional interaction between herpes simplex virus immediate‐early proteins during infection: Gene expression as a consequence of ICP27 and different domains of. ICP4. J. Virol. 69:5705‐5715.
   Samaniego, L., Wu, N., and DeLuca, N.A. 1997. The herpes simplex virus immediate‐early protein ICP0 affects transcription from the viral genome and infected ‐cell survival in the absence of ICP4 and ICP27. J. Virol. 71 4614‐4625.
   Samaniego, L.A., Neiderhiser, L., and DeLuca, N.A. 1998. Persistence and expression of the herpes simplex virus genome in the absence of immediate‐early proteins. J. Virol. 72:.3307‐3320.
   Shapira, M., Homa, F.L., Glorioso, J.C., and Levine, M. 1987. Regulation of the herpes simplex virus type 1 late (γ 2) glycoprotein C gene: Sequences between base pairs −34 to +29 control transient expression and responsiveness to transactivation by the products of immediate early (α) 4 and 0 genes. Nucl. Acids Res. 15:3097‐3111.
   Soares, M.K., Hwang, D.‐Y., Ramakrishnan, R., Schmidt, M.C., Fink, D.J., and Glorioso, J.C. 1996. Cis‐acting elements involved in transcriptional regulation of the herpes simplex virus type 1 latency‐associated promoter 1 (LAP1) in vitro and in vivo. J. Virol. In press.
   Southern, E.M. 1975. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98:503‐517.
   Spaete, R.R. and Frankel, N. 1982. The herpes simplex virus amplicon: A new eukaryotic defective‐virus cloning‐amplifying vector. Cell 30:295‐304.
   Spivack, J.G. and Fraser, N.W. 1987. Detection of herpes simplex virus type 1 transcripts during latent infection in mice. J. Virol. 61:3841‐3847.
   Stevens, J.G., Wagner, E.K., Devi‐Rao, G.B., Cook, M.L., and Feldman, L.T. 1987. RNA complementary to a herpes viruses a gene mRNA is prominent in latently infected neurons. Science 255:1056‐1059.
   Stevens, J.G. 1989. Human herpes viruses: A consideration of the latent state. Microbiol. Rev. 53:318‐332.
   Wolfe, D., Goins, W.F., Kaplan, T., Capuano, S., Fradette, J., Murphey‐Corb, M., Cohen, J.B., Robbins, P., and Glorioso, J.C. 2001. Systemic accumulation of biologically active nerve growth factor folowing intra‐articular herpes virus gene transfer. Mol. Ther. 3:61‐69.
   Wu, N., Watkins, S.C., Schaffer, P.A., and DeLuca, N.A. 1996. Prolonged gene expression and cell survival after infection by a herpes simplex virus mutant defective in the immediate‐early genes encoding ICP4, ICP27, and ICP22. J. Virol. 70:6358‐6368.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library