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CRM1-mediated Nuclear Export of Dengue Virus RNA Polymerase NS5 Modulates Interleukin-8 Induction and Virus Production

2009; Elsevier BV; Volume: 284; Issue: 23 Linguagem: Inglês

10.1074/jbc.m808271200

1083-351X

Stephen M. Rawlinson, Melinda J. Pryor, P.J. Wright, David A. Jans,

HIV Research and Treatment

Although all established functions of dengue virus NS5 (nonstructural protein 5) occur in the cytoplasm, its nuclear localization, mediated by dual nuclear localization sequences, is essential for virus replication. Here, we have determined the mechanism by which NS5 can localize in the cytoplasm to perform its role in replication, establishing for the first time that it is able to be exported from the nucleus by the exportin CRM1 and hence can shuttle between the nucleus and cytoplasm. We define the nuclear export sequence responsible to be residues 327–343 and confirm interaction of NS5 and CRM1 by pulldown assay. Significantly, greater nuclear accumulation of NS5 during infection due to CRM1 inhibition coincided with altered kinetics of virus production and decreased induction of the antiviral chemokine interleukin-8. This is the first report of a nuclear export sequence within NS5 for any member of the Flavivirus genus; because of its high conservation within the genus, it may represent a target for the treatment of diseases caused by several medically important flaviviruses. Although all established functions of dengue virus NS5 (nonstructural protein 5) occur in the cytoplasm, its nuclear localization, mediated by dual nuclear localization sequences, is essential for virus replication. Here, we have determined the mechanism by which NS5 can localize in the cytoplasm to perform its role in replication, establishing for the first time that it is able to be exported from the nucleus by the exportin CRM1 and hence can shuttle between the nucleus and cytoplasm. We define the nuclear export sequence responsible to be residues 327–343 and confirm interaction of NS5 and CRM1 by pulldown assay. Significantly, greater nuclear accumulation of NS5 during infection due to CRM1 inhibition coincided with altered kinetics of virus production and decreased induction of the antiviral chemokine interleukin-8. This is the first report of a nuclear export sequence within NS5 for any member of the Flavivirus genus; because of its high conservation within the genus, it may represent a target for the treatment of diseases caused by several medically important flaviviruses. The four serotypes of dengue virus (DENV-1–4) 2The abbreviations used are:DENVdengue virusNLSnuclear localization sequenceImpimportinNESnuclear export sequenceLMBleptomycin BIL-8interleukin-8GFPgreen fluorescent proteinGSTglutathione S-transferaseCLSMconfocal laser scanning microscopy. 2The abbreviations used are:DENVdengue virusNLSnuclear localization sequenceImpimportinNESnuclear export sequenceLMBleptomycin BIL-8interleukin-8GFPgreen fluorescent proteinGSTglutathione S-transferaseCLSMconfocal laser scanning microscopy. are the causative agents of the most common arthropod-borne viral disease, dengue fever, and its more severe and potentially deadly dengue hemorrhagic fever form (1.Gubler D.J. Arch. Med. Res. 2002; 33: 330-342Crossref PubMed Scopus (722) Google Scholar). DENV is a member of the genus Flavivirus within the family Flaviviridae. Like all flaviviruses, DENV possesses an ∼11-kb, positive-sense, single-stranded RNA genome that is translated as one long polyprotein and cleaved into 10 viral proteins: three structural (capsid, pre-membrane/membrane, and envelope) and seven nonstructural (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) proteins (2.Lindenbach B.D. Rice C.M. Flaviviridae: The Viruses and Their Replication. Lippincott Williams & Wilkins, Philadelphia2001Google Scholar). Flavivirus replication takes place in the cytoplasm, whereby several viral NS and host proteins are believed to constitute the replication complex, the proposed replication machinery of flaviviruses (3.Mackenzie J.M. Westaway E.G. J. Virol. 2001; 75: 10787-10799Crossref PubMed Scopus (255) Google Scholar). Two key enzymes in replication, NS3 and NS5, the RNA helicase and RNA-dependent RNA polymerase, respectively, interact within the cytoplasm of infected cells (4.Kapoor M. Zhang L. Ramachandra M. Kusukawa J. Ebner K.E. Padmanabhan R. J. 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NS5 contains an N-terminal S-adenosylmethyltransferase domain (5.Egloff M.P. Benarroch D. Selisko B. Romette J.L. Canard B. EMBO J. 2002; 21: 2757-2768Crossref PubMed Scopus (469) Google Scholar) and a C-terminal RNA-dependent RNA polymerase domain (8.Ackermann M. Padmanabhan R. J. Biol. Chem. 2001; 276: 39926-39937Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar, 9.Bartholomeusz A.I. Wright P.J. Arch. Virol. 1993; 128: 111-121Crossref PubMed Scopus (83) Google Scholar, 10.Tan B.H. Fu J. Sugrue R.J. Yap E.H. Chan Y.C. Tan Y.H. Virology. 1996; 216: 317-325Crossref PubMed Scopus (217) Google Scholar) separated by an "interdomain linker region" (see Fig. 1). Despite all well established functions of NS5 occurring within the cytoplasm (2.Lindenbach B.D. Rice C.M. Flaviviridae: The Viruses and Their Replication. Lippincott Williams & Wilkins, Philadelphia2001Google Scholar), NS5 is predominantly nuclear in DENV-2-infected cells (4.Kapoor M. Zhang L. Ramachandra M. 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Within the nucleus, the cargo-NLS-Imp complex is dissociated through binding of Ran-GTP to Imp-β, releasing the cargo into the nucleoplasm. Analogously, proteins containing nuclear export sequences (NESs) interact with Imp-β homologs termed exportins, which, when complexed with Ran-GTP, mediate translocation out of the nucleus and into the cytoplasm (14.Fried H. Kutay U. CMLS Cell. Mol. Life Sci. 2003; 60: 1659-1688Crossref PubMed Scopus (402) Google Scholar, 15.Kutay U. Guttinger S. Trends Cell Biol. 2005; 15: 121-124Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar, 16.Mosammaparast N. Pemberton L.F. Trends Cell Biol. 2004; 14: 547-556Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar). The best characterized of these is CRM1 (exportin 1), which typically binds hydrophobic/leucine-rich NESs (17.Fornerod M. Ohno M. Yoshida M. Mattaj I.W. Cell. 1997; 90: 1051-1060Abstract Full Text Full Text PDF PubMed Scopus (1724) Google Scholar, 18.la Cour T. Kiemer L. Molgaard A. Gupta R. Skriver K. Brunak S. Protein Eng. Des. Sel. 2004; 17: 527-536Crossref PubMed Scopus (621) Google Scholar), such as those of the human immunodeficiency virus Rev protein (19.Fischer U. Huber J. Boelens W.C. Mattaj I.W. Luhrmann R. Cell. 1995; 82: 475-483Abstract Full Text PDF PubMed Scopus (976) Google Scholar) or the protein kinase A inhibitor PKI (20.Wen W. Meinkoth J.L. Tsien R.Y. Taylor S.S. Cell. 1995; 82: 463-473Abstract Full Text PDF PubMed Scopus (991) Google Scholar). The antibiotic leptomycin B (LMB), able to bind CRM1 specifically and to prevent CRM1-NES interaction (21.Kudo N. Matsumori N. Taoka H. Fujiwara D. Schreiner E.P. Wolff B. Yoshida M. Horinouchi S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 9112-9117Crossref PubMed Scopus (835) Google Scholar, 22.Kudo N. Wolff B. Sekimoto T. Schreiner E.P. Yoneda Y. Yanagida M. Horinouchi S. Yoshida M. Exp. Cell Res. 1998; 242: 540-547Crossref PubMed Scopus (700) Google Scholar, 23.Nishi K. Yoshida M. 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Previously, we showed that DENV-2 NS5 possesses two NLSs within the interdomain region (see Fig. 1) (29.Forwood J.K. Brooks A. Briggs L.J. Xiao C.Y. Jans D.A. Vasudevan S.G. Biochem. Biophys. Res. Commun. 1999; 257: 731-737Crossref PubMed Scopus (83) Google Scholar). The C-terminal NLS (amino acids 369–405) or "aNLS" is recognized by Imp-α/β with high affinity and is able to target β-galactosidase to the nucleus in either microinjected or mechanically perforated rat hepatoma cells (29.Forwood J.K. Brooks A. Briggs L.J. Xiao C.Y. Jans D.A. Vasudevan S.G. Biochem. Biophys. Res. Commun. 1999; 257: 731-737Crossref PubMed Scopus (83) Google Scholar). In contrast, the N-terminal NLS (amino acids 320–368) or "bNLS" is able to bind either Imp-β or NS3 directly in a competitive fashion (30.Brooks A.J. Johansson M. John A.V. Xu Y. Jans D.A. Vasudevan S.G. J. Biol. Chem. 2002; 277: 36399-36407Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, 31.Johansson M. Brooks A.J. Jans D.A. Vasudevan S.G. J. Gen. Virol. 2001; 82: 735-745Crossref PubMed Scopus (147) Google Scholar). We demonstrated recently that when NS5 nuclear import is impaired by mutation of the viral genome, the virus is no longer viable, indicating that NS5 nuclear import is essential for virus replication (11.Pryor M.J. Rawlinson S.M. Butcher R.E. Barton C.L. Waterhouse T.A. Vasudevan S.G. Bardin P.G. Wright P.J. Jans D.A. Davidson A.D. Traffic. 2007; 8: 795-807Crossref PubMed Scopus (137) Google Scholar). This is attributable, at least in part, to the role of nuclear NS5 in inhibiting induction of the antiviral chemokine interleukin-8 (IL-8) during DENV infection; reduced NS5 nuclear accumulation correlates with increased virus production (11.Pryor M.J. Rawlinson S.M. Butcher R.E. Barton C.L. Waterhouse T.A. Vasudevan S.G. Bardin P.G. Wright P.J. Jans D.A. Davidson A.D. Traffic. 2007; 8: 795-807Crossref PubMed Scopus (137) Google Scholar, 32.Medin C.L. Fitzgerald K.A. Rothman A.L. J. Virol. 2005; 79: 11053-11061Crossref PubMed Scopus (98) Google Scholar). At least one of the roles of nuclear NS5 is thus to reduce the magnitude of the antiviral response in terms of IL-8 induction. The undisputed role of NS5 is in replication in the cytoplasm. Here, we shed light for the first time on how despite its efficient nuclear localization ability, NS5 is able to fulfill this role. We report the ability of NS5 to be exported from the nucleus in a CRM1-dependent fashion in both transfected and DENV-2-infected cells and identify the NES responsible. Importantly, we show that inhibition of CRM1 during DENV-2 infection results in increased nuclear NS5, reduced IL-8 induction, and increased virus production, underlining the importance of NS5 nuclear export to DENV infection. Our results thus establish for the first time the ability of NS5 to shuttle between the nucleus and cytoplasm and its importance to modulation of the host antiviral response and virus replication. Vero, 293, and HEK-293T cells were cultured in Dulbecco's modified Eagle's medium with 10% fetal calf serum. C6/36 mosquito (Aedes albopictus) cells used for infection were cultured in basal Eagle's medium containing 10% fetal calf serum. DENV-2 New Guinea C strain-infected Vero cells were maintained in Dulbecco's modified Eagle's medium containing 2% fetal calf serum. Where indicated, 5 ng/ml LMB (provided by M. Yoshida) was added to the culture medium at 0 h post-infection. At specific time points post-infection, the culture medium was collected for virus titration, and coverslips were seeded with Vero cells fixed for indirect immunofluorescence (see below). Virus titers, calculated as plaque-forming units/ml, were determined in plaque assays using C6/36 cells (33.Gualano R.C. Pryor M.J. Cauchi M.R. Wright P.J. Davidson A.D. J. Gen. Virol. 1998; 79: 437-446Crossref PubMed Scopus (132) Google Scholar). DNA transfection of Vero cells was performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Where indicated, 2.8 ng/ml LMB was added to the culture medium 15–19 h post-transfection, and live cell imaging was performed (see below) at 20–24 h post-transfection. Constructs expressing full-length and truncated NS5 forms fused in-frame with GFP at the N terminus (see Fig. 1) were generated using GatewayTM cloning technology (Invitrogen) according to the manufacturer's instructions. Briefly, NS5 PCR fragments from the DENV-2 Townsville strain (TSV01) were generated using primers designed to include attB1 or attB2 sites to allow integration into the Gateway system. These truncations were inserted into the pDONR207 vector by the BP-Reaction (Invitrogen) and subsequently into the mammalian GFP fusion protein construct pEPI-GFP (34.Ghildyal R. Ho A. Wagstaff K.M. Dias M.M. Barton C.L. Jans P. Bardin P. Jans D.A. Biochemistry. 2005; 44: 12887-12895Crossref PubMed Scopus (89) Google Scholar) or the pDEST27 mammalian GST fusion protein expression construct using the LR-Reaction (Invitrogen). Hydrophobic regions (HR1–HR3) (Fig. 1) were selected as putative NES regions based on their similarity to known NESs (35.la Cour T. Gupta R. Rapacki K. Skriver K. Poulsen F.M. Brunak S. Nucleic Acids Res. 2003; 31: 393-396Crossref PubMed Scopus (190) Google Scholar). Leucine/isoleucine/valine residues within these regions were mutated to alanine to generate mHR1–mHR3, whereby primers containing the appropriate mutations were designed and used in overlap extension PCR as described previously (36.Ho S.N. Hunt H.D. Horton R.M. Pullen J.K. Pease L.R. Gene (Amst.). 1989; 77: 51-59Crossref PubMed Scopus (6771) Google Scholar). Overlap extension PCR products encoding either full-length NS5 or NS5-(1–368) containing the appropriate mutations were inserted into pDONR207 and then pEPI-GFP using the BP-Reaction and LR-Reaction, respectively.