CRM1-mediated nuclear export of dengue virus RNA polymerase NS5 modulates interleukin-8 induction and virus production

Abstract

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.

FIGURE 1.

Top, DENV-2 NS5 possesses two domains, the N-terminal methyltransferase (MET) domain and C-terminal RNA-dependent RNA polymerase (RdRp) domain. The interdomain linker region (residues 320–405, containing two adjacent NLSs) is highlighted. bNLS (residues 320–368) and aNLS (residues 369–405) bind Imp-β/NS3 and Imp-α/β, respectively. Bottom, shown are the GFP-NS5 constructs made in this study. Encoded proteins are shown with the N terminus (residues 1–319; white boxes), bNLS (dotted boxes), aNLS (black boxes), and C terminus (residues 406–900; striped boxes) marked. Hydrophobic regions (termed HR1–HR3) identified as putative NESs are highlighted, and the corresponding alanine substitutions (underlined) generated in this study are indicated (mHR1–mHR3).

FIGURE 2.

NS5 is exported from the nucleus via a CRM1-dependent nuclear export pathway dependent on residues 1–368. A, shown are CLSM images of Vero cells transfected to express the indicated GFP-NS5 constructs in the presence or absence of LMB and imaged at 20–24 h post-transfection. Human immunodeficiency virus Rev (GFP-Rev) and GFP were used as positive and negative controls, respectively. B, quantitative analysis of CLSM images such as those in A was performed to determine the F_n/c values (see “Experimental Procedures”). The results represent the means ± S.E., where significant differences (p values) are indicated between LMB-treated and untreated cells (n ≥ 25).

FIGURE 3.

Mapping of the NS5 NES. A, shown are CLSM images of Vero cells transfected to express wild-type (WT) GFP-NS5-(1–368) and mutant derivatives thereof treated with or without LMB. B, quantitative analysis of CLSM images such as those in A was performed to determine the F_n/c values. C, shown are CLSM images of Vero cells transfected to express wild-type GFP-NS5-(1–900), GFP-NS5-(1–900)-mHR3, and GFP-NS5-(1–900)-aNLS-A1+A2 treated with or without LMB. D, quantitative analysis was performed as described for B. Significant differences (p values) are indicated between LMB-treated and untreated cells (n ≥ 27).

FIGURE 4.

NS5 complexes with CRM1 in vivo. Non-expressing and GST-, GST-NS5-, and GST-NS5-(1–368)-expressing HEK-293T cells were lysed, and GST co-precipitation assays were performed (see “Experimental Procedures”). Immunoblotting with antibodies to CRM1 and GST was used to detect co-precipitated proteins in combination with horseradish peroxidase-conjugated secondary antibodies for visualization by enhanced chemiluminescence. Molecular mass markers are indicated. WB, Western blot.

FIGURE 5.

Inhibition of CRM1 during DENV-2 infection of Vero cells increases nuclear accumulation of NS5, but not capsid protein. A, CLSM images of fixed mock- and DENV-2-infected Vero cells immunostained for NS5 and capsid protein (C) at the indicated time points post-infection and treated with or without LMB. Quantitative analysis to determine F_n/c values for NS5 (B) and F_n/c and F_nu/c values for capsid protein (C) was performed as described in the legend to Fig. 2. Results represent the means ± S.E., where significant differences (p values) are indicated between LMB-treated and untreated cells (n ≥ 48).

FIGURE 6.

Virus production and suppression of IL-8 induction. Virus production (A) and IL-8 induction (B) throughout DENV-2 infection as determined by plaque assay and enzyme-linked immunosorbent assay, respectively, were investigated in the presence or absence of LMB. Results for virus production represent the means ± S.D. of three independent experiments performed in triplicate, whereas those for IL-8 production represent the means ± S.E. of two independent experiments performed in duplicate. PFU, plaque-forming units.

FIGURE 7.

Conservation of the NS5 NES within the interdomain linker region (residues 320–405) in Flavivirus members. The NS5 NES (residues 327–343) is highlighted, with the critical residues mutated in this study shown in boldface. The GenBank^TM accession numbers of the following sequences are as indicated: DENV-2 TSV01 (DEN-2; AY037116), DENV-1 (DEN-1; U88535), DENV-3 (DEN-3; M93130), DENV-4 (DEN-4; AF32657), yellow fever (YF; X15062), West Nile (WN; M12294), Kunjin (KUN; D00246), Japanese encephalitis virus (JE; M55506), Murray Valley encephalitis virus (MVE; AF161266), and tick-borne encephalitis virus (TBE; U27495).