Gottwein Eva, Cai Xuezhong, Cullen Bryan R
Department of Molecular Genetics and Microbiology, Box 3025, Duke University Medical Center, Durham, NC 27710, USA.
J Virol. 2006 Jun;80(11):5321-6. doi: 10.1128/JVI.02734-05.
MicroRNAs (miRNAs) are a class of approximately 22-nucleotide noncoding RNAs that inhibit the expression of specific target genes at the posttranscriptional level. Recently, 11 miRNAs encoded by the pathogenic human herpesvirus Kaposi's sarcoma-associated herpesvirus (KSHV) were cloned from latently infected cells. While the expression of these miRNAs has been confirmed by Northern analysis, their ability to inhibit target gene expression has not been demonstrated. We have devised a novel assay for miRNA function that uses lentiviral indicator vectors carrying two perfectly complementary target sites for each given miRNA in the 3' untranslated region of the Renilla luciferase gene. This assay allowed us to demonstrate the activity of each viral miRNA upon cotransduction of cells with the Renilla luciferase indicator vector together with a firefly luciferase control vector. In KSHV-infected BC-1 and BCBL-1 cells, but not uninfected control cells, Renilla luciferase expression was selectively reduced up to 10-fold. Interestingly, one of the viral miRNAs (miR-K5) exhibited much higher activity in BC-1 cells than in BCBL-1 cells. Sequence analysis of both viral genomes revealed a single nucleotide polymorphism in the miR-K5 precursor stem-loop, which inhibits the expression of mature miR-K5 in BCBL-1 cells. We show that the primary miR-K5 sequence present in BCBL-1 results in diminished processing by Drosha both in vivo and in vitro. This is the first report of a naturally occurring sequence polymorphism in an miRNA precursor that results in reduced processing and therefore lower levels of mature miRNA expression and function.
微小RNA（miRNA）是一类约22个核苷酸的非编码RNA，它们在转录后水平抑制特定靶基因的表达。最近，从潜伏感染的细胞中克隆出了由致病性人类疱疹病毒卡波西肉瘤相关疱疹病毒（KSHV）编码的11种miRNA。虽然通过Northern分析已证实了这些miRNA的表达，但它们抑制靶基因表达的能力尚未得到证实。我们设计了一种用于miRNA功能的新型检测方法，该方法使用慢病毒指示载体，在海肾荧光素酶基因的3'非翻译区为每个给定的miRNA携带两个完全互补的靶位点。该检测方法使我们能够在将海肾荧光素酶指示载体与萤火虫荧光素酶对照载体共转导细胞时，证明每种病毒miRNA的活性。在KSHV感染的BC-1和BCBL-1细胞中，而不是未感染的对照细胞中，海肾荧光素酶的表达选择性降低了多达10倍。有趣的是，其中一种病毒miRNA（miR-K5）在BC-1细胞中的活性比在BCBL-1细胞中高得多。对两种病毒基因组的序列分析揭示了miR-K5前体茎环中的一个单核苷酸多态性，这抑制了BCBL-1细胞中成熟miR-K5的表达。我们表明，BCBL-1中存在的初级miR-K5序列在体内和体外都会导致Drosha的加工减少。这是关于miRNA前体中自然发生的序列多态性导致加工减少，从而导致成熟miRNA表达和功能水平降低的首次报道。
