Molecular Genetics and Microbiology, The University of Texas at Austin, Austin, TX 78712-0162, USA.
RNA. 2010 Aug;16(8):1540-58. doi: 10.1261/rna.1967910. Epub 2010 Jun 21.
Kaposi's sarcoma-associated herpesvirus (KSHV) is a human tumor virus that encodes 12 precursor microRNAs (pre-miRNAs) that give rise to 17 different known approximately 22-nucleotide (nt) effector miRNAs. Like all herpesviruses, KSHV has two modes of infection: (1) a latent mode whereby only a subset of viral genes are expressed and (2) a lytic mode during which the full remaining viral genes are expressed. To date, KSHV miRNAs have been mostly identified via analysis of cells that are undergoing latent infection. Here, we developed a method to profile small RNAs ( approximately 18-75 nt) from populations of cells undergoing predominantly lytic infection. Using two different next-generation sequencing platforms, we cloned and sequenced both pre-miRNAs and derivative miRNAs. Our analysis shows that the vast majority of viral and host 5p miRNAs are co-terminal with the 5' end of the cloned pre-miRNAs, consistent with both being defined by microprocessor cleavage. We report the complete repertoire (25 total) of 5p and 3p derivative miRNAs from all 12 previously described KSHV pre-miRNAs. Two KSHV pre-miRNAs, pre-miR-K12-8 and pre-miR-K12-12, encode abundant derivative miRNAs from the previously unreported strands of the pre-miRNA. We identify several novel small RNAs of low abundance, including viral miRNA-offset-RNAs (moRNAs), and antisense viral miRNAs (miRNA-AS) that are encoded antisense to previously reported KSHV pre-miRNAs. Finally, we observe widespread antisense transcription relative to known coding sequences during lytic replication. Despite the enormous potential to form double-stranded RNA in KSHV-infected cells, we observe no evidence for the existence of abundant viral-derived small interfering RNAs (siRNAs).
卡波济肉瘤相关疱疹病毒（KSHV）是一种人类肿瘤病毒，它编码 12 个前体 microRNAs（pre-miRNAs），这些前体 microRNAs 产生 17 种不同的已知约 22 个核苷酸（nt）的效应 microRNAs。与所有疱疹病毒一样，KSHV 有两种感染模式：（1）潜伏模式，只有一部分病毒基因表达；（2）裂解模式，在此期间，所有剩余的病毒基因都表达。迄今为止，KSHV 的 microRNAs 主要通过分析处于潜伏感染的细胞来鉴定。在这里，我们开发了一种方法来分析处于主要裂解感染的细胞群体中的小 RNA（约 18-75 nt）。使用两种不同的下一代测序平台，我们克隆和测序了 pre-miRNAs 和衍生 microRNAs。我们的分析表明，绝大多数病毒和宿主 5p microRNAs 与克隆的 pre-miRNA 的 5' 端末端共终止，这与两者均由 microprocessor 切割定义一致。我们报告了所有 12 个先前描述的 KSHV pre-miRNAs 的 5p 和 3p 衍生 microRNAs 的完整 repertoire（共 25 个）。两个 KSHV pre-miRNAs，pre-miR-K12-8 和 pre-miR-K12-12，从以前未报道的 pre-miRNA 链上编码丰富的衍生 microRNAs。我们鉴定了一些新的低丰度小 RNA，包括病毒 miRNA-offset-RNAs（moRNAs）和反义病毒 microRNAs（miRNA-AS），它们是反义于以前报道的 KSHV pre-miRNAs 编码的。最后，我们观察到在裂解复制过程中相对于已知编码序列广泛的反义转录。尽管 KSHV 感染细胞中形成双链 RNA 的潜力巨大，但我们没有发现大量存在的病毒衍生的 small interfering RNAs（siRNAs）的证据。
