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. 2016 Aug;22(8):1228–1238. doi: 10.1261/rna.057299.116

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© 2016 Eckwahl et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society

This article is distributed exclusively by the RNA Society for the first 12 months after the full-issue publication date (see http://rnajournal.cshlp.org/site/misc/terms.xhtml). After 12 months, it is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/.

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FIGURE 5. — HIV-1 packages pre-tRNAs. (A) To determine whether specific pre-tRNAs were encapsidated selectively, Northern blotting was used to compare the levels of the precursor and mature forms of tRNA-Ser-UGA in RNA from HIV-infected CEM-SS cells (lane 2) and virions (lane 4). As controls, RNA from uninfected cells (lane 1) and culture media (lane 3) was examined. The probe detects sequences present in both the mature tRNA and its precursor. Virion enrichment was normalized to 7SL RNA. (B–D) Northern blotting was used to determine the levels of precursor and mature forms of tRNA-Ile-UAU (B), tRNA-Tyr-GUA (C), and tRNA-Leu-CAA (D) in cells (lane 2) and virions (lane 4) produced in HEK293T cells after transfection with a proviral plasmid lacking env. RNA from mock-transfected cells (lane 1) and media (lane 3) was also examined. Intron-containing forms of pre-tRNA-Ile-UAU (B) and pre-tRNA-Leu-CAA (D) were detected with oligonucleotides complementary to their intervening sequences (middle panels), while spliced forms were detected with oligonucleotides complementary to the spliced anticodon stem–loop (bottom panels). As the tRNA-Tyr-GUA probe is complementary to 3′ exon sequences, it detects both unspliced and spliced tRNAs. Consistent with a report that mature tRNA-Ile-UAU is highly enriched in HIV virions relative to its cellular concentration (Pavon-Eternod et al. 2010), we found that tRNA-Ile-UAU was more selectively incorporated into virions than the other mature tRNAs examined.