FIG. 1.

Northern analysis of the RNA expression from lentivirus vectors. Three pHR2 vectors carrying an expression cassette for the same transgene (truncated low-affinity NGFR) and driven by three different promoters (PGK, CMV, and retroviral MFG) were analyzed in producer and transduced cells. Total RNA was extracted and analyzed by Northern blotting with a probe specific for the transgene sequence. (A) Schematic of the vector construct depicts the species of RNA driven by the internal promoter (Prom.; broken arrow, shorter transcript) and the viral LTR (solid arrows, longer transcripts; the two species differ for the splicing of the viral intron). The splice donor and acceptor sites (SD and SA), the packaging sequence (Ψ), the truncated gag sequence (GA), and the RRE are indicated. (B) The vector constructs were transfected in 293T cells without or with the packaging construct. (C) Vector particles produced by the 293T transfectants were used to transduce HeLa cells. In the absence of the viral transactivators, supplied by the core packaging construct only in the producer cells, vector expression occurs mainly from the internal promoter. Note the dramatic enhancement of the upstream transcription and the accumulation of unspliced RNA (carrying the Ψ sequence) in the presence of the packaging construct. In the transduced cells, the LTR is silenced. Note that the three expression cassettes differ in the size of the promoters and 5′ untranslated sequence. In each case, the smallest RNA species represents transcripts initiated from the internal promoter, while the intermediate-size and larger species correspond to spliced and unspliced LTR-driven RNAs, respectively.