Since vSP-1 has been found to interact with component II of the PARS motif, it is likely that vSP-1 binds to the E3 docking site, thereby blocking RTA ubiquitination. pathway. As a consequence, vSP-1 facilitates KSHV gene expression and lytic replication. This obtaining reveals a novel mechanism of gene regulation in the viral life cycle. INTRODUCTION As a herpesvirus, Kaposi’s sarcoma-associated herpesvirus (KSHV) has two modes of contamination: latency and productive lytic replication. The switch of KSHV between latency and lytic replication is usually controlled by a virally encoded transcriptional activator, namely, RTA (replication and transcription activator). RTA expression is necessary and sufficient to disrupt latency and initiate viral lytic replication. It activates a number of viral and cellular promoters by different mechanisms, including (i) directly binding to specific motifs in some promoters, (ii) piggybacking on other cellular proteins bound on certain promoters, and (iii) promoting the degradation of transcriptional repressors (reviewed in reference 1). In addition, RTA Hoechst 34580 has also been found to promote the degradation of several proteins, cellular and viral (including itself), through the ubiquitin-proteasome pathway by serving as an intrinsic E3 ubiquitin ligase (2). Therefore, defining the mechanism that regulates RTA expression and activity is crucial for understanding Rabbit Polyclonal to GANP the molecular switch of the KSHV life cycle. Genomewide analyses of the KSHV transcriptome have revealed that nearly the entire viral genome, including both DNA strands, is usually transcribed. Ganem and colleagues observed extensive transcription from noncoding regions, including both intergenic regions and noncoding regions antisense to known open reading frames (ORFs) (3). Currently no biological function has been demonstrated to be associated with Hoechst 34580 these noncoding RNAs. The same phenomenon is also seen in mammalian genomes. Although only 2% of the total human genomic sequence consists of protein-coding genes, 90% of the human genome is usually transcribed, as revealed by large-scale cDNA cloning projects (4, 5) and genomewide tiling arrays (6C8). The majority of the transcripts are long RNAs with little or no protein-coding capacity (with the criterion that only open reading frames with at least 100 codons are annotated) (9). Although Hoechst 34580 the functions of the majority of these noncoding RNAs have not been revealed, several potential functions are beginning to emerge, including inducing chromatin remodeling to affect gene expression either in on neighboring genes (10) or in (11), serving as antisense RNAs to generate endogenous small interfering RNA (endo-siRNA) (12, 13), binding to specific protein partners to modulate protein activity (14, 15), serving as a structural component to form an RNA-protein complex that regulates cell functions (16), and serving as precursors to small RNAs, including microRNA (miRNA) and Piwi-interacting RNA (piRNA) (17C20). A 3.0-kb polyadenylated RNA (designated T3.0) that is transcribed from the opposite strand of ORF50 in the KSHV genome has been identified in KSHV-infected cells and has been annotated as a noncoding RNA because no large open reading frame was found in the transcript (21). Since T3.0 RNA is potentially able to base-pair with ORF50 mRNA, which specifies RTA, we wondered if T3.0 modulates RTA expression, either positively or negatively. Here we report that T3.0 indeed upregulates RTA expression. However, T3.0 exerts this function by encoding a small peptide that complexes with RTA and prevents it from being degraded by the ubiquitin-proteasome pathway, representing a novel mechanism underlying RTA regulation and KSHV reactivation. This obtaining also demonstrates a novel paradigm for the function of so-called noncoding RNAs in cells. MATERIALS AND METHODS Cells. The primary effusion lymphoma cell line BCBL-1 was obtained from the NIH AIDS Research and Reference Reagent Program. The cells were produced in RPMI 1640 medium (Gibco-BRL, Gaithersburg, MD) supplemented with 10% fetal bovine serum (Gibco-BRL) and penicillin-streptomycin (50 U/ml). Human embryonic kidney (HEK) 293T cells were obtained from the ATCC and were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, 2 Hoechst 34580 mM l-glutamine, and antibiotics (penicillin-streptomycin and amphotericin B [Fungizone]). Constructs. The pCR3.1-ORF50 plasmid, RTA internal deletion mutants, and the promoter-luciferase reporter plasmids [pK8-DE250 and pOrilyt.
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