The top schematic diagram shows HIV-1-derived Gag-PolD64V, a lentiviral packaging vector encoding Gag (matrix [MA], capsid [CA], nucleocapsid [NC], and p6 domain), and the alternative reading frame encoding the enzymatic components, such as protease (PR), reverse transcriptase (RT), and an inactivated integrase (INTD64V) as reference. The initial ENLVPE design is based on a codon-optimized Gag with L21S, which improves membrane-targeting, combined with the grafting of a PP7 coat protein (PCP) into the second zinc-finger motif within the NC domain to enable binding to PP7-aptamer-tagged guide RNAs. The first zinc-finger motif was inactivated by C>S mutations. The bottom illustration shows the additional insertions of a concatenation of nuclear localization (NLS) and export sequences (NES) to facilitate Gag shuttling between the nucleus and cytosol.
