FIG. 2.

Structure of the HIV-1 proviral genome and the third-generation lentiviral vector system. (A) The HIV-1 proviral genome contains nine genes and several cis-acting sequences. (B) To reduce the risk of generating replication-competent viruses, essential viral genes are provided in trans and separated onto three helper plasmids, which prevents them being incorporated into the vector genome. Third-generation lentiviral vectors are generated with four plasmids: the packaging plasmid, envelope plasmid, Rev plasmid, and transfer plasmid. (C) The transfer plasmid, which carries the viral vector genome, contains only essential cis-acting viral sequences. In the example shown the vector would encode a CMV-driven transgene and an H1-driven shRNA cassette. In this plasmid the 5′ LTR U3 region is replaced by a heterologous promoter such as the RSV promoter, whereas on the 3′ LTR the U3 region bears a deletion that removes transcriptional activity; after reverse transcription in the transduced cell the LTRs on the double-stranded DNA viral genome have the structure of the plasmid 3′ LTR. CMV, cytomegalovirus promoter; cPPT, central polypurine tract; cTS, central termination sequence; dLTR, long terminal repeat from which 400 bp in the U3 region has been deleted; GLS, gag leader sequence; ψ, encapsidation sequence; LTR, long terminal repeat; Poly-A, polyadenylylation signal; RRE, Rev response element; RSV, Rous sarcoma virus promoter; shRNA, short hairpin RNA; WPRE, woodchuck hepatitis virus posttranscriptional regulatory element. Color images available online at www.liebertpub.com/hgtb