Pan-viral-microRNA screening identifies interferon inhibition as a common function of diverse viruses.

Diverse viruses encode regulatory RNAs called microRNAs (miRNAs). Despite much progress, the functions of the majority of viral miRNAs remain unknown. Most previous studies have used biochemical methods to uncover targets of viral miRNAs, but it is unclear what fraction of these targets is functionally important. Here, we apply an alternative strategy based on the premise that assorted viral miRNAs will share functionality. Screening a library of >70 human viral miRNAs showed that three unrelated miRNAs from distantly related herpesviruses significantly inhibited IFN signaling. Strikingly, each of these miRNAs directly reduced expression of the cyclic AMP-responsive element-binding protein (CBP), which as part of the p300-CBP complex, mediates IFN signaling. We show that both 5' and 3' derivatives from Epstein-Barr virus (EBV) encoded miR-BART-18 precursor miRNA (pre-miRNA) and the orthologous pre-miRNA from Rhesus lymphocryptovirus contribute to reducing IFN signaling. Thus, through both convergent and divergent evolutionary mechanisms, varied herpesviral miRNAs share the ability to decrease IFN signaling. Restoring miR-BART-18 to cells infected with an EBV miRNA mutant conveyed a cellular growth advantage upon IFN treatment, and relevant miRNAs from other herpesviruses were able to complement this activity. Blocking miR-BART-18 function in an EBV(+) tumor cell line renders cells more susceptible to IFN-mediated effects. These findings provide a mechanism that can at least partially explain the resistance of some EBV-associated tumors to IFN therapy. Our work suggests that similar pan-viral-miRNA functional-based screening strategies are warranted for determining relevant activities of other viral miRNAs.

Comprehensive mapping and analysis of Kaposi's sarcoma-associated herpesvirus 3' UTRs identify differential posttranscriptional control of gene expression in lytic versus latent infection.

3' untranslated regions (UTRs) are known to play an important role in posttranscriptional regulation of gene expression. Here we map the 3' UTRs of Kaposi's sarcoma-associated herpesvirus (KSHV) using next-generation RNA sequencing, 3' rapid amplification of cDNA ends (RACE), and tiled microarray analyses. Chimeric reporters containing the KSHV 3' UTRs show a general trend toward reduced gene expression under conditions of latent infection. Those 3' UTRs with a higher GC content are more likely to be associated with reduced gene expression. KSHV transcripts display an extensive use of shared polyadenylation sites allowing for partially overlapping 3' UTRs and regulatory activities. In addition, a subset of KSHV 3' UTRs is sufficient to convey increased gene expression under conditions of lytic infection. These results suggest a role for viral 3' UTRs in contributing to differential gene expression during latent versus lytic infection.

Reporter-based assays for analyzing RNA interference in mammalian cells.

RNA interference (RNAi) is a process whereby small RNAs serve as effectors to direct posttranscriptional regulation of gene expression. The effector small RNAs can arise from various sources including plasmids that express short-hairpin RNAs (shRNAs) or microRNA (miRNAs), or alternatively, from synthetic small-interfering RNAs (siRNAs). These small RNAs enter a protein complex that binds directly to mRNA targets and this results in transcript-specific inhibition of protein expression. Though the key core components of the mammalian RNAi processing and effector complexes have been identified, accessory and regulatory factors are less well-defined. Reporter assays that can quantitatively assess RNAi activity can be used to identify modulators of RNAi. We present two methods to quantitatively analyze RNAi activity that have overlapping and distinct utility. The first method uses an eGFP reporter in transiently transfected cells to identify RNAi modulators. The second method uses cells that express luciferase-based reporters in a stable fashion. This assay can easily be conducted in 96-well plate format. Both methods can be used to identify novel proteins or small molecules that modulate RNAi activity.

Detection of viral microRNAs by Northern blot analysis.

microRNAs (miRNAs) of host and viral origin have been suggested to play important roles in the viral infectious cycle. The discovery of new viral miRNAs, and understanding how viral infection alters host miRNAs, has been greatly aided by Northern blot analysis. The Northern blot method is used to detect specific RNAs that have been separated by size and immobilized onto a membrane. This method can provide specific information regarding the size of a miRNA and possible precursor structures. Thus, it represents a valuable tool in the discovery and validation of new miRNAs. Viral infection can sometimes present special challenges to utilizing Northern blot analysis. These challenges may include low miRNA expression levels, high GC content, and abundant background signal from nonspecific RNA degradation fragments triggered by the stress of lytic infection. We present a protocol for small RNA Northern blot analysis that we have successfully used to detect viral miRNAs from cells undergoing lytic infection from members of the Herpes and Polyoma virus families. Included are optimization strategies and a protocol for using radiolabeled oligonucleotides to detect larger RNAs.

Kaposi's sarcoma herpes virus taps into a host microRNA regulatory network.

Kaposi's sarcoma-associated herpes virus (KSHV), the causative agent of several neoplasms, has recently been shown to encode 12 microRNAs. mir-155 is a host-encoded microRNA associated with lymphomagenesis. Two new papers provide strong evidence that a KSHV-encoded microRNA and mir-155 share a common set of mRNA targets and binding sites, implying a possible link between viral- and non-viral-mediated tumorigenesis.