COS-7 cells were transfected with DNAs containing mutations in the NCp7 sequences of human immunodeficiency virus. Selective incorporation into the virus of tRNA Lys was measured by two-dimensional polyacrylamide gel electrophoresis, and Pr160 gag-pol incorporation into the virus was detected in Western blots of viral protein. Mutations tested included cysteine and histidine mutations in either of the Cys-His boxes, as well as mutations in the N-and C-terminal flanking regions and in the linker

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... region between the two Cys-His boxes. Of 10 mutations tested, only 2 inhibited tRNA Lys incorporation: a P31L mutation in the linker region and a deletion which removed both Cys-His boxes and the linker region (⌬K14-T50). The P31L mutation prevents the incorporation of Pr160 gag-pol into the virus. Cotransfection of COS cells with both P31L DNA and a plasmid coding only for unprocessed Pr160 gag-pol resulted in the viral incorporation of Pr160 gag-pol and the rescue of selective packaging of tRNA Lys into the virion. In the ⌬K14-T50 mutant, Pr160 gag-pol is incorporated into the virus. Selective tRNA Lys packaging is not rescued by cotransfection with a plasmid coding for Pr160 gag-pol but is rescued by cotransfection with DNA coding for wild-type Pr55 gag . Since Pr55 gag does not by itself selectively package tRNA Lys , the ⌬K14-T50 mutation may be affecting tRNA Lys binding to a cytoplasmic Pr55 gag /Pr160 gag-pol complex.