Nucleocytoplasmic shuttling of the West Nile virus RNA ‐dependent RNA polymerase NS5 is critical to infection

Abstract

West Nile virus (WNV) is a single-stranded, positive sense RNA virus of the family Flaviviridae and is a significant pathogen of global medical importance. Flavivirus replication is known to be exclusively cytoplasmic, but we show here for the first time that access to the nucleus of the WNV strain Kunjin (WNV ) RNA-dependent RNA polymerase (protein NS5) is central to WNV virus production. We show that treatment of cells with the specific nuclear export inhibitor leptomycin B (LMB) results in increased NS5 nuclear accumulation in WNV -infected cells and NS5-transfected cells, indicative of nucleocytoplasmic shuttling under normal conditions. We used site-directed mutagenesis to identify the nuclear localisation sequence (NLS) responsible for WNV NS5 nuclear targeting, observing that mutation of this NLS resulted in exclusively cytoplasmic accumulation of NS5 even in the presence of leptomycin B. Introduction of NS5 NLS mutations into FLSDX, an infectious clone of WNV , resulted in lethality, suggesting that the ability of NS5 to traffic into the nucleus in integral to WNV replication. This study thus shows for the first time that NLS-dependent trafficking into the nucleus during infection of WNV NS5 is critical for viral replication. Excitingly, specific inhibitors of NS5 nuclear import reduce WNV virus production, proving the principle that inhibition of WNV NS5 nuclear import is a viable therapeutic avenue for antiviral drug development in the future.

The Potential Role of the ZIKV NS5 Nuclear Spherical-Shell Structures in Cell Type-Specific Host Immune Modulation during ZIKV Infection

“…Nuclear export can also play a role in the subcellular localization of proteins as has been reported for several virus proteins [24,[49][50][51], including flavivirus NS5 which has a conserved nuclear export sequence (NES) ( Figure 1). But broader validation studies are needed to evaluate the functional role of nuclear export in flaviviruses, especially in the context of ZIKV NS5 which forms supramolecular spherical shell-like structures in the nucleus [21].…”

“…Flavivirus NS5 subcellular localization has been best studied in the context of dengue virus (DENV) [15,[17][18][19][20]22,23,43], trace amounts of DENV2 NS5 have been found co-localising with dsRNA, in addition to the NS5 detected in the nucleus [23]. Other flavivirus NS5 such as Zika virus (ZIKV), the main subject of this review, Japanese encephalitis virus (JEV), yellow fever virus (YFV) and West Nile virus (WNV) [16,21,24,44] have also been reported to accumulate in the nucleus. The regulation and function of the sub-cellular localization NS5 has to be tightly controlled in order for it to perform all its functions in the cell, but this is not well elucidated.…”

“…Additionally, it is also believed to contain a guanylyltransferase activity required for RNA cap formation [41,42]. While these flavivirus functions occur in the cytoplasm of infected cells, the NS5 protein from several flaviviruses are already present in the nucleus of the infected cells from earliest detectable time points post-infection [15][16][17][18][19][20][21][22][23][24] (Figure 1). Immunofluorescence assay studies have shown that >95% of fluorescence signal associated with NS5 can be located in the nucleus from around 24 h post-infection.…”

“…Apart from these functions, flavivirus NS5 has other viral and cellular roles during virus infection, including the modulation of the host immune response. Interestingly, the subcellular localization of NS5 proteins is different amongst the flaviviruses when examined by immunofluorescence assays, as they have been found in the nucleus and/or cytoplasm [15][16][17][18][19][20][21][22][23][24]. In particular, the observation of these flavivirus NS5 proteins in the nucleus suggests that NS5 plays important cellular and virus roles there, despite its major virus enzymatic role in virus RNA replication in the cytoplasm.…”

Polymerase Activity, Protein-Protein Interaction, and Cellular Localization of the Usutu Virus NS5 Protein

“…The primary amino acid sequence of the flaviviral NS5 protein has conserved nuclear localization signals (NLS) (12,13). Although the function of NS5 in the nucleus remains controversial, some authors have proposed the nucleocytoplasmic trafficking of NS5 as a target for drug design (14)(15)(16)(17)(18)(19).…”