Expression and Processing of a Small Nucleolar RNA from the Epstein-Barr Virus Genome

Abstract

Small nucleolar RNAs (snoRNAs) are localized within the nucleolus, a sub-nuclear compartment, in which they guide ribosomal or spliceosomal RNA modifications, respectively. Up until now, snoRNAs have only been identified in eukaryal and archaeal genomes, but are notably absent in bacteria. By screening B lymphocytes for expression of non-coding RNAs (ncRNAs) induced by the Epstein-Barr virus (EBV), we here report, for the first time, the identification of a snoRNA gene within a viral genome, designated as v-snoRNA1. This genetic element displays all hallmark sequence motifs of a canonical C/D box snoRNA, namely C/C′- as well as D/D′-boxes. The nucleolar localization of v-snoRNA1 was verified by in situ hybridisation of EBV-infected cells. We also confirmed binding of the three canonical snoRNA proteins, fibrillarin, Nop56 and Nop58, to v-snoRNA1. The C-box motif of v-snoRNA1 was shown to be crucial for the stability of the viral snoRNA; its selective deletion in the viral genome led to a complete down-regulation of v-snoRNA1 expression levels within EBV-infected B cells. We further provide evidence that v-snoRNA1 might serve as a miRNA-like precursor, which is processed into 24 nt sized RNA species, designated as v-snoRNA124pp. A potential target site of v-snoRNA124pp was identified within the 3′-UTR of BALF5 mRNA which encodes the viral DNA polymerase. V-snoRNA1 was found to be expressed in all investigated EBV-positive cell lines, including lymphoblastoid cell lines (LCL). Interestingly, induction of the lytic cycle markedly up-regulated expression levels of v-snoRNA1 up to 30-fold. By a computational approach, we identified a v-snoRNA1 homolog in the rhesus lymphocryptovirus genome. This evolutionary conservation suggests an important role of v-snoRNA1 during γ-herpesvirus infection.

A Cluster of Virus-Encoded MicroRNAs Accelerates Acute Systemic Epstein-Barr Virus Infection but Does Not Significantly Enhance Virus-Induced Oncogenesis In Vivo

“…However, the magnitude of the CD8 ϩ T cell response to latent antigens during acute infection is less robust than the response to lytic antigens and peaks during convalescence (29). In vitro, BHRF1 miRNAs have been shown to reduce expression of latent viral proteins in infected B cells, which may facilitate immune evasion of latently infected B cells in vivo (30). B cells infected with the ⌬123 mutant in vitro not only exhibit blood cells (cell-associated DNA) and plasma (cell-free DNA) of WT/REV (n ϭ 10) and ⌬123 (n ϭ 11) virus-exposed mice was determined with real-time PCR.…”

“…slower cell growth kinetics but also display increased expression of latent viral proteins compared to their WT/REV virus-infected counterparts (30). Therefore, we next wanted to examine the CD8 ϩ T cell response to EBV infection in the peripheral blood of mice exposed to the WT/REV and ⌬123 viruses.…”

Dual function of C/D box small nucleolar RNAs in rRNA modification and alternative pre-mRNA splicing

“…However, numerous SNORDs without obvious target RNAs have been identified (7)(8)(9)(10) and are termed "orphan snoRNAs." Genome-wide deep sequencing experiments identified shorter but stable SNORD fragments that were found in all species tested, ranging from mammals to the protozoan Giardia lamblia (11) and EpsteinBarr virus (12). Fragments longer than 27 nt generated by SNORDs will likely not bind argonaute proteins that bind to 21-to 22-nt-long microRNAs.…”

NcRNA-microchip analysis

“…8 Interestingly, v-snoRNA1 exhibits sequence complementarity to the 3'-UTR of the viral DNA polymerase BALF5 mRNA. In addition, it has been demonstrated that v-snoRNA1 is processed into a miRNA-like species, which might target BALF5 mRNA, as it was also reported for EBV-encoded miRNA-BART2.…”

“…3 Until now, 28 non-protein coding RNA (ncRNA) genes have been mapped to the EBV genome, including Epstein-Barr virus encoded RNAs (EBER) 1 and 2, 4 25 micro-RNAs (miRNAs) [5][6][7] and a single small nucleolar RNA (snoRNA), designated as v-snoRNA1. 8 EBV employs viral encoded small ncRNAs from infection of B cells: for example, EBER 1 binds to double-stranded RNAactivated protein kinase (PKR), a key mediator of the antiviral effect of interferon (IFN)-α. Binding of EBER1 to PKR prevents PKR-mediated phosphorylation of the α-subunit of epstein-Barr virus (eBV) infection of human B cells requires the presence of noncoding RNAs (ncRNAs), which regulate expression of viral and host genes.…”