ROLE OF PR160(GAG-POL) IN MEDIATING THE SELECTIVE INCORPORATION OF TRNA(LYS) INTO HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 PARTICLES

COS-7 cells transfected with human immunodeficiency virus type 1 (HIV- 1) proviral DNA produce virus in which three tRNA species are most abu ndant in the viral tRNA population. These tRNAs have been identified t hrough RNA sequencing techniques as tRNA(3)(Lys) the primer tRNA in HI V-1, and members of the tRNA(1,2)(Lys) isoacceptor family. These RNAs represent 60% of the low-molecular-weight RNA isolated from virus part icles, while they represent only 6% of the low-molecular-weight RNA is olated from the COS cell cytoplasm. Thus, tRNA(Lys) is selectively inc orporated into HIV-1 particles. We have measured the ratio of tRNA(3)( Lys) molecules to copies of genomic RNA in viral RNA samples and have calculated that HIV-1 contains approximately eight molecules of tRNA(3 )(Lys) per two copies of genomic RNA. We have also obtained evidence t hat the Pr160(gag-pol) precursor is involved in primer tRNA(3)(Lys) in corporation into virus. First, selective tRNA(Lys) incorporation and w ild-type amounts of tRNA(3)(Lys) were maintained in a protease-negativ e virus unable to process Pr55(gag) and Pr160(gag-pol) precursors, ind icating that precursor processing was not required for primer tRNA inc orporation. Second, viral particles containing only unprocessed Pr55(g ag) protein did not selectively incorporate tRNA(Lys), while virions c ontaining both unprocessed Pr55(gag), and Pr160(gag-pol) proteins demo nstrated select tRNA(3)(Lys) packaging. Third, studies with a proviral mutant containing a deletion of most of the reverse transcriptase seq uences and approximately one-third of the integrase sequence in the Pr 160(gag-pol) precursor resulted in the loss of selective tRNA incorpor ation and an eightfold decrease in the amount of tRNA(3)(Lys) per two copies of genomic RNA. We have also confirmed herein finding of a prev ious study which indicated that the primer binding site is not require d for the selective incorporation of tRNA(Lys).