Epstein–Barr Virus MicroRNAs Are Evolutionarily Conserved and Differentially Expressed

Abstract

The pathogenic lymphocryptovirus Epstein–Barr virus (EBV) is shown to express at least 17 distinct microRNAs (miRNAs) in latently infected cells. These are arranged in two clusters: 14 miRNAs are located in the introns of the viral BART gene while three are located adjacent to BHRF1. The BART miRNAs are expressed at high levels in latently infected epithelial cells and at lower, albeit detectable, levels in B cells. In contrast to the tissue-specific expression pattern of the BART miRNAs, the BHRF1 miRNAs are found at high levels in B cells undergoing stage III latency but are essentially undetectable in B cells or epithelial cells undergoing stage I or II latency. Induction of lytic EBV replication was found to enhance the expression of many, but not all, of these viral miRNAs. Rhesus lymphocryptovirus, which is separated from EBV by ≥13 million years of evolution, expresses at least 16 distinct miRNAs, seven of which are closely related to EBV miRNAs. Thus, lymphocryptovirus miRNAs are under positive selection and are likely to play important roles in the viral life cycle. Moreover, the differential regulation of EBV miRNA expression implies distinct roles during infection of different human tissues. Vertebrate cells express a large family of diverse small RNAs, called microRNAs, that can inhibit the expression of specific target genes. Recently, it has become apparent that several pathogenic human viruses, and in particular herpes viruses, also encode microRNAs that these viruses likely use to prevent infected cells and individuals from mounting effective antiviral responses. Here, we demonstrate that Epstein–Barr virus (EBV), which causes infectious mononucleosis and also some cancers in humans, makes 17 different microRNAs in infected human cells. These microRNAs are found in two clusters in the viral genome, one of three microRNAs, the second of 14 microRNAs, that are differentially expressed in different kinds of EBV-induced human tumors. Analysis of the closely related rhesus lymphocryptovirus shows that seven of these EBV microRNAs have been conserved in this simian virus across >13 million years of divergent evolution. This argues that these microRNAs likely play an important role in EBV replication and represents the first demonstration of the evolutionary conservation of viral microRNAs.