CRM1dependent nuclear export of dengue virus type 2 NS5

Melinda J. Pryor, Stephen M. Rawlinson, Peter J. Wright* and David A. Jans1

Department of Biochemistry and Molecular Biology, and * Department of Microbiology Monash University, Victoria 3800, Australia

Abstract. The dengue virus multidomain RNA polymerase NS5 has been observed in the nucleus in mammalian infected cell systems. We previously showed that NS5 nuclear localization is mediated by two nuclear targeting signals within the NS5 interdomain region that are recognized by distinct members of the importin superfamily of intra-cellular transporters. Intriguingly, we have recently found that NS5 also possesses the ability to be exported from the nucleus by the importin family member CRM1 (exportin 1) both in Vero cells transfected to express NS5, and in dengue virus type 2 infected Vero cells, based on use of the CRM1-specific inhibitor leptomycin B (LMB). LMB treatment of Vero cells resulted in increased nuclear accumulation in both systems, and interestingly in the latter, resulted in an alteration in the kinetics of virus production. Our results imply that subcellular trafficking of NS5 at particular times in the infectious cycle may be central to the kinetics of virus production; perturbing this trafficking may represent a viable approach to develop new antiviral therapeutics.

2006 New treatment strategies for dengue and other flaviviral diseases. Wiley, Chichester (Novartis Foundation Symposium 277) p 149—163

The dengue virus non-structural protein NS5 is the largest protein encoded by the viral genome and the most highly conserved of the flavivirus proteins. It is a multifunctional protein comprising an N-terminal methyltransferase (MTase) (Egloff et al 2002) and a C-terminal RNA-dependent RNA polymerase (RdRp) (Bartholomeusz & Wright 1993, Tan et al 1996, Ackermann & Padmanabhan 2001) domains, separated by an interdomain region containing functional nuclear targeting signals (Forwood et al 1999, Brooks et al 2002) (see Fig. 1). Similar to other animal RNA viruses, flavivirus RNA replication occurs in the cytoplasm of host cells in membrane-induced perinuclear vesicles (Westaway et al 1999). A hypo-phosphorylated form of dengue virus type 2 (DENV-2) NS5 has been shown to

1 This paper was presented at the symposium by David A. Jans, to whom correspondence should be addressed.

FIG. 1. Schematic diagram of the NS5 protein highlighting the interdomain region (residues 320-405). The single letter amino acid code is used with the bNLS highlighted and aNLS underlined. The basic residues in the aNLS are shown in bold. The binding sites for importin P, NS3 and importin ap are indicated.

FIG. 1. Schematic diagram of the NS5 protein highlighting the interdomain region (residues 320-405). The single letter amino acid code is used with the bNLS highlighted and aNLS underlined. The basic residues in the aNLS are shown in bold. The binding sites for importin P, NS3 and importin ap are indicated.

interact with NS3 in the cytoplasm (Kapoor et al 1995), constituting part of the proposed replication complex that also includes the other non-structural proteins NS1, NS2A and NS4A (Mackenzie et al 1998). The 5' end of the DENV-2 RNA genome has been shown to contain a type 1 cap (Cleaves & Dubin 1979). Capping of cellular mRNA is normally performed in the cell nucleus, but viruses like dengue that replicate in the cytoplasm must encode their own cytoplasmic capping enzymes. NS5 possess the nucleoside-2'-0-methyltransferase activity (Egloff et al

2002), one of the four enzymatic activities required for the capping process.

All the known functions of DENV-2 NS5 are believed to occur in the cytosol of infected cells, but a hyperphosphorylated form of NS5, that is unable to interact with NS3, has been reported in the nucleus of DENV-2 infected mammalian cells late in infection (Kapoor et al 1995). These results suggest that phosphorylation is involved in modulating NS5 subcellular localization; the NS5/NS5A proteins belonging to all three genera of the Flaviviridae are known to be phosphorylated (Reed et al 1998). The role of DENV-2 NS5 within the nucleus has yet to be established, but it has been speculated to be involved in the regulation of cellular genes expressed in response to viral infection (Kapoor et al 1995), and promoting a shift from RNA replication to the packaging phase of infection (Butcher

Nuclear import of proteins >45 kDa is generally dependent on transport receptors called importins that bind to proteins and transport them through the nuclear envelope localized nuclear pore complexes (NPCs) (Paschal 2002). There are two classes of well characterized classical nuclear localization sequences (NLSs) that confer recognition by importins; these include monopartite NLSs containing a single cluster of basic amino acids, and bipartite NLSs comprising two clusters of basic residues separated by a variable spacer region (Jans et al 2000). Additional non-basic NLSs have also been described. NLS-containing proteins are conventionally recognized in the cytoplasm by a heterodimer composed of importin a and ß1 (Jans et al 2000). Importin a binds directly to the NLS (Conti et al 1998) and importin ß1 is responsible for the translocation of the NLS-containing protein-importin complex into the nucleus by interaction with the protein constituents of the NPC (Gorlich & Kutay 1999). It has been shown that nuclear import can also be mediated by importin ß1 or importin ß homologues exclusively, whereby importin ß1/the importin ß homologue is able to bind the NLS directly without the need for an adaptor protein such as importin a, (Jans et al 2000). Dissociation of the NLS-containing protein—importin complex is facilitated by binding to importin ß1/the importin ß homologue by the nuclear guanine nucleotide binding protein Ran in activated GTP-bound form (Pemberton & Paschal 2005). Analogously, nuclear export of proteins is mediated by transport receptors of the importin ß superfamily, called exportins, in association with Ran-GTP (Pemberton & Paschal 2005). The best studied exportin, CRM1, binds to leucine-rich nuclear export sequences (NESs) (Fornerod et al 1997), including that of HIV-Rev (Fischer et al 1995). Leptomycin B (LMB), a specific inhibitor of CRM1, has been shown to block this nuclear export pathway specifically by preventing NES-CRM1 interaction (Fukuda et al 1997).

The interdomain linker region of DENV-2 NS5 between the methyltransferase and polymerase domains has been shown to contain two functional NLSs (Fig. 1). NS5 residues 369—405 were initially demonstrated to contain a functional NLS recognized by the importin aß heterodimer (Forwood et al 1999). Site-directed mutagenesis established that the two basic regions K371K372 and K388K389 separated by a 14 amino acid linker were critical for nuclear localization, referred to as the aNLS (Brooks et al 2002). Using the yeast two-hybrid system, residues 320—368 adjacent to the previously characterized NLS were found to confer interaction with importin ß1 (Johansson et al 2001). This region was subsequently shown to be able to target ß-galactosidase to the nucleus of mammalian cells in an in vitro reconstituted system, albeit at slower rates than that observed for aNLS (Brooks et al 2002). This region is referred to as the bNLS, although the critical residues of the NLS have yet to be identified.

The overall objective of this work was to investigate further the subcellular localization of DENV-2 NS5 in virus-infected and transfected mammalian cells. Confocal laser scanning microscopy (CLSM) of immunostained DENV-2-infected Vero cells was used to monitor the localization of NS5 during infection. Surprisingly, NS5 was detected in the nucleus early in infection, with this localization continuing throughout the course of infection. Using the CRM1-specific inhibitor LMB, we were able to establish that NS5 possesses the ability to be exported from the nucleus, and that disruption of nuclear export in virus-infected cells altered the kinetics of virus production. This latter implies that the subcellular trafficking of NS5 during infection may be important to the kinetics of virus production. Antiviral drugs designed to disrupt the trafficking of NS5 may represent a viable strategy to control the progression of dengue virus infection.

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