Here we report for the first time that DENV-2 NS5 RNA polymerase possesses the ability to be exported from the nucleus by the importin family member CRM1. Previously, we demonstrated that NS5 possesses two functional NLSs, recognized by importin ap and p1, which mediate its nuclear import. Together, these results imply that NS5 has the ability to shuttle between nuclear and cytoplasmic compartments through distinct NLS- and NES-dependent nuclear transport pathways. From as early as 12 h p.i., we observed DENV-2 NS5 predominantly localized in the nucleus, suggesting a specific role for NS5 in the nuclear compartment,

FIG. 4. The kinetics of DENV-2 NGC virus production are altered by LMB treatment. Vero cells were infected with DENV-2 NGC at a MOI of 10, ± LMB (5 ng/ml) at 0 h p.i. Virus titration of the culture supernatant at the indicated times post infection were performed on C6/36 cells. The mean titre (±SD) is graphed against time (hour p.i.) for each sample.

additional to its cytoplasmic role in viral replication. This role remains unclear, but in a similar fashion to other cytoplasmic RNA viruses, DENV-2 NS5 may be involved in assisting replication, inhibiting antiviral responses and/or interfering with host cell functions (Hiscox 2003).

We also show that both the nuclear import and export of NS5 is independent of other DENV-2 proteins, including the capsid protein that has also been identified in the nucleus of dengue virus infected cells (Bulich & Aaskov 1992, Makino et al 1989). The control over NS5's distribution in the nucleus and cytoplasm through its competing NLS and NES sequences remains to be determined, but may involve protein phosphorylation. The NS5/NS5A proteins belonging to all three genera of the Flaviviridae are known to be phosphorylated (Reed et al 1998); in the case of dengue, the nuclear form of NS5 is hyperphosphorylated by comparison with the cytoplasmic form (Kapoor et al 1995). It thus does not seem inconceivable that phosphorylation may represent a switch between the competing import and export mechanisms.

Blocking the CRM1 export pathway in DENV-2-infected cells was observed here to increase NS5 nuclear accumulation in parallel with the apparent acceleration of virus production. Although these data imply that CRM1-dependent nuclear export is important to the kinetics of virus production, we have not formally shown that this is due to effects on NS5 specifically; clearly LMB will inhibit nuclear export of a number of host factors which could impact on infection kinetics. Demonstrating that NS5 nuclear export is a key factor in dengue infection will require identification of the sequences responsible and reverse genetic approaches. In conclusion, the development of antiviral drugs designed to disrupt the trafficking of NS5, specifically by blocking NS5 interaction with importin ap, importin P, CRM1 or interfering with the phosphorylation of NS5, may represent a viable strategy to control the progression of dengue virus infection. The results here with respect to the alteration of the kinetics dengue virus replication by LMB treatment represent an initial proof-of-principle in this regard.

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