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. 2014 Mar 5:11:21.
doi: 10.1186/1742-4690-11-21.

MLV requires Tap/NXF1-dependent pathway to export its unspliced RNA to the cytoplasm and to express both spliced and unspliced RNAs

Lucie Pessel-Vivares, Mireia Ferrer, Sébastien Lainé  1 , Marylène Mougel
Affiliations

MLV requires Tap/NXF1-dependent pathway to export its unspliced RNA to the cytoplasm and to express both spliced and unspliced RNAs

Lucie Pessel-Vivares et al. Retrovirology. .

Abstract

Background: Eukaryotic cells have evolved stringent proofreading mechanisms to ensure that intron-containing mRNAs do not leave the nucleus. However, all retroviruses must bypass this checkpoint for replication. Indeed, their primary polycistronic transcript (Full-Length) must reach the cytoplasm to be either translated or packaged as genomic RNA in progeny viruses.Murine leukemia virus (MLV) is a prototype of simple retroviruses with only two well-regulated splicing events that directly influence viral leukemogenic properties in mice. Several cis-elements have been identified in the FL RNA that regulate its cytoplasmic accumulation. However, their connection with an export mechanism is yet unknown. Our goal was to identify the cellular pathway used by MLV to export its RNAs into the cytoplasm of the host cells.

Results: Since other retroviruses use the CRM1 and/or the Tap/NXF1 pathways to export their unspliced RNA from the nucleus, we investigated the role of these two pathways in MLV replication by using specific inhibitors. The effects of export inhibition on MLV protein synthesis, RNA levels and RNA localization were studied by Western blotting, RT-qPCR, fluorescence microscopy and ribonucleoprotein immunoprecipitation assays. Taken together, our results show for the first time that MLV requires the Tap/NXF1-mediated export pathway, and not the CRM1 pathway, for the expression of its spliced and unspliced RNAs and for FL RNA nuclear export.

Conclusions: By contrast to HIV-1, MLV recruits the same pathway for the cytoplasmic expression of its spliced and unspliced RNAs. Thus, MLV RNA expression depends upon coordinated splicing/export processes. In addition, FL RNA translation relies on Tap/NXF1-dependent export, raising the critical question of whether the pool of FL RNA to be packaged is also exported by Tap/NXF1.

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Figures

Figure 1
Figure 1
Effects of CRM1 and Tap inhibition on MLV expression. (A) RNAs produced by MLV and their encoded proteins. Black arrows correspond to the RT primers while positions of the RT-qPCR primers used to amplify the different RNAs are indicated by red arrows. (B) Investigation of the CRM1 pathway by Western blot analysis of MLV or HIV proteins produced in LMB-treated NIH3T3 or 293 T cells, respectively. Cells were transfected with 2 μg of pMov9.1 or pNL4.3 plasmid, the molecular clones of Mo-MLV and HIV-1 respectively, for 48 h and incubated for the last 8 h with 20 nM LMB (LC Laboratories) before harvesting. (C) Activity of the Tap siRNA. 1 μg of TAPΔC was cotransfected in NIH3T3 cells with 100 pmol of Tap Stealth RNAi (invitrogen) or control siRNA (Eurogentec). Cell were collected 48 h p.t. and Tap expression analyzed by Western blotting with anti-GFP antibodies (Roche). (D) Investigation of the Tap pathway by Western blot analysis of MLV proteins produced in NIH3T3 cells transfected for 48 h with 1 μg pMov9.1 with either 1 μg of peGFP-TapΔC [19], 1.5 μg of p3-CCCC (4-CTE) [18] or 1.5 μg of empty pcDNA3.1 vector. In all experiments, MLV:inhibitor ratios are 1:3 for peGFP-TapΔC and p3-CCCC (or pcDNA3.1). For RNA interference, NIH3T3 cells were co-transfected for 48 h with 2 μg pMov9.1 and 100 pmol of either Tap stealth RNAi as described in [18], or control siRNA. Viruses were collected by ultracentrifugation from culture supernatant and loaded on SDS-PAGE. Procedure and antibodies used to detect viral proteins are described in [16]. Actin was detected with a rabbit polyclonal antibody (1/300) (Sigma) and a peroxidase-conjugated (HRP) goat anti-rabbit antibody (1:4000) (Sigma). (E) Band integrated densities were determined with the ImageJ software and normalized to actin. Intensities are presented as mean +/− SEM.
Figure 2
Figure 2
Effects of Tap inhibitors on MLV RNA levels. Transfections were performed with pMov9.1Δenv, a Mo-MLV molecular clone deleted of the HpaI segment (1.38Kb) in the env gene in order to prevent re-infection events and the inhibition conditions were as in Figure  1. At 12 h (A), 24 h (B) and 48 h (C), RNAs were extracted, DNAse treated, and analyzed by RT-qPCR with an MLV standard curve as described in [15]. Primers used are indicated in Figure  1A and their sequences are given in Additional file 1 Table S1. The RT reaction was performed using oligodT or specific antisense primer and followed by different specific qPCR amplifications. Controls were systematically performed to check for DNA contamination by running an RT reaction without enzyme. Background RNA copies measured in untransfected cells were removed from values and then normalized to the GAPDH mRNA control monitored with specific probes as in [16]. Real time PCR was performed with the FastStart SYBRGreen (Roche) on a RotorGene (Corbett Research). Ribosomal RNAs were loaded on agarose gel and detected with SybrGreen staining as controls (D).
Figure 3
Figure 3
Analysis of MLV export by fluorescence microscopy. (A) Map of the reporter MS2- RNA. Red arrows correspond to the Cyan3-labelled probe used in FISH. (B) Reporter MS2-RNA was expressed in NIH3T3 or packaging GPE cell lines transfected or not with peGFP-TapΔC and fixed in 4% paraformaldehyde between 8-12 h p.t for FISH analysis using Cy3-MS2 probes as described [24]. Cells were imaged on a 100× NA 1.4 wide-field microscope (Zeiss AxioimagerZ1) equipped with a Coolsnap HQ2 camera. Stacks of images were recorded for Cy3 (MS2 RNA), GFP (TapΔC) and Hoechst (nucleus) fluorescence over the depth of the cell using a Z step of 0.3 μm, and represented as stacked images. Scale bar is 10 μm. All images were processed using the ImageJ software. (C) For quantitative analysis of viral RNA localization, filtered images (by subtraction of the blurred image using a sigma radius of 20) were stacked and automatically thresholded. Values for the integrated density (Mean Fluorescence intensity × Area corresponding to Cy3 signal) in the different regions of interest, nucleus and cytoplasm, were calculated using the Particle Analysis plugin in ImageJ. Data are expressed as the percentage of nuclear signal, with the mean +/− SEM values of 32 cells for each condition.
Figure 4
Figure 4
Co-immunoprecipitation of spliced or unspliced MLV RNA with TapΔC factor. NIH3T3 cells were cotransfected with 10 μg of peGFP-TAPΔC and 10 μg of pMov9.1 or 7 μg of peGFP-TAPΔC and 10,5 μg of p3-CCCC (4-CTE). At 24 h p.t., cells were extracted with 1 ml of lysis buffer (1% triton X-100, 150 mM NaCl, 50 mM Tris–HCl, complete anti-protease cocktail from Roche) and precleared with Protein A sepharose beads (invitrogen) during one hour at 4°C. 100 μl of cell lysates were saved to control transfection efficiencies by monitoring RNA levels (input). 900 μl of cell lysates were incubated 3 hours at 4°C with beads that specifically bind GFP (GFP-Trap® Chromotek) in order to co-immunoprecipitate GFP-TapΔC/RNA complexes. After disruption of the RNP complexes, RT-PCRs were performed on the precipitated RNA and the input to specifically amplify MLV RNA, or 4-CTE RNA, and two cellular RNA controls: actinB mRNA and U6 snRNA, the nuclear export of which rely on the Tap and CRM1 pathways, respectively. Control PCR experiments were systematically performed without prior RT reaction as a control for DNA contamination of RNA extracts. Amplicons were analyzed on 1% agarose gel.
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