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 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 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 (deltaK14-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 deltaK14-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 deltaK14-T50 mutation may be affecting tRNA(Lys) binding to a cytoplasmic Pr55(gag)/Pr160(gag-pol) complex.
Huang
Y
Y
Jewish General Hospital, and Department of Microbiology and Immunology, McGill University, Montreal, Quebec, Canada.
Khorchid
A
A
Wang
J
J
Parniak
M A
MA
Darlix
J L
JL
Wainberg
M A
MA
Kleiman
L
L
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002213:Capsid / Q000502:physiology*; D036022:Capsid Proteins; D002522:Chlorocebus aethiops; D015180:Electrophoresis, Gel, Two-Dimensional; D015683:Gene Products, gag / Q000378:metabolism* / Q000502:physiology*; D015497:HIV-1 / Q000737:chemistry / Q000254:growth & development* / Q000648:ultrastructure; D008969:Molecular Sequence Data; D016296:Mutagenesis; D019251:Nucleocapsid / Q000502:physiology*; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D011498:Protein Precursors / Q000378:metabolism*; D012357:RNA, Transfer, Lys / Q000378:metabolism*; D013329:Structure-Activity Relationship; D014764:Viral Proteins; D014771:Virion / Q000648:ultrastructure*; D014779:Virus Replication; D054301:gag Gene Products, Human Immunodeficiency Virus; D054302:pol Gene Products, Human Immunodeficiency Virus
D036022:Capsid Proteins; D015683:Gene Products, gag; C071884:NCP7 protein, Human immunodeficiency virus 1; D011498:Protein Precursors; D012357:RNA, Transfer, Lys; D014764:Viral Proteins; D054301:gag Gene Products, Human Immunodeficiency Virus; D054302:pol Gene Products, Human Immunodeficiency Virus; C091950:pr160 gag-pol precursor protein, Human immunodeficiency virus 1; D010749:Phosphoprotein Phosphatases