Kaposi's sarcoma-associated herpesvirus ORF57 promotes escape of viral and human interleukin-6 from microRNA-mediated suppression.
ABSTRACT Kaposi's sarcoma-associated herpesvirus (KSHV) lytic infection increases the expression of viral and human interleukin-6 (vIL-6 and hIL-6, respectively), an important factor for cell growth and pathogenesis. Here, we report genome-wide analysis of viral RNA targets of KSHV ORF57 by a novel UV-cross-linking and immunoprecipitation (CLIP) assay. We identified 11 viral transcripts as putative ORF57 targets and demonstrate that vIL-6 mRNA is an authentic target of ORF57. Disrupting the ORF57 gene in the KSHV genome leads to inefficient expression of vIL-6. With transient transfection, the expression of vIL-6 could be enhanced greatly in the presence of ORF57 in a dose-dependent manner. We found that the open reading frame (ORF) region of vIL-6 RNA contains an MRE (MTA [ORF57]-responsive element) composed of two motifs, MRE-A and MRE-B, and binding of ORF57 to these two motifs stabilizes vIL-6 RNA and promotes vIL-6 translation. We demonstrate that vIL-6 MRE-B bears an miR-1293 binding site and that, mechanistically, ORF57 competes with miR-1293 for the same binding site to interact with vIL-6 RNA, thereby preventing vIL-6 RNA from association with the miR-1293-specified RNA-induced silencing complex (RISC). Consistent with this, ORF57 also interacts with an miR-608 binding site in the hIL-6 ORF and prevents miR-608 repression of hIL-6. Collectively, our results identify a novel function of ORF57 in being responsible for stabilization of viral and human IL-6 RNAs and the corresponding enhancement of RNA translation. In addition, our data provide the first evidence that a tumor virus may use a viral protein to interfere with microRNA (miRNA)-mediated repression of an miRNA target to induce cell proliferation and tumorigenesis during virus infection.
Article: Stability of a long noncoding viral RNA depends on a 9-nt core element at the RNA 5' end to interact with viral ORF57 and cellular PABPC1.[show abstract] [hide abstract]
ABSTRACT: Kaposi sarcoma-associated herpesvirus (KSHV) ORF57, also known as Mta (mRNA transcript accumulation), enhances viral intron-less transcript accumulation and promotes splicing of intron-containing viral RNA transcripts. In this study, we identified KSHV PAN, a long non-coding polyadenylated nuclear RNA as a main target of ORF57 by a genome-wide CLIP (cross-linking and immunoprecipitation) approach. KSHV genome lacking ORF57 expresses only a minimal amount of PAN. In cotransfection experiments, ORF57 alone increased PAN expression by 20-30-fold when compared to vector control. This accumulation function of ORF57 was dependent on a structured RNA element in the 5' PAN, named MRE (Mta responsive element), but not much so on an ENE (expression and nuclear retention element) in the 3' PAN previously reported by other studies. We showed that the major function of the 5' PAN MRE is increasing the RNA half-life of PAN in the presence of ORF57. Further mutational analyses revealed a core motif consisting of 9 nucleotides in the MRE-II , which is responsible for ORF57 interaction and function. The 9-nt core in the MRE-II also binds cellular PABPC1, but not the E1B-AP5 which binds another region of the MRE-II. In addition, we found that PAN RNA is partially exportable in the presence of ORF57. Together, our data provide compelling evidence as to how ORF57 functions to accumulate a non-coding viral RNA in the course of virus lytic infection.International journal of biological sciences 01/2011; 7(8):1145-60. · 2.70 Impact Factor