MULTIPLE FORMS OF TRNA(LYS3) IN HIV-1

tRNA(Lys3) is the primer for HIV-1 reverse transcriptase (RT) and is s electively incorporated into HIV-1 during viral assembly. While whole cell extracts of uninfected or infected cells contain only one detecta ble form of tRNA(Lys3), multiple forms of tRNA(Lys3) are detected in t he virus released into the cell culture media. These tRNA(Lys3) isoacc eptors are found in HIV-1 produced from newly infected cord blood lymp hocytes and from cells chronically infected with HIV-1. such as the ly mphocytic cell line H9 and the monocytic cell lines U937 and PLB. They can be detected through the use of either RPC-5 column chromatography of tRNA aminoacylated with radioactive lysine or northern blot analys is using a tRNA(Lys3)-specific DNA hybridization probe. Both RPC-5 chr omatography and northern blot analysis show the cytoplasmic form of tR NA(Lys3) to be the major abundance form of tRNA(Lys3) in the virus. St arting with the viral RNA isolated from HIV (PLB), the tRNA(Lys3) spec ies resolved by RPC-5 into peaks 2, 3, and 4 were deacylated and 3' en d-labeled by heat-annealing the RNA in each peak to synthetic HIV geno mic RNA, and extending the hybridized species one base using HIV-1 RT and radioactive dCTP. An electrophoretic comparison of the partial T1 digest pattern of purified human placental tRNA(Lys3) with those of th e RPC-5 resolved species showed that the labeled RNA species in each p eak was tRNA(Lys3)). These radioactive tRNA(Lys3) species retained the ir relative mobilities when rechromatograped on RPC-5. When total HIV (PLB) RNA was used as the source of primer/template, and similarly ext ended with RT in the presence of radioactive dCTP, the major priming t RNA resolved by RPC-5 bad a chromatographic mobility identical to peak 3. This tRNA primer has a T1 digest pattern identifying it as tRNA(Ly s3). These results indicate that the major tRNA(Lys3) species present in the virus is also the major tRNA(Lys3) isoacceptor used as the prim er for reverse transcription. (C) 1996 Academic Press, Inc.