Identification of candidate miRNAs encoded by GU-rich tract in HIV LTR and is highly conserved in HIV-1.

<p><b>A</b>: Small RNAs processed from HIV-1 LTR region observed by SOLiD Deep Sequencing. Left peak shows small RNAs derived from TAR stem (miR TAR). Right peak shows a hotspot for small RNAs derived from R and U5 stem region. The GU-rich tract (46 nt) encodes a family of viral miRs including vmiR88 and vmiR99. Modified from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106006#pone.0106006-Schopman1" target="_blank">[14]</a>. <b>B</b>: shRNA mirs are intermediates in biogenesis of mature vmiRs. shRNA reported for 43/9175 TAR (left). UNAFold software predicts folding of shRNAs vmir88 (middle) and vmir99 (right), which suggests the structures of intermediates in the biogenesis of the mature vmiR-TAR (black rectangle), vmiR88 (blue rectangle) and vmiR99 (red rectangle). UNAFold's thermodynamic calculations predict that all three shRNAs fold spontaneously (Δ<i>G</i><0) into stable hairpins (high melting temperature, <i>T</i>_M>53.8°C in 1M Na⁺). <b>C</b>: To delineate the boundaries and sequences of mature viral miRNAs, cell extracts and exosomal extracts were analyzed. Sample cell extracts were <i>in vitro</i>-infected AM (healthy AM+HIV), HIV-positive U1 macrophages stimulated by PMA (U1+PMA). Exosomal extracts were from exosomes of HIV+ human serum (HIV+ serum 10 b). Total RNA was amplified by qRT-PCR, cloned into pCR4-TOPO vector and DNA was sequenced. Sequences of vmiR88 and vmiR99 PCR products were aligned with sequences of plasmid (vector) and HIV-BaL strain. The polyadenylation signal (PA signal) and polyadenylation site (PA site) were reported <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106006#pone.0106006-Bhnlein1" target="_blank">[29]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106006#pone.0106006-Berkhout1" target="_blank">[36]</a>.