Zika Virus NS5 Forms Supramolecular Nuclear Bodies That Sequester Importin-α and Modulate the Host Immune and Pro-Inflammatory Response in Neuronal Cells

Abstract

The Zika virus (ZIKV) epidemic in the Americas was alarming because of its link with microcephaly in neonates and Guillain-Barrésyndrome in adults. The unusual pathologies induced by ZIKV infection and the knowledge that the flaviviral nonstructural protein 5 (NS5), the most conserved protein in the flavivirus proteome, can modulate the host immune response during ZIKV infection prompted us to investigate the subcellular localization of NS5 during ZIKV infection and explore its functional significance. A monopartite nuclear localization signal (NLS) sequence within ZIKV NS5 was predicted by the cNLS Mapper program, and we observed localization of ZIKV NS5 in the nucleus of infected cells by immunostaining with specific antibodies. Strikingly, ZIKV NS5 forms spherical shell-like nuclear bodies that exclude DNA. The putative monopartite NLS 390 KRPR 393 is necessary to direct FLAG-tagged NS5 to the nucleus as the NS5 390 ARPA 393 mutant protein accumulates in the cytoplasm. Furthermore, coimmunostaining experiments reveal that NS5 localizes with and sequesters importin-α, but not importin-β, in the observed nuclear bodies during virus infection. Structural and biochemical data demonstrate binding of ZIKV NS5 with importin-α and reveal important binding determinants required for their interaction and formation of complexes that give rise to the supramolecular nuclear bodies. Significantly, we demonstrate a neuronal-specific activation of the host immune response to ZIKV infection and a possible role of ZIKV NS5's nuclear localization toward this activation. This suggests that ZIKV pathogenesis may arise from a tissue-specific host response to ZIKV infection.

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

“…The so‐called α/β NLS (nuclear localization signal) region, including residues around 369–389 , as well as the C‐terminal stretch around amino acids 883–900 , are described to be important for nuclear‐targeting ability and importin‐α/β‐binding activity in the various flaviviruses. Our recent studies of ZIKV NS5 and previous work done by Grant et al . revealed that the protein forms supramolecular nuclear spherical bodies with importin‐α and describes the possible importance of ZIKV NS5's nuclear localization toward neuronal cell‐specific activation of innate immune and proinflammatory pathways.…”

“…7A), 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 [8,56], while DENV-2 NS5 exhibited more diffused-nuclear localization [6]. 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 where it is predominantly nuclear for DENV‐2, ‐3, and ‐4, and mostly cytoplasmic for DENV‐1 . 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 . Interestingly, previous works showed that ZIKV NS5 formed higher order oligomers in solution in which in part residues Y25, K28, and K29 have been proposed to be involved in .…”

“…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.…”

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