Epstein-Barr virus noncoding RNAs are confined to the nucleus, whereas their partner, the human La protein, undergoes nucleocytoplasmic shuttling

Abstract

The Epstein-Barr virus (EBV) noncoding RNAs, EBV-encoded RNA 1 (EBER1) and EBER2, are the most abundant viral transcripts in all types of latently infected human B cells, but their function remains unknown. We carried out heterokaryon assays using cells that endogenously produce EBERs to address their trafficking, as well as that of the La protein, because EBERs are quantitatively bound by La in vivo. Both in this assay and in oocyte microinjection assays, EBERs are confined to the nucleus, suggesting that their contribution to viral latency is purely nuclear. EBER1 does not bind exportin 5; therefore, it is unlikely to act by interfering with microRNA biogenesis. In contrast, La, which is a nuclear phosphoprotein, undergoes nucleocytoplasmic shuttling independent of the nuclear export protein Crm1. To ensure that small RNA shuttling can be detected in cells that are negative for EBER shuttling, we demonstrate the shuttling of U1 small nuclear RNA.

Figure 1.

EBER1 and EBER2 do not shuttle in HKB5cl8 cells. (A) EBER1 and EBER2 expression in HKB5cl8 cells. A Northern blot of 5 μg total RNA from HKB5cl8 (lane 1), BJAB (lane 2), and BJAB-B1 (lane 3) was sequentially probed for EBER1, the U6 loading control, and EBER2. (B) Lack of EBER nucleocytoplasmic shuttling. Heterokaryons were prepared by fusing HKB5cl8 cells transfected with a plasmid producing hnRNP A1-GFP and mouse NIH3T3 cells for 6–7 h in the presence of cycloheximide. Heterokaryons were identified by the shuttling of hnRNP A1-GFP (2 and 5, green) into mouse nuclei, identified by punctate DAPI staining (1 and 4). Human (H) and mouse (M) nuclei of the heterokaryons are labeled. A total of 14 heterokaryons were analyzed. Endogenous EBER1 (3) and EBER2 (6) were detected using DIG-labeled probes (yellow).

Figure 2.

Detection of α2 U1 RNA shuttling. (A) Heterokaryons were prepared as in Fig.1, except that an α2 U1–expressing plasmid, rather than an hnRNP A1-GFP–expressing plasmid, was transfected into HKB5cl8 cells and no cycloheximide was added. The two human (H) and one mouse (M) nuclei in the heterokaryon are labeled as identified by DAPI (1). The α2 U1 RNA (red) shuttled into the mouse nucleus (3), whereas the endogenous EBER1 (green) did not (2). A total of seven heterokaryons were analyzed. (B) Turnover rates of EBER1 compared with U1, 7SL, and Y1 RNAs. HKB5cl8 (open symbols) and BJAB-B1 (closed symbols) cells were treated with actinomycin D, and the indicated RNAs were detected by Northern blotting. Each time point is the mean of three independent experiments, with error bars indicating the SD.

Figure 3.

Lack of oocyte nuclear export and Exp5 binding by EBER1. (A) Oocyte microinjections. A mixture of T7-transcribed, α-[³²P]UTP–labeled U6, tRNA^Phe, and either wild-type EBER1 or mutant EBER1 lacking its 3′ polyU terminus (0.5–1 fmol per oocyte) was microinjected into the geminal vesicles of whole X. laevis oocytes. After either a 0.5-h (wild-type, lanes 2 and 3; mutant, lanes 7 and 8) or 2.5-h (wild-type, lanes 4 and 5; mutant, lanes 9 and 10) incubation at RT, 5–6 oocytes were fractionated. RNAs extracted from the nuclear (N), cytoplasmic (C), or total fractions were resolved on a urea polyacrylamide gel and visualized by autoradiography. The percentages of RNA in the nucleus are indicated. (B) Electrophoretic mobility shift assays performed on binding reactions containing 4.5 fmol of labeled VARdm RNA, 1 pmol of recombinant Exp5 (lanes 2–10) and RanQ69LGTP (see Materials and methods), and the indicated amounts of unlabeled competitor RNA: VARdm (lanes 3 and 4), EBER1 (lanes 5–7), or U6 (lanes 8–10). Lane 1 contained no protein.

Figure 4.

Human La protein undergoes nucleocytoplasmic shuttling in multiple cell lines. Heterokaryons were made by fusing HKB5cl8 (1–6), HeLa (7–9), or HEK293 (10–12) cells, which were transfected with a plasmid producing either the shuttling hnRNP A1-GFP (1–3) or the nonshuttling hnRNP C1-GFP (4–12), with mouse NIH3T3 cells. After 4 h, human La protein (red) was detected with a monoclonal anti-La antibody specific for human La, which is demonstrated by the absence of signal in the unfused mouse cells labeled m (7, 8, 10, and 11). Human (H) and mouse (M) nuclei are labeled as in Fig. 1. More than 20 heterokaryons were analyzed.

Figure 5.

Shuttling of the human La protein is not blocked by LMB. Heterokaryons were made by fusing HEK293 cells transfected with plasmids producing Flag-PP32 (1–3) or Flag-PP32 and hnRNP A1-GFP (4–11) with mouse NIH3T3 cells as described in Fig. 4, except that no LMB (1–3) or 30 ng/ml LMB (4–11) was included during fusion. Detection of the human La protein and labeling of the nuclei are as described in Fig. 4. La is shown in red (2) or in pseudocolor orange (5 and 9). Flag-PP32 is in green (3) or in pseudocolor deep red for infrared (6 and 10). 2 of 11 heterokaryons analyzed are shown.