J . Virol . and Lawrence Kleiman Juliana Gabor , Shan Cen , Hassan Javanbakht , Meijuan Niu Incorporation , and Viral Infectivity upon Its Annealing to Viral RNA , GagPol Human Immunodeficiency Virus

Human immunodeficiency virus type 1 (HIV-1) uses tRNA 3 Lys as a primer for reverse transcription and, during viral assembly, this tRNA is selectively packaged into the virus along with the other major tRNA Lys , tRNA 1,2 Lys. Increasing the cytoplasmic concentration of tRNA 3 Lys through transfection of cells with a plasmid containing both HIV-1 proviral DNA and a tRNA 3 Lys gene results in a greater incorporation of tRNA 3 Lys into virions, which is accompanied by increased annealing of tRNA 3 Lys to the viral genome and increased infectivity of the viral population. Increased viral tRNA 3 Lys is accompanied by decreased viral tRNA 1,2 Lys , with the total tRNA Lys /virion and the GagPol/Gag ratios remaining unchanged. Viral tRNA Lys can be doubled, with increases in both tRNA 3 Lys and tRNA 1,2 Lys concentrations, by overexpressing lysyl tRNA synthetase. This also results in increased tRNA 3 Lys annealing to the viral RNA and increased viral infectivity but, again, no change in the GagPol/Gag ratio was observed. This result indicates that GagPol, whose interaction is required during packaging, is not a limiting factor during tRNA Lys incorporation into HIV-1, whereas LysRS is. During retroviral assembly, particular species of cellular tRNA are selectively packaged into the virus, where they are placed onto the primer binding site (PBS) of the viral genome and are used to initiate the reverse transcriptase (RT)-cata-lyzed synthesis of minus-strand cDNA. The primer tRNA for members of the avian sarcoma and leukosis virus group is tRNA Trp (1, 4, 16, 19, 23, 24), and it is tRNA Pro for murine leukemia virus (MuLV) (3, 15, 21). In mammalian cells, there are three major tRNA Lys isoacceptors (18). tRNA 1,2 Lys , representing two tRNA Lys isoacceptors differing by 1 bp in the anticodon stem, is the primer tRNA for several mammalian retroviruses, including Mason-Pfizer monkey virus and human foamy virus, while tRNA 3 Lys is the primer tRNA for lentivi-ruses, including human immunodeficiency virus type 1 (HIV-1) (10). Selective packaging of primer tRNA is defined as an increase in the percentage of the low-molecular-weight RNA population representing primer tRNA in moving from the cy-toplasm to the virus. For example, in avian myeloblastosis virus, the relative concentration of tRNA Trp changes from 1.4% in the cytoplasm to 32% in the virus (23). In HIV-1 produced from COS7 cells transfected with HIV-1 proviral DNA, …