Multiple forms of tRNA(Lys3) in HIV-1.
tRNALys3 is the primer for HIV-1 reverse transcriptase (RI) and is selectively incorporated into HIV-1 during viral assembly. While whole cell extracts of uninfected or infected cells contain only one detectable form of tRNALys3, multiple forms of tRNALys3 are detected in the virus released into the cell culture media. These tRNALys3 isoacceptors are found in HIV-1 produced from newly infected cord blood lymphocytes and from cells chronically infected with HIV-1, such as the lymphocytic 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 analysis using a tRNALys3-specific DNA hybridization probe. Both RPC-5 chromatography and northern blot analysis show the cytoplasmic form of tRNALys3 to be the major abundance form of tRNALys3 in the virus. Starting with the viral RNA isolated from HIV (PLB), the tRNALys3 species resolved by RPC-5 into peaks 2, 3, and 4 were deacylated and 3' end-labeled by heat-annealing the RNA in each peak to synthetic HIV genomic 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 tRNALys3 with those of the RPC-5 resolved species showed that the labeled RNA species in each peak was tRNALys3. These radioactive tRNALys3 species retained their relative mobilities when rechromatographed on RPC-5. When total HIV (PLB) RNA was used as the source of primer/template, and similarly extended with RT in the presence of radioactive dCTP, the major priming tRNA resolved by RPC-5 had a chromatographic mobility identical to peak 3. This tRNA primer has a T1 digest pattern identifying it as tRNALys3. These results indicate that the major tRNALys3 species present in the virus is also the major tRNALys3 isoacceptor used as the primer for reverse transcription.
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Lady Davis Institute for Medical Research, Jewish General Hospital, Montreal, Quebec, Canada.
Shalom
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A
Huang
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Mak
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J
Arts
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E
Wainberg
M A
MA
Kleiman
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L
D001483:Base Sequence; D015152:Blotting, Northern; D002460:Cell Line; D002848:Chromatography, DEAE-Cellulose; D002852:Chromatography, Ion Exchange; D005260:Female; D005312:Fetal Blood; D015497:HIV-1 / Q000235:genetics*; D006801:Humans; D007231:Infant, Newborn; D015345:Oligonucleotide Probes; D010920:Placenta / Q000737:chemistry; D011247:Pregnancy; D012346:RNA, Transfer, Amino Acyl / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D012357:RNA, Transfer, Lys / Q000737:chemistry* / Q000302:isolation & purification; D012367:RNA, Viral / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D014407:Tumor Cells, Cultured
D015345:Oligonucleotide Probes; D012346:RNA, Transfer, Amino Acyl; D012357:RNA, Transfer, Lys; D012367:RNA, Viral