EFFECT OF MUTATIONS IN THE NUCLEOCAPSID PROTEIN (NCP7) UPON PR160(GAG-POL) AND TRNA(LYS) INCORPORATION INTO HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1

COS-7 cells were transfected with DNAs containing mutations in the NCp 7 sequences of human immunodeficiency virus. Selective incorporation i nto the virus of tRNA(Lys) was measured by two-dimensional polyacrylam ide gel electrophoresis, and Pr160(gag-pol) incorporation into the vir us was detected in Western blots of viral protein. Mutations tested in cluded cysteine and histidine mutations in either of the Cys-His boxes , as well as mutations in the N- and C-terminal flanking regions and i n the linker region between the two Cys-His boxes. Of 10 mutations tes ted, 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 (Delta K14-T50). The P31L mutation prevents the incorpor ation of Pr160(gag-pol) into the virus, Cotransfection of COS cells wi th both P31L DNA and a plasmid coding only for unprocessed Pr160(gag-p ol) resulted in the viral incorporation of Pr160(gag-pol) and the resc ue of selective packaging of tRNA(Lys) into the virion. In the Delta K 14-T50 mutant, Pr160(gag-pol) is incorporated into the virus. Selectiv e tRNA(Lys) packaging is not rescued by cotransfection with a plasmid coding for Pr160(gag-pol) but is rescued by cotransfection with DNA co ding for wild-type Pr55(gag). Since pr55(gag) does not by itself selec tively package tRNA(Lys), the Delta K14-T50 mutation may be affecting tRNA(Lys) binding to a cytoplasmic Pr55(gag)/Pr160(gag-pol) complex.