IDENTIFICATION OF TRANSFER-RNAS INCORPORATED INTO WILD-TYPE AND MUTANT HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1

We have identified the tRNAs which are incorporated into both wild-typ e human immunodeficiency virus type 1 strain IIIB (HIV-1IIIB) produced in COS-7 cells transfected with HIV-1 proviral DNA and mutant, noninf ectious HIV-1Lai particles produced in a genetically engineered Vero c ell line. The mutant proviral DNA contains nucleotides 678 to 8944; i. e., both long terminal repeats and the primer binding site are absent. As analyzed by two-dimensional polyacrylamide gel electrophoresis, bo th mutant and wild-type HIV-1 contain four major-abundance tRNA specie s, which include tRNA1/2Lys, tRNA3Lys (the putative primer for HIV-1 r everse transcriptase) and tRNA(Ile). Identification was accomplished b y comparing the electrophoretic mobilities and RNase T1 digests with t hose of tRNA3Lys and tRNA1/2Lys purified from human placenta and compa ring the partial nucleotide sequence at the 3' end of each viral tRNA species with published tRNA sequences. Thus, the absence of the primer binding site in the mutant virus does not affect tRNA(Lys) incorporat ion into HIV-1. However, only the wild-type virus contains tRNA3Lys ti ghtly associated with the viral RNA genome. The identification of the tightly associated tRNA as tRNA3Lys is based upon an electrophoretic m obility identical to that of tRNA3Lys and the ability of this RNA to h ybridize with a tRNA3Lys-specific DNA probe. In addition to the four w ild-type tRNA species, the mutant HIV-1-like particle contains two tRN A(His) species and three tRNA-sized species that we have been unable t o identify. Their absence in wild-type virus makes it unlikely that th ey are required for viral infectivity.