Construction and characterization of bacterial artificial chromosomes containing HSV-1 strains 17 and KOS.

Bacterial artificial chromosomes (BACs) were constructed containing full-length, infectious DNA of HSV-1 strains 17 and KOS. To generate BACs without altering viral genes, sequences required for selection and propagation of the BAC were placed between the U(L)37 and U(L)38 genes, and flanked by LoxP sites. The system was tested by studying multiple properties of these HSV-1 BAC constructs in vitro and in vivo following propagation in bacteria, virus reconstitution from HSV-BAC DNA in eukaryotic cells, and Cre-recombinase-mediated excision of the BAC backbone. Based on in vitro growth in mouse embryo fibroblasts and in vivo growth in mouse corneas and trigeminal ganglia, the strain KOS BAC-derived virus behaved similarly to wild-type. Small changes in neurovirulence were, however, observed. The strain 17 BAC-derived virus exhibited modest decreases in growth and virulence compared to wild-type. Modest differences were observed in reactivation from latency with both strain KOS and 17 BAC-derived viruses. In addition, the system was further validated by performing mutagenesis of the BACs by allelic exchange in E. coli. These BACs are suitable for the rapid generation of recombinant viruses for pathogenesis and other studies, but as with all mutagenesis systems, care must be taken in their construction and repair.

Neuronal expression and regulation of CGRP promoter activity following viral gene transfer into cultured trigeminal ganglia neurons.

We have examined the regulation of calcitonin gene-related peptide (CGRP) promoter activity in primary cultures of rat trigeminal ganglia neurons. A viral vector was used to circumvent the potential complication of examining only a small subpopulation of cells in the heterogeneous cultures. Infection with high titers of recombinant adenovirus containing 1.25 kb of the rat CGRP promoter linked to the beta-galactosidase reporter gene (AdCGRP-lacZ) yielded expression in about 50% of the CGRP-expressing neurons. The CGRP-lacZ reporter gene was preferentially expressed in neurons, with 91% co-expression with endogenous CGRP. In contrast, an adenoviral vector containing a CMV-lacZ reporter was predominantly expressed in non-neuronal cells, with only 29% co-expression with CGRP. We then asked whether the CGRP promoter in the viral vector could be regulated by serotonin receptor type 1 (5-HT(1)) agonists. Promoter activity was decreased two- to threefold by treatment with five 5-HT(1B/D) agonists, including the triptan drugs sumatriptan, eletriptan, and rizatriptan that are used for migraine treatment. As controls, CMV promoter activity was not affected, and 5-HT(1B/D) receptor antagonists blocked the repression caused by sumatriptan and eletriptan. Thus, adenoviral gene transfer can be used in trigeminal ganglia neurons for studying the mechanisms of triptan drug action on CGRP synthesis.