The C-terminal 18 Amino Acid Region of Dengue Virus NS5 Regulates its Subcellular Localization and Contains a Conserved Arginine Residue Essential for Infectious Virus Production

Abstract

Dengue virus NS5 is the most highly conserved amongst the viral non-structural proteins and is responsible for capping, methylation and replication of the flavivirus RNA genome. Interactions of NS5 with host proteins also modulate host immune responses. Although replication occurs in the cytoplasm, an unusual characteristic of DENV2 NS5 is that it localizes to the nucleus during infection with no clear role in replication or pathogenesis. We examined NS5 of DENV1 and 2, which exhibit the most prominent difference in nuclear localization, employing a combination of functional and structural analyses. Extensive gene swapping between DENV1 and 2 NS5 identified that the C-terminal 18 residues (Cter18) alone was sufficient to direct the protein to the cytoplasm or nucleus, respectively. The low micromolar binding affinity between NS5 Cter18 and the nuclear import receptor importin-alpha (Impα), allowed their molecular complex to be purified, crystallised and visualized at 2.2 Å resolution using x-ray crystallography. Structure-guided mutational analysis of this region in GFP-NS5 clones of DENV1 or 2 and in a DENV2 infectious clone reveal residues important for NS5 subcellular localization. Notably, the trans conformation adopted by Pro-884 allows proper presentation for binding Impα and mutating this proline to Thr, as present in DENV1 NS5, results in mislocalizaion of NS5 to the cytoplasm without compromising virus fitness. In contrast, a single mutation to alanine at NS5 position R888, a residue conserved in all flaviviruses, resulted in a completely non-viable virus, and the R888K mutation led to a severely attenuated phentoype, even though NS5 was located in the nucleus. R888 forms a hydrogen bond with Y838 that is also conserved in all flaviviruses. Our data suggests an evolutionarily conserved function for NS5 Cter18, possibly in RNA interactions that are critical for replication, that is independent of its role in subcellular localization.

Zika virus nonstructural protein 5 residue R681 is critical for dimer formation and enzymatic activity

“…A), suggesting that the R681 mutations disrupting the dimer–dimer interface do not affect NS5 protein stability in cells. ZIKV NS5 WT has been demonstrated to localize into distinct spherical bodies within the nucleus , while DENV‐2 NS5 exhibited more diffused‐nuclear localization . Interrogation of the ZIKV NS5 mutants’ subcellular localization by CLSM revealed that the R681A mutant exhibited a mix phenotype of diffused‐nuclear localization similar to DENV‐2 NS5, and distinct nuclear body formation in the transfected cells (Fig.…”

“…Differential subcellular localization of NS5 has been reported for DENV [4,6] where it is predominantly nuclear for DENV-2, -3, and -4, and mostly cytoplasmic for DENV-1 [6]. More recently, it was demonstrated that the closely related flavivirus, ZIKV NS5 formed supramolecular nuclear bodies that were distinct from DENV-2 NS5 despite being nuclear localized [8,9,56].…”

“…Although replication occurs in the cytoplasm, NS5 of several flaviviruses are known to be transported into the nucleus by utilizing the host nuclear transport machinery made-up of importin-a and -b [3][4][5][6]. The so-called a/b NLS (nuclear localization signal) region, including residues around 369-389 [2,5,7], as well as the C-terminal stretch around amino acids 883-900 [6], are described to be important for nuclear-targeting ability and importin-a/b-binding activity in the various flaviviruses. Our recent studies of ZIKV NS5 [8] and previous work done by Grant et al [9] revealed that the protein forms supramolecular nuclear spherical bodies with importin-a and describes the possible importance of ZIKV NS5's nuclear localization toward neuronal Abbreviations CLSM, confocal laser scanning microscopy; DENV, dengue virus; DLS, dynamic light; DTT, dithiothreitol; GA, genetic algorithm; GTP, guanosine triphosphate; JEV, Japanese encephalitis virus; MM, molecular mass; MTase, methyltransferase; NLS, nuclear localization signal; NS3, nonstructural protein 3; NS5, nonstructural protein 5; NSD, normalized spatial discrepancy; NTP, nucleotide triphosphate; RC, replication complex; RdRp, RNA-dependent RNA polymerase; SAH, S-adenosyl-homocysteine; SAXS, small-angle X-ray scattering; WNV, West Nile virus; WT, wild-type; YFV, yellow fever virus; ZIKV, Zika virus.…”

Substrate‐induced structural alterations of Mycobacterial mycothione reductase and critical residues involved

“…Such mechanism of oligomer variations is common e.g. for the family of peroxiredoxins, which alter between catalytically active dimers and decamers or even dodecamers depending on redox regulation , the Dengue nonstructural protein NS5 proposed to switch from a monomer to dimer during the process of the replication complex , or the mycobacterial aspartate decarboxylase PanD, required for pantothenate and CoenzymeA (CoA) biosynthesis, described to occur in a tetrameric and octameric state . Furthermore, NADPH induces tetramer formation with inactivation of the human dihydrolipoamide dehydrogenase or causing an increase in activity of the related glutathione reductase of Cyanobacterium and Spirulina maxima have been reported.…”

Biochemistry and Molecular Biology of Flaviviruses

“…312 Elegant structural and reverse genetic studies indicate the possibility of NS5 dimerization 313 and position the C-terminal 18 residues close to the thumb subdomain, which could be important for initiation of RNA synthesis. 314 …”