Development of herpes simplex virus type-1 as an efficient vector for foreign gene expression in the nervous system

Qiu Sue Huang, University of Pennsylvania


A simple, rapid and highly efficient method was developed for introducing specific DNA sequences into a defined locus of the herpes simplex virus type 1 (HSV-1) genome by restriction enzyme cleavage and ligation. The HSV-1 strain HFEM does not contain any PacI site, thus two unique PacI sites flanking an HSV-1 ICP6 promoter-LacZ cassette were engineered into the latency-associated transcript (LAT) region to generate a recombinant virus HFEM/ICP6-LacZ which produced blue plaques in the presence of X-gal. HFEM/ICP6-LacZ was then used to test the in vitro ligation and transfection system by digesting HFEM/ICP6-LacZ with PacI and SwaI (an endogenous unique site upstream of the LAT promoter locus) and inserting a PacI-SwaI fragment containing LAT promoter-LacZ cassette into HFEM/ICP6-LacZ genome. The new recombinant virus HFEM/LAT-LacZ was selected as white plaques in the presence of X-gal since $\beta$-galactosidase expression, when driven by the LAT promoter, is not detectable in tissue culture (Ho et al., 1989). The high yield (approximately 100%) of the recombinant virus obtainable from this system coupled with a blue-white detection scheme makes this a powerful method for constructing HSV-1 vectors. The life-long expression of the latency-associated transcripts (LATs) in neurons latently infected with HSV-1 has been tempting researchers to explore the feasibility of LAT promoter-driven foreign gene expression for neuronal gene therapy. In order to understand the mechanism involved in regulation of LAT-promoter driven gene expression, in situ hybridization and histochemical staining assays were employed in trigeminal ganglia (TGs) acutely and latently infected with HFEM/LAT-lacZ, in which the LacZ reporter gene was placed downstream of the LAT promoter at the LAT locus. It was found that there were 89 fold more cells positive for $\beta$-gal transcript than cells positive for $\beta$-gal enzyme in acutely infected TGs and 10 fold difference in latently-infected TGs. Thus, there was a big discordance between LacZ mRNA and enzyme expression in TGs of HFEM/LAT-lacZ. It suggested that a block between transcription and translation may cause inefficient translation of $\beta$-galactosidase.

Subject Area

Cellular biology|Molecular biology|Microbiology|Neurology

Recommended Citation

Huang, Qiu Sue, "Development of herpes simplex virus type-1 as an efficient vector for foreign gene expression in the nervous system" (1995). Dissertations available from ProQuest. AAI9543090.