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 > or =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.

Figure 1. Genomic Location of Selected EBV and rLCV miRNAs

(A) Schematic of a segment of the EBV genome, extending from 137,490 to 152,641, with EBV genes located on the antisense strand and BART mRNA exons located on the sense strand indicated. The location of the EBV BART miRNAs identified in this report is indicated. Also shown is the extent of the BART gene deletion found in the EBV B95–8 strain. Not shown are the three miRNAs encoded within the EBV BHRF1 miRNA cluster, which extends from 41,474 to 42,990, or the miR-BART2 miRNA, which is located 3′ to the other BART miRNAs between positions 152,747 and 152,768. (B) Schematic of a similar segment of the rLCV genome, extending from position 131,487 to 148,684, with known rLCV homologs of EBV genes indicated. The rLCV miRNAs identified in this report are indicated and the miRNAs conserved in EBV highlighted. Although the BART gene is thought to be conserved in this region of the rLCV genome, based on sequence analysis, the BART exons in rLCV have not been mapped. Not shown are the rLCV miR-rL1-1 and miR-rL1-2 miRNAs, which are encoded 3′ to position 35,323 and 36,709, respectively.

Figure 2. Analysis of EBV miRNA and mRNA Expression in Tumor-Derived Cells

(A) Northern analysis of selected EBV miRNAs in total RNA samples derived from the indicated cell lines and tumors. The EBV uninfected PEL cell line BCBL-1 served as a negative control and U6 RNA as a loading control. (B) Northern analysis of BART mRNA expression. The total RNA samples analyzed here are the same ones used in (A). The probe used was specific for the invariant exon 7 of the alternatively spliced BART miRNAs. GAPDH mRNA expression was used as a loading control. The mobility of 28S and 18S rRNA is indicated. (C) RT-PCR analysis using primers specific for transcripts initiating at the viral Cp, Wp, and Qp promoters. This analysis used oligo(dT)-primed cDNA preparations. Primers specific for the cellular GAPDH mRNA were used as a control.

Figure 3. Induction of Lytic Replication Can Increase EBV miRNA Expression

The EBV-infected B-cell lines Daudi and MUTU I were either cultured under normal conditions or treated with TPA (30 ng/ml) and n-butyrate (300 ng/ml) for 48 h. At this point cell samples were analyzed for entry into lytic EBV replication by immunofluorescent detection of Zebra expression (see Figure S4) or used for RNA preparation and Northern analysis, as described in Figure 2. In the case of miR-BHRF1–2, we here show a larger panel that also includes the approximately 59-nt pre-miRNA precursor, as this was more readily detected than the mature approximately 23-nt miR-BHRF1–2 miRNA.

Figure 4. Analysis of rLCV miRNA Expression

Northern analysis of selected rLCV miRNAs in the rLCV-infected rhesus B-cell lines 211–98 and 309–98. The human B-cell line BJAB served as a negative control.

Figure 5. Sequence Comparison of miRNAs That Are Evolutionarily Conserved in EBV and rLCV and Expressed in Virus-Infected Cells

All the indicated miRNAs were cDNA cloned from infected cells except miR-rL1-2, whose existence was predicted in silico and then confirmed by Northern analysis (Figure 4).

Figure 6. Sequence Comparison of the Predicted Primary miRNA Stem-Loop Structures of the Indicated rLCV and EBV miRNAs

The stem-loop shown is the rLCV sequence, with the changes observed in EBV indicated. The mature miRNA sequences are shown in red. In some cases, the miRNA precursors give rise to two mature miRNAs. A “+” sign indicates an insertion.