A defective HSV-1 vector expresses Escherichia coli beta-galactosidase in cultured peripheral neurons

Abstract

A defective herpes simplex virus 1 (HSV-1) vector, pHSVlac, has been developed that contains a transcription unit that places the Escherichia coli lacZ gene under the control of the HSV-1 immediate early 4/5 promoter. The vector pHSVlac was propagated with the HSV-1 temperature-sensitive mutant ts K as helper virus. Infection of neurons from rat superior cervical ganglia and dorsal root ganglia in primary culture resulted in stable expression of high levels of beta-galactosidase without cell death. These HSV-1 vectors should be useful for introducing genes into postmitotic cells, such as neurons, in vitro and in vivo.

Fig. 1

The structure of pHSVlac. The clear region contains the HSV-1 a segment, nucleotides 127 to 1132, the packaging site (). The crosshatched region symbolizes the HSV-1 c region, nucleotides 47 to 1066 (). pHSVlac was constructed () from pCH110 ().

Fig. 2

Expression of β-galactosidase from pHSVlac in cells from dorsal root ganglia (A to C) and superior cervical ganglia (D to F) in primary culture. Virus stock containing pHSVlac was prepared () with HSV-1 strain 17 ts K () as helper virus. The titer of the virus stock was 1 × 10⁶ plaque-forming units (PFU) of ts K per milliter and 8 × 10⁵ infectious particles of pHSVlac per milliter. Dissociated cell cultures () were prepared from newborn rat dorsal root ganglia or superior cervical ganglia and treated for 24 hours with 10⁻⁵M cytosine arabinoside. After 10 days in vitro, the cultures contained 3 to 8 × 10⁵ cells per 35-mm plate; cultures were then infected with 0.1 ml of pHSVlac virus stock and incubated for 24 hours at 37°C. Cells were fixed with 0.5% glutaraldehyde and stained for β-galactosidase activity with X-gal (). The width of the photomicrograph represents 230 μm.

Fig. 3

Immunofluorescent colocalization of β-gal-IR and either A2B5-IR or Nf-IR in cultured sensory neurons infected with pHSVlac. pHSVlac virus stock and cultures of dorsal root ganglia were prepared as described in Fig. 2 except that cultures were prepared on 13-mm glass cover slips coated with 0.8 μg of laminin. Cultures (5 × 10⁴ cells in 0.5 ml) were infected with 0.1 ml of pHSVlac virus stock and incubated for 24 hours at 37°C. Fixation with 4% paraformaldehyde in 0.1M NaPO₄ (pH 7.0) and immunohistochernistry were done () with a rabbit antibody to E. coli β-galactosidase (Cooper Biomedical, Malvern, Pennsylvania) diluted 1:800 and either mouse monoclonal to rat neurofilament () (SMI 33, Sternberger-Meyer) diluted 1:800 or mouse monoclonal A2B5 () supernatant diluted 1:2 as primary antibodies. Fluorescein isothiocyanate–conjugated goat F(ab′)₂ (fragment-antigen) antibody to mouse F(ab′)₂ (Cooper Biomedical) diluted 1:200 and rhodamine isothiocyanate–conjugated goat F(ab′)₂ antibody to rabbit F(ab′)a diluted 1:250 (Cooper Biomedical) were used as secondary antibodies. Cover slips were mounted in phosphate-buffered saline and glycerol (1:1) containing 0.4% n-propyl gallate. The width of the photomicrograph represents 438 μm. (A) A2B5-IR. (B) β-Gal-IR. (C) Phase-contrast photomicrograph of the same field. (D) Nf-IR. (E) β-Gal-IR. (F) Phase-contrast photomicrograph of the same field. (G) Preimmune mouse serum, fluorescein isothiocyanate–conjugated second antibody. (H) Preimmune rabbit serum, rhodamine isothiocyanate–conjugated second antibody. (I) Phase-contrast photomicrograph of the same field.

Fig. 4

Stable expression of β-galactosidase (A to C) and persistence of pHSVlac DNA (D) in cultured sensory neurons for 2 weeks. Cultures () of dorsal root ganglia (9 × 10⁴ cells in 1.5 ml) were infected with 0.05 ml pHSVlac virus stock and incubated for 2 weeks at 37°C. Cultures were assayed for β-galactosidase activity as described in Fig. 2. Alternatively, cultures were infected with 5 × 10⁵ PFU of ts K () and incubated for 2 days at 31°C. The resulting virus stock was passaged three times on 2 × 10⁶ CV1 monkey fibroblasts at 31°C to yield virus stocks DRG1 and DRG2. CV1 cells (1 × 10⁷) were infected with 5 × 10⁷ PFU of virus stock (DRG1, DRG2, ts K alone, or mock-infected) and incubated at 31°C for 24 hours. Total cellular DNA (5 μg) was prepared () or 2 × 10⁻⁴ μg of pHSVlac DNA isolated from E. coli (Stds) was digested with Eco RI, resolved on 0.7% agarose gels, and transferred to Genetran (Fiasco) (). Hybridization was performed with the 5.9-kb Eco RI fragment from the plasmid pCH110 () radiolabeled with ³²P (). This 5.9-kb fragment contains pBR sequences and most of the lacZ gene, lacking only 133 bp at the 3′ end (). pHSVlac contains three Eco RI sites (), one at each end of the pBR segment and a third in the lacZ gene 133 bp from the 3′ end of the gene. The 4.3-kb band contains most of the transcription unit in pHSVlac, and the 2.3-kb band contains the pBR sequences. The 1.5-kb fragment of pHSVlac contains the 3′ end of the lacZ gene, the SV40 early region polyadenylation site, and the a sequence; it is not homologous to the probe.