Confirmation of Novel miRNAs of EBV, rLCV and JMHV by Northern Blot

4. RESULTS

4.1. Identification of Novel miRNAs Within the γ-Herpesvirus Family

4.1.3. Confirmation of Novel miRNAs of EBV, rLCV and JMHV by Northern Blot

count: 25, min. stem-loop length: 50, max. stem-loop-length: 220) (for details see 3.6.1)) were further analyzed in confirmatory Northern blots. The confirmation was performed by using RNA derived from cell lines latently infected with the corresponding virus and expressing the miRNAs (EBV and rLCV). For JMHV no latently infected cell lines were available and therefore confirmation of miRNAs was performed using transfected cells. A vector expressing all miRNAs encoded by JMHV was generated (see below and 2.7.1.2). The prediction of pre-miRNA hairpins in general allows a good estimation where the mature miRNA is located. The location of mature miRNA sequences is estimated to compasse 21 nt adjacently to the terminal loop. Probes were designed for either 5p and 3p

Results

nt. However, secondary structure prediction is not accurate, especially for the size of the terminal loop and therefore exact shape and processing by Drosha and Dicer is not known.

EBV miRNAs

All hairpins of EBV type I and II that passed the threshold, were analyzed in Northern hybridization to either confirm novel pre-miRNA hairpins or to exclude false positive predictions. Since it has been shown that miRNA expression varies between cell lines and also differs based on the hairpin structures, RNA from different EBV positive B-cell lines Raji and Jijoye and an EBV positive epithelial cell line the nasopharyngeal carcinoma cell line C666-1, were used. The EBV negative B-cell line BJAB was used as negative control.

VMirscore

BHRF locus BART locus

VMirscore

BHRF locus BART locus

Figure 4-4 VMir Prediction of EBV Pre-miRNA Hairpins

Hairpins are shown as blue triangles and are encoded in the direct orientation of the genome. Dark blue circles display known conserved pre-miRNAs, and light blue circles display known non-conserved pre-miRNAs.

Analyzed pre-miRNAs in this work are highlighted as orange and yellow circles for putative conserved and non-conserved pre-miRNAs, respectively.

The latency genes are encoded in direct orientation. According to this, the pre-miRNAs investigated are also predicted in the direct orientation. From the 15 novel predicted BART miRNA precursors, only two could be confirmed to be processed into mature miRNAs by Northern blotting (figure 4-4).

They were named miR-BART-21, for which miRNAs from both arms of the hairpin could be detected, and miR-BART-22, which is shown to express just the 5p miRNA at detectable levels.

Figure 4-5 Confirmatory Northern Blot for Novel EBV miRNAs.

Four different cell lines were used: EBV negative B cell line BJAB (1) and three EBV positive cell lines, the epithelial cell line C666-1 (2), and two B cell lines Jijoye (3) and Raji (4) (Walz et al., 2010, modified). (figure:

In all Northern blots, the epithelial cell line C666-1 showed the highest expression of mature miRNAs.

The two miRNAs were also abundantly expressed in the B-cell line Jijoye but were not detectable in the B-cell line Raji. This is in good correlation with previous findings for other EBV miRNAs (Cai et al., 2006). The identification of two novel EBV miRNAs, which were missed in diverse previous analysis, argues for the high probability to detect novel miRNAs also in rLCV and JMHV.

rLCV miRNAs

The pre-miRNA hairpins predicted at the BHRF and BART loci of rLCV were investigated. Beneath several putative conserved pre-miRNA hairpins, non-conserved good candidates were also found. The analysis was therefore extended, to include all good performing pre-miRNA hairpins.

VMirscore

BHRF locus BART locus

VMirscore

BHRF locus BART locus

Figure 4-6 VMir Prediction of rLCV Pre-miRNA Hairpins

Analyzed pre-miRNAs in this work are highlighted as orange and yellow circles for putative conserved and

non-Results

Altogether 20 pre-miRNA hairpins were analyzed by Northern blotting including one candidate found to be located in reverse direction and being conserved to miR-BART-9. On closer inspection, a hairpin was folded in reverse orientation to the gene product, but failed to pass the threshold. But a suboptimal folding with a slightly lower energy of 0.5 kcal/mol represents a good hairpin and so this one was included in the analysis.

RNA from EBV and rLCV negative B-cell line BJAB was used as a negative control and two rLCV positive B-cell lines, 211-98 and 260-98, were used to confirm rLCV miRNAs. In addition the EBV positive and rLCV negative cell line C666-1 was integrated to show cross reactivity of probes for conserved miRNAs. 17 out of 20 predicted precursors could be confirmed to be processed to mature miRNAs. Overall 22 novel mature miRNAs were identified (fig. 4-7).

Figure 4-7 Confirmatory Northern Blots of Novel rLCV miRNAs.

Different cell lines were used for the detection: rLCV negative B cell line BJAB (1), rLCV positive B cell lines 211-98 (2) and 260-98 (3) and a rLCV negative but EBV positive epithelial cell line C666-1 (4). miRNAs which were further sequenced are shown in A, whereas a Northern Blot with a potential miRNA band for hairpin MD1517, which failed to be sequenced, is shown in B (Walz et al., 2010, modified). (figure: Copyright © American Society for Microbiology, [J Virol, 84, 716-728, 2010])

In some cases, i.e. hairpins rL1-19, rL1-27 and rL1-32 3p, the degree of conservation is represented by the cross-reaction of probes in the EBV positive cell line C6661-1 (fig. 4-7 A). The expression level varies for the different miRNAs. EBV and rLCV encoded miRNAs are expressed from one primary transcript, suggesting an equal expression level. Since the hairpins are different in structure, processing through Drosha and Dicer is affected. Therefore, some hairpins are more efficiently processed than others. One pre-miRNA hairpin (MD1517) leads to a faint reproducible band in the Northern blots, but further analysis (see below) could not confirm the existence of that miRNA.

Nevertheless, it was included for completeness (fig. 4-7 B).

JMHV miRNAs

The Vmir prediction for JMHV miRNAs revealed only novel, putative, conserved pre-miRNA hairpins (figure 4-8), since the virus has been shown to be highly conserved to RRV (figure 1-1) and no viral miRNAs have been identified from JMHV so far. As for EBV and rLCV, the miRNAs are oriented in the same direction as the latent gene products (figure 4-8).

VMirscoreVMirscoreVMirscoreVMirscore

Figure 4-8 VMir Prediction of JMHV Pre-miRNA Hairpins

The upper graph shows all predicted pre-miRNA hairpins, that passed filter criteria. The VMir score is given on the y-axis, the genomic position on the x-axis. The graphs below depict the enlarged miRNA encoding region between ORF69 and ORF 71. Hairpins are shown as green diamonds and are encoded in the reverse orientation of the genome. Analyzed miRNAs in this work are highlighted as orange circles for putative conserved pre-miRNAs.

Two circumstances hampered the further identification of JMHV miRNAs. First, no stably infected established cell lines were available for JMHV and, secondly, the amount of RNA from primary rhesus fibroblast infected with JMHV was restricted. To obtain sufficient amount of RNA, 1*10⁸ primary cells had to be infected with JMHV, which makes the procedure very expensive. For that reason, the genomic region probably encoding for miRNAs was subcloned into the pcDNA3-GFP vector and used for transfection of 293T cells.

Results

Two vectors were generated, pcGJmiR and pcGJmiRΔ containing the regions ranging from nt 111105 - 116751 and nt 111105 - 116283, respectively. The first contained a poly-A site at the beginning of the miRNA encoding region that was excluded in the second expression plasmid (see also chapter 2.7.1.2). The vector containes a CMV promotor that is Pol II driven and encodes for gfp and miRNAs.

If the poly-A site is functional, some or all transcripts might be shortend by restriction and polyadenylation 10-30 nt behind this site and that would lead to a lower expression of the miRNAs.

However, miRNAs were expressed from both vectors, but indeed the vector pcGJmiRΔ expresses miRNAs at a higher level (figure 4-9).

Figure 4-9 miRNA Locus of JMHV

The JMHV genome is shown at the top, location of polymerase gene is indicated by an black arrow. The miRNA locus is enlarged below the genome and open reading frames of adjacent genes are highlighted as large arrows.

The region cloned into an expression vector is given by the nt positions of the miRNA locus. Northern blot analysis of transfected 293T cells with control, pcGJmiRΔ and pcGJmiR for one miRNA is shown on the right.

To confirm the predicted JMHV miRNAs, 293T cells were either transfected with the vector pcGJmiRΔ or the control vector pcDNA3-GFP. Twenty μg RNA was used for Northern Blots.

A total of 21 different pre-miRNA hairpins were tested and 14 of them were shown to express mature miRNAs detectable in the Northern blot. Out of these 14 pre-miRNAs at least 9 expressed mature miRNAs from both arms as seen in figure 4-10. For miRNAs miR-rJ1-4, -7, -9, -10 and -14, only one mature miRNA could be detected in the Northern blot.

75 nt

25 nt

75 nt

25 nt

miR-rJ1-7

5p 3p 5p 3p 3p

5p 3p

miR-rJ1-1 miR-rJ1-2 miR-rJ1-3 miR-rJ1-4

5p 3p

miR-rJ1-5

- + - + - + - + - + - + - + - + - + - +

5p 3p

miR-rJ1-6 - +

5p 3p 5p 3p 3p

5p 3p

miR-rJ1-1

5p 3p

miR-rJ1-1 miR-rJ1-2 miR-rJ1-3 miR-rJ1-4

5p 3p

miR-rJ1-5

5p 3p

miR-rJ1-5

- + - + - + - + - + - + - + - + - + - +

5p 3p

miR-rJ1-6

5p 3p

miR-rJ1-6 - + - +

5p - + - +

5p 3p 5p 3p

miR-rJ1-8 miR-rJ1-9

5p 3p

miR-rJ1-12

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miR-rJ1-12 miR-rJ1-10

5p 3p

miR-rJ1-13

5p 3p

miR-rJ1-13 - +

- + - + - +- +

5p 3p

miR-rJ1-15

5p 3p

miR-rJ1-15 - + - + - + - +

miR-rJ1-14 3p - +

3p - + - + - +

- + - + - + - +

- + - +

Figure 4-10 Confirmatory Northern Blots for JMHV miRNAs

293T cells were transfected with either pcDNA3-GFP (-) or pcGJmiRΔ (+) plasmid. RNA was isolated 48 h post transfection and 20 μg RNA were used and blots were exposed to auoradiographic films for 4 d.

Viral miRNAs encoded by RRV have been shown to increase in expression upon lytic replication. As mentioned before, no stably latent infected cell lines are available for JMHV. Infection of rhesus primary fibroblast cells with JMHV, however, results in lytic replication. Therefore it was tested, if miRNAs can be detected in JMHV infected rhesus primary fibroblast cells. A Northern blot containing 14 μg RNA of rhesus primary fibroblast cells infected with JMHV for 24, 48 and 72 h was probed with two different and abundantly expressed miRNAs (figure 4-11).

miR-rJ1-7 5p miR-rJ1-14 3p 75 nt

25 nt

- 24 h p.i. 48 h p.i. 72 h p.i. - 24 h p.i. 48 h p.i. 72 h p.i.

- 24 h p.i. 48 h p.i. 72 h p.i.

Figure 4-11 Confirmatory Northern Blot for miRNAs in JMHV Infected Rhesus Primary Fibroblast Cells Rhesus primary fibroblast cells were either mock or JMHV infected for 24, 48 and 72 h, respectively. Shown are

Results

Both tested miRNAs miR-rJ7-5p and miR-rJ14-3p were abundantly expressed and expression increased over the time of infection.

Im Dokument Identification and Analysis of microRNAs Encoded by gamma-Herpesviruses (Seite 87-94)