Generation of a recombinant herpes simplex virus type 1 expressing the rat corticotropin- releasing hormone precursor: endoproteolytic processing, intracellular targeting and biological activity.
We describe the generation of a recombinant herpes simplex virus type 1 (HSV1) vector, tsK/CRH10, derived from the temperature-sensitive mutant tsK, expressing rat pre-procorticotropin-releasing hormone (ppCRH). In hypothalamic neurons, within the paraventricular and supraoptic nuclei, this neuropeptide precursor is processed to mature CRH (1-41), the key modulator of the hypothalamic-pituitary-adrenal stress response. We used the recombinant HSV1 tsK/CRH10 to study posttranslational processing, intracellular localization and biological activity of proCRH (pCRH) within neuronal, glial and epithelial cell lines. We showed that CRH-like immunoreactivity expressed in neuronal, glial and epithelial cells infected with tsK/CRH10 was biologically active, could be detected intracellularly and was also secreted. Our data also show that within Neuro2a and NG115 cells, the CRH precursor is cleaved to yield a CRH-like immunoreactive fragment of approximately 4.75 kD which could account for mature CRH (1-41). No endoproteolytic processing of the precursor takes place within the astrocytic 1321 NI cell line. Using immunocytochemistry techniques we detected CRH-like immunoreactivity within the endoplasmic reticulum-Golgi region in all cells and within secretory vesicles of Neuro2a and NG115 cells, suggesting correct targeting to the regulated secretory pathway within these cells. Our results demonstrate that the HSV1 recombinant vector expressing the full-length CRH precursor molecule constitutes an excellent delivery system for both cell lines and postmitotic neurons in vitro, which has enabled the study of targeting, endoproteolytic processing and biological activity of this neuropeptide precursor. Furthermore, it can also be used to generate transient transgenesis of the CRH precursor in vivo, to study neuroendocrine-immune interactions within the mammalian central nervous system.
Tomasec
P
P
Molecular Medicine Unit, Department of Medicine, University of Manchester, Manchester, UK.
Preston
C M
CM
Linton
E A
EA
Ahmed
I
I
Lowenstein
P R
PR
Castro
M G
MG
D000818:Animals; D000906:Antibodies; D001254:Astrocytoma; D001932:Brain Neoplasms; D016466:CHO Cells; D019556:COS Cells; D003001:Cloning, Molecular; D003346:Corticotropin-Releasing Hormone / Q000032:analysis / Q000235:genetics* / Q000276:immunology; D006224:Cricetinae; D017077:Culture Media, Conditioned; D018076:DNA, Complementary; D004847:Epithelial Cells / Q000737:chemistry / Q000502:physiology / Q000821:virology; D015967:Gene Expression Regulation, Viral; D005822:Genetic Vectors; D005910:Glioma; D006561:Herpes Simplex / Q000235:genetics; D018259:Herpesvirus 1, Human / Q000235:genetics*; D006801:Humans; D006822:Hybrid Cells; D051379:Mice; D009447:Neuroblastoma; D009457:Neuroglia / Q000737:chemistry / Q000502:physiology / Q000821:virology; D009474:Neurons / Q000737:chemistry / Q000502:physiology / Q000821:virology; D011401:Promoter Regions, Genetic / Q000502:physiology; D011499:Protein Processing, Post-Translational / Q000502:physiology; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics*; D014407:Tumor Cells, Cultured
D000906:Antibodies; D017077:Culture Media, Conditioned; D018076:DNA, Complementary; D011994:Recombinant Proteins; D003346:Corticotropin-Releasing Hormone