MLV requires Tap/NXF1-dependent pathway to export its unspliced RNA to the cytoplasm and to express both spliced and unspliced RNAs
© Pessel-Vivares et al.; licensee BioMed Central Ltd. 2014
Received: 15 November 2013
Accepted: 4 February 2014
Published: 5 March 2014
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.
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.
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.
Cellular mRNAs are fully spliced prior to their export from the nucleus. The quality of gene expression is assured by proofreading mechanisms that eliminate unprocessed or irregular pre-mRNAs [1, 2]. Retroviral RNA, however, needs to be exported to the cytoplasm in a partially spliced or totally unspliced, full-length (FL), form in order to serve as a template for protein synthesis. Furthermore, in addition to producing the structural proteins (Gag) and enzymes (GagPol), unspliced RNA also acts as genomic RNA to be packaged into virions. To achieve this nuclear export of incompletely spliced and FL RNAs, complex retroviruses, such as HIV, encode an adaptor protein (Rev) that bridges the FL RNA, via its Rev-responsive element (RRE), and the CRM1 nuclear export factor . Simpler retroviruses such as Mason-Pfizer monkey virus (MPMV), recruit the global mRNA export pathway mediated by the cellular Tap factor (also called NXF1) that directly binds a constitutive transport element (CTE) in the FL RNA . The scenario appears to be more complex for the export of avian retroviral FL RNA. Indeed, Rous sarcoma virus (RSV), another simple retrovirus, relies on the CRM1 pathway using Gag protein as an adapter  and on the Tap pathway via two direct repeat (DR) sequences in the FL RNA .
The murine leukemia virus (MLV) was among the first retroviruses to be studied and constitutes the prototype for simple retroviruses. Moloney-MLV, in particular, has been key in our understanding of basic cellular processes such as the discovery of oncogenes , eukaryotic gene regulation, viral pathogenesis , and therapeutic gene transfer trials with MLV-based vectors . Although MLV has been extensively studied, the export pathway used by MLV RNAs to reach the cytoplasm remains unidentified. Several cis-elements, such as the Psi motifs and R region sequences in the 5' UTR of FL RNA, have been reported to promote the cytoplasmic accumulation of FL RNA [10–13]. However, the fact that these cis-acting elements also modulate RNA splicing efficiency, RNA stability and virus assembly has made it difficult to elucidate the nuclear export pathway that they use (reviewed in ).
In this study, we have investigated the nuclear export of MLV RNAs. We show that inhibition of the Tap-pathway dramatically decreases viral protein production and simultaneously decreases the levels of spliced and unspliced MLV RNAs. A sensitive and specific fluorescence detection method was used to study the export of unspliced viral RNA. RNA imaging and ribonucleoprotein (RNP) immunoprecipitation assays (RIP) provided strong evidence for the recruitment of the Tap-pathway by MLV to export its RNAs to the cytoplasm.
MLV expression is dependent on the Tap pathway
Tap-pathway inhibition decreases the spliced and unspliced MLV RNA levels
TapΔC inhibits nuclear export of viral unspliced RNA
TapΔC interacts with spliced and unspliced MLV RNAs
In this report, different strategies were used to block the Tap-pathway, in cis with siRNA or excess of 4-CTE RNA competitor and in trans with a dominant-negative Tap mutant (TapΔC). We have demonstrated for the first time that Tap is required for MLV expression and that it interacts with both spliced and unspliced MLV RNAs. When interacting with TapΔC, the unspliced RNA was restricted to the nucleus, indicating that MLV RNA export requires the Tap-dependent pathway. Our results support the notion that MLV recruits the same pathway to export its spliced and unspliced RNAs. In contrast to HIV, which uses different export pathways to transport its fully-spliced and unspliced RNAs, MLV regulates the expression of its FL RNA through a highly coordinated splicing/export process. Furthermore, the Tap-pathway is a prerequisite to the translation of the two MLV RNA species, since Gag and Env protein levels were sensitive to TapΔC. Unlike HIV-1, MLV has two distinct pools of FL RNA with two different destinies: translation and packaging into new particles . In this scenario, our results support the notion that the translated RNA pool is exported by the Tap pathway. Whether the packageable FL RNA is exported by the same Tap mechanism remains to be established although this will be difficult to do. It will be interesting to determine whether MLV uses a similar replication strategy to RSV, another simple retrovirus. Interestingly, it has been postulated that RSV, after exporting a fraction of FL RNA from the nucleus to produce large amounts of structural Gag proteins for virus formation, uses excess Gag proteins to fetch the remainder of the FL RNA in the nucleus and to route it to the virus assembly sites where it serves as genome in new virus particles. Complex retroviruses regulate this temporal switch between early and late steps of replication differently, by using early and late gene expression (for review ). It must now be established whether nuclear export pathway use regulates cytosolic RNA fate.
The authors acknowledge the technical assistance of C. Chamontin. We are grateful to the Montpellier RIO Imaging staff and to E. Bertrand's team for advice on FISH and RIP experiments. We want to thank Drs H. Wodrich, J. Bohne, S.F. Flint, E. Izaurralde E.Bertrand and M.Biard for providing reagents. Many thanks to J.M. Jacqué and I. Robbins for critical reading of the manuscript.
This work was supported by institutional grants from the CNRS and the Universities of Montpellier (UMI&UMII). LPV was supported by fellowships from the Ministère de l'Enseignement Supérieur et de la Recherche and La Ligue contre le Cancer and MFA from French National Agency for Research on AIDS and Viral hepatitis.
- Le Hir H, Nott A, Moore MJ: How introns influence and enhance eukaryotic gene expression. Trends Biochem Sci. 2003, 28: 215-220. 10.1016/S0968-0004(03)00052-5.View ArticlePubMedGoogle Scholar
- Behm-Ansmant I, Kashima I, Rehwinkel J, Sauliere J, Wittkopp N, Izaurralde E: mRNA quality control: an ancient machinery recognizes and degrades mRNAs with nonsense codons. FEBS Lett. 2007, 581: 2845-2853. 10.1016/j.febslet.2007.05.027.View ArticlePubMedGoogle Scholar
- Malim MH, Hauber J, Le SY, Maizel JV, Cullen BR: The HIV-1 rev trans-activator acts through a structured target sequence to activate nuclear export of unspliced viral mRNA. Nature. 1989, 338: 254-257. 10.1038/338254a0.View ArticlePubMedGoogle Scholar
- Bray M, Prasad S, Dubay JW, Hunter E, Jeang KT, Rekosh D, Hammarskjold ML: A small element from the Mason-Pfizer monkey virus genome makes human immunodeficiency virus type 1 expression and replication Rev- independent. Proc Natl Acad Sci USA. 1994, 91: 1256-1260. 10.1073/pnas.91.4.1256.PubMed CentralView ArticlePubMedGoogle Scholar
- Gudleski N, Flanagan JM, Ryan EP, Bewley MC, Parent LJ: Directionality of nucleocytoplasmic transport of the retroviral gag protein depends on sequential binding of karyopherins and viral RNA. Proc Natl Acad Sci USA. 2010, 107: 9358-9363. 10.1073/pnas.1000304107.PubMed CentralView ArticlePubMedGoogle Scholar
- LeBlanc JJ, Uddowla S, Abraham B, Clatterbuck S, Beemon KL: Tap and Dbp5, but not Gag, are involved in DR-mediated nuclear export of unspliced Rous sarcoma virus RNA. Virology. 2007, 363: 376-386. 10.1016/j.virol.2007.01.026.PubMed CentralView ArticlePubMedGoogle Scholar
- Dudley JP: Tag, you're hit: retroviral insertions identify genes involved in cancer. Trends Mol Med. 2003, 9: 43-45. 10.1016/S1471-4914(03)00003-0.View ArticlePubMedGoogle Scholar
- Fan H: Leukemogenesis by Moloney murine leukemia virus: a multistep process. Trends Microbiol. 1997, 5: 74-82. 10.1016/S0966-842X(96)10076-7.View ArticlePubMedGoogle Scholar
- Cavazzana-Calvo M, Fischer A: Gene therapy for severe combined immunodeficiency: are we there yet?. J Clin Invest. 2007, 117: 1456-1465. 10.1172/JCI30953.PubMed CentralView ArticlePubMedGoogle Scholar
- Smagulova F, Maurel S, Morichaud Z, Devaux C, Mougel M, Houzet L: The highly structured encapsidation signal of MuLV RNA is involved in the nuclear export of its unspliced RNA. J Mol Biol. 2005, 354: 1118-1128. 10.1016/j.jmb.2005.10.021.View ArticlePubMedGoogle Scholar
- Basyuk E, Boulon S, Skou Pedersen F, Bertrand E, Vestergaard Rasmussen S: The packaging signal of MLV is an integrated module that mediates intracellular transport of genomic RNAs. J Mol Biol. 2005, 354: 330-339. 10.1016/j.jmb.2005.09.071.View ArticlePubMedGoogle Scholar
- Trubetskoy AM, Okenquist SA, Lenz J: R region sequences in the long terminal repeat of a murine retrovirus specifically increase expression of unspliced RNAs. J Virol. 1999, 73: 3477-3483.PubMed CentralPubMedGoogle Scholar
- King JA, Bridger JM, Gounari F, Lichter P, Schulz TF, Schirrmacher V, Khazaie K: The extended packaging sequence of MoMLV contains a constitutive mRNA nuclear export function. FEBS Lett. 1998, 434: 367-371. 10.1016/S0014-5793(98)00948-X.View ArticlePubMedGoogle Scholar
- Jouvenet N, Lainé S, Pessel-Vivares L, Mougel M: Cell biology of retroviral RNA packaging. RNA Biol. 2011, 8: 1-9. 10.4161/rna.8.1.15302.View ArticleGoogle Scholar
- Houzet L, Battini JL, Bernard E, Thibert V, Mougel M: A new retroelement constituted by a natural alternatively spliced RNA of murine replication-competent retroviruses. EMBO J. 2003, 22: 4866-4875. 10.1093/emboj/cdg450.PubMed CentralView ArticlePubMedGoogle Scholar
- Wolff B, Sanglier JJ, Wang Y: Leptomycin B is an inhibitor of nuclear export: inhibition of nucleo-cytoplasmic translocation of the human immunodeficiency virus type 1 (HIV-1) Rev protein and Rev-dependent mRNA. Chem Biol. 1997, 4: 139-147. 10.1016/S1074-5521(97)90257-X.View ArticlePubMedGoogle Scholar
- Yatherajam G, Huang W, Flint SJ: Export of adenoviral late mRNA from the nucleus requires the Nxf1/Tap export receptor. J Virol. 2010, 85: 1429-1438.PubMed CentralView ArticlePubMedGoogle Scholar
- Wodrich H, Schambach A, Krausslich HG: Multiple copies of the Mason-Pfizer monkey virus constitutive RNA transport element lead to enhanced HIV-1 Gag expression in a context-dependent manner. Nucleic Acids Res. 2000, 28: 901-910. 10.1093/nar/28.4.901.PubMed CentralView ArticlePubMedGoogle Scholar
- Bachi A, Braun IC, Rodrigues JP, Pante N, Ribbeck K, von Kobbe C, Kutay U, Wilm M, Gorlich D, Carmo-Fonseca M, Izaurralde E: The C-terminal domain of TAP interacts with the nuclear pore complex and promotes export of specific CTE-bearing RNA substrates. RNA. 2000, 6: 136-158. 10.1017/S1355838200991994.PubMed CentralView ArticlePubMedGoogle Scholar
- Vyboh K, Ajamian L, Mouland AJ: Detection of viral RNA by fluorescence in situ hybridization (FISH). J Vis Exp. 2012, 63: e4002-PubMedGoogle Scholar
- Mougel M, Zhang Y, Barklis E: Cis-active structural motifs involved in specific encapsidation of Moloney Murine Leukemia Virus RNA. J Virol. 1996, 70: 5043-5050.PubMed CentralPubMedGoogle Scholar
- Mougel M, Barklis E: A role for two hairpin structures as a core RNA encapsidation signal in murine leukemia virus virions. J Virol. 1997, 71: 8061-8065.PubMed CentralPubMedGoogle Scholar
- Basyuk E, Galli T, Mougel M, Blanchard JM, Sitbon M, Bertrand E: Retroviral genomic RNAs are transported to the plasma membrane by endosomal vesicles. Dev Cell. 2003, 5: 161-174. 10.1016/S1534-5807(03)00188-6.View ArticlePubMedGoogle Scholar
- Boireau S, Maiuri P, Basyuk E, de la Mata M, Knezevich A, Pradet-Balade B, Backer V, Kornblihtt A, Marcello A, Bertrand E: The transcriptional cycle of HIV-1 in real-time and live cells. J Cell Biol. 2007, 179: 291-304. 10.1083/jcb.200706018.PubMed CentralView ArticlePubMedGoogle Scholar
- Fury MG, Zieve GW: U6 snRNA maturation and stability. Exp Cell Res. 1996, 228: 160-163. 10.1006/excr.1996.0311.View ArticlePubMedGoogle Scholar
- Dorman N, Lever A: Comparison of viral genomic RNA sorting mechanisms in human immunodeficiency virus type 1 (HIV-1), HIV-2, and Moloney murine leukemia virus. J Virol. 2000, 74: 11413-11417. 10.1128/JVI.74.23.11413-11417.2000.PubMed CentralView ArticlePubMedGoogle Scholar
- Stake MS, Bann DV, Kaddis RJ, Parent LJ: Nuclear trafficking of retroviral RNAs and Gag proteins during late steps of replication. Viruses. 2013, 5: 2767-2795. 10.3390/v5112767.PubMed CentralView ArticlePubMedGoogle Scholar
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