Bel-independent HFV vector system.

The structure of the wild-type (wt) HFV genome and relevant portions of packaging plasmid pCGPES and vector plasmids pCGPMAPΔBel and pCGPMscvF are shown. Arrows below each map represent transcription start sites. The bel1 gene in wt HFV is transcribed initially from the internal promoter, after which the Bel1 protein transactivates both the wt HFV LTR and internal promoters.18-20 In the Bel-independent plasmid constructs, cytomegalovirus (CMV) promoter sequences replace the 5′ HFV LTR U3 region, to generate a constitutive CMV/HFV fusion promoter.25 The packaging plasmid pCPGES expressesgag, pol, and env followed by an SV40 polyadenylation site (SV pA). The vector plasmids express AP or GFP reporter genes from internal MLV LTR or murine stem cell virus (MSCV) promoters, respectively. After reverse transcription, both LTRs in the vector provirus are derived from Bel-1–dependent HFV sequences so thegag and pol genes contained in vector genomes are not expressed in transduced cells. Recent studies show that only the most 5′ portion of gag and 3′ portion of pol are required in cis for vector production42 43 (our unpublished results, July 1998), so future vectors may contain significantly fewer viral sequences. The locations of relevant restriction sites, PCR primers, and the probe fragment used for Southern blot analysis are shown above the pCGPMAPΔBel map.