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. 2023 Jan 31;97(1):e0177322.
doi: 10.1128/jvi.01773-22. Epub 2022 Dec 8.

Nucleo-Cytoplasmic Transport of ZIKV Non-Structural 3 Protein Is Mediated by Importin-α/β and Exportin CRM-1

Luis Adrián De Jesús-González  1   2 , Selvin Noé Palacios-Rápalo  1 , José Manuel Reyes-Ruiz  3   4 , Juan Fidel Osuna-Ramos  1   5 , Carlos Noe Farfán-Morales  1 , Carlos Daniel Cordero-Rivera  1 , Bulmaro Cisneros  6 , Ana Lorena Gutiérrez-Escolano  1 , Rosa María Del Ángel  1
Affiliations

Nucleo-Cytoplasmic Transport of ZIKV Non-Structural 3 Protein Is Mediated by Importin-α/β and Exportin CRM-1

Luis Adrián De Jesús-González et al. J Virol. .

Abstract

Flaviviruses have a cytoplasmic replicative cycle, and crucial events, such as genome translation and replication, occur in the endoplasmic reticulum. However, some viral proteins, such as C, NS1, and NS5 from Zika virus (ZIKV) containing nuclear localization signals (NLSs) and nuclear export signals (NESs), are also located in the nucleus of Vero cells. The NS2A, NS3, and NS4A proteins from dengue virus (DENV) have also been reported to be in the nucleus of A549 cells, and our group recently reported that the NS3 protein is also located in the nucleus of Huh7 and C636 cells during DENV infection. However, the NS3 protease-helicase from ZIKV locates in the perinuclear region of infected cells and alters the morphology of the nuclear lamina, a component of the nuclear envelope. Furthermore, ZIKV NS3 has been reported to accumulate on the concave face of altered kidney-shaped nuclei and may be responsible for modifying other elements of the nuclear envelope. However, nuclear localization of NS3 from ZIKV has not been substantially investigated in human host cells. Our group has recently reported that DENV and ZIKV NS3 alter the nuclear pore complex (NPC) by cleaving some nucleoporins. Here, we demonstrate the presence of ZIKV NS3 in the nucleus of Huh7 cells early in infection and in the cytoplasm at later times postinfection. In addition, we found that ZIKV NS3 contains an NLS and a putative NES and uses the classic import (importin-α/β) and export pathway via CRM-1 to be transported between the cytoplasm and the nucleus. IMPORTANCE Flaviviruses have a cytoplasmic replication cycle, but recent evidence indicates that nuclear elements play a role in their viral replication. Viral proteins, such as NS5 and C, are imported into the nucleus, and blocking their import prevents replication. Because of the importance of the nucleus in viral replication and the role of NS3 in the modification of nuclear components, we investigated whether NS3 can be localized in the nucleus during ZIKV infection. We found that NS3 is imported into the nucleus via the importin pathway and exported to the cytoplasm via CRM-1. The significance of viral protein nuclear import and export and its relationship with infection establishment is highlighted, emphasizing the development of new host-directed antiviral therapeutic strategies.

Keywords: NES; NLS; ZIKV; flavivirus; nuclear export; nuclear export signal; nuclear localization; nuclear localization signal; viral proteases.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

FIG 1
FIG 1
ZIKV NS3 protein localizes in the nucleus early in infection and in the cytoplasm at later times postinfection in Huh-7 cells. (A) The localization of NS3 and E proteins in mock-infected or ZIKV-infected Huh7 cells at 8, 12, 16, and 24 h postinfection (hpi) was analyzed by confocal microscopy using anti-NS3 (red) and anti-E antibodies (green) and Hoechst staining of nuclei (blue). To determine the nuclear localization of ZIKV NS3, clipped three-dimensional images (yz clip 3D images) were analyzed, as mentioned in the Materials and Methods. (B and C) The MFI (B) was determined for selected regions of interest in the nucleus and cytoplasm (30 cells per condition), and the Fn/Fc ratio (C) was determined for the selected regions in each immunofluorescence confocal microscopy image; *, P = 0.05; **, P = 0.001; ***, P = 0.0001; ****, P < 0.0001.
FIG 2
FIG 2
The ZIKV NS3 protein is present in the nucleus of Huh7 cells during ZIKV infection. (A to C) Mock-infected (A) or ZIKV-infected Huh7 cells were fixed and processed for TIM using an anti-NS3 antibody at 12 hpi (B) and at 24 hpi (C). (D) NS3 immunolabeling in arbitrary units was determined by selected regions of interest in the nucleus and cytoplasm (6 cells per condition). Anti-NS3 in the cellular compartment was divided by the total cellular anti-NS3 of each condition. The NS3-specific tagging signal is shown as dark dots and is indicated by an arrowhead; Ne, nuclear envelope; N, nucleus; ER, endoplasmic reticulum; M, mitochondria; N, XYZ; ns, not significant; *, P = 0.05; **, P = 0.001; ***, P = 0.0001; ****, P < 0.0001.
FIG 3
FIG 3
Schematic of the localization of the ZIKV NS3 protein during inhibition of the importin-α and exportin CRM-1 pathways using IVM and LMB, respectively. (A) Localization of NS3 at 12 hpi and during importin-α inhibition with IVM. (B) NS3 localization at 24 hpi and during CRM-1 exportin inhibition with LMB.
FIG 4
FIG 4
Ivermectin inhibits nuclear import of ZIKV NS3 protein in Huh-7 cells. (A) Localization of NS3 and NLS-SV40-tetraGFP proteins in untreated or IVM-treated Huh7 cells was analyzed by confocal microscopy using anti-NS3 (red) and Hoechst staining of nuclei (blue). To determine the nuclear localization of ZIKV NS3 at 12 hpi, three-dimensional clipped images (yz clip 3D images) were analyzed, as mentioned in the Materials and Methods. (B) MFI was determined for selected regions of interest in the nucleus and cytoplasm (30 cells per condition). (C) The Fn/Fc ratio was determined for the latter selected regions of interest in each immunofluorescence confocal microscopy image in the different conditions; *, P = 0.05; **, P = 0.001; ***, P = 0.0001; ****, P < 0.0001.
FIG 5
FIG 5
Leptomycin B inhibits nuclear export of ZIKV NS3 in Huh-7 cells. (A) Localization of NS3 and CCNB1 proteins in mock-infected, LMB-treated, or ZIKV-infected Huh7 cells at 24 h postinfection (hpi) was analyzed by confocal microscopy using anti-NS3 (red) and anti-CCNB1 antibodies (green) and Hoechst staining of nuclei (blue). To confirm the nuclear localization of ZIKV NS3 protein, three-dimensional clipped images (yz clip 3D images) were analyzed, as mentioned in the Materials and Methods. (B) MFI was determined for selected regions of interest in the nucleus and cytoplasm (30 cells per condition). (C) The Fn/Fc ratio was determined for these latter selected regions in each immunofluorescence confocal microscopy image in the different conditions; *, P = 0.05; **, P = 0.001; ***, P = 0.0001; ****, P < 0.0001.
FIG 6
FIG 6
In silico prediction of nuclear localization signals (NLSs) in ZIKV NS3 protein. (A) Three-dimensional structure of NS3 showing the location of the putative NLS identified with the cNLS Mapper software and WREGEX. (B) Structure and sequence of putative NLS 210. (C) Putative NLS 169. (D) Putative NLS 583. (E) ClustalW alignment of putative NLSs from other flaviviruses.
FIG 7
FIG 7
In silico prediction of nuclear export signals (NESs) in ZIKV NS3 protein. (A) Three-dimensional structure of NS3 showing the location of the putative NES identified with LocNES and WREGEX software. (B) Structure and sequence of putative NES 85. (C) Putative NES 248. (D) ClustalW alignment of putative NESs of different flaviviruses.
FIG 8
FIG 8
Molecular docking of NS3 ZIKV-importin-α. (A) Molecular docking of NS3 ZIKV-importin-α. (B) Binding of the armadillo domains of importin-α and the putative NLS 210 of the ZIKV NS3 protein. Molecular docking analysis was performed using HDOCK software. The 3D protein structure of importin-α used was PDB ID 4WV6. (C) MM/GBSA analysis of the binding of the armadillo domains of importin-α and the putative NLS 210 of the ZIKV NS3 protein, predicting the free energy of binding of the protein-protein complex for each residue using HawkDock software.
FIG 9
FIG 9
Mutation in the putative NLS 210 of the ZIKV NS3 protein prevents its nuclear localization. (A) Localization of mutated NS2B3-WT or NLS 210 and importin-α proteins in Huh7 cells was analyzed 48 h posttransfection by confocal microscopy using anti-NS3 (red) and anti-importin-α (green) antibodies and Hoechst staining of nuclei (blue). To determine the nuclear localization of ZIKV NS3, three-dimensional clipped images (yz clip 3D images) were analyzed, as mentioned in the Materials and Methods. (B) MFI was determined for selected regions of interest in the nucleus and cytoplasm (30 cells per condition). (C) We determined the Fn/Fc ratio for these regions of interest in each immunofluorescence confocal microscopy image. (D) Image analysis results presented as the mean and SEM of the Person’s correlation coefficients (30 cells at each time point); *, P = 0.05; **, P = 0.001; ***, P = 0.0001; ****, P < 0.0001.
FIG 10
FIG 10
Putative NES 248 deletion of the ZIKV NS3 protein retains NS3 in the nucleus. (A) Localization of NS2B3-WT, NES-Mut85, or NES-248 proteins and CRM-1 exportin in Huh7 cells was analyzed 48 h posttransfection by confocal microscopy using anti-NS3 (red) and anti-CRM-1 (green) and Hoechst staining of nuclei (blue). To determine the nuclear localization of ZIKV NS3, three-dimensional clipped images (yz clip 3D images) were analyzed, as mentioned in the Materials and Methods. (B) MFI was determined for selected regions of interest in the nucleus and cytoplasm (30 cells per condition). (C) We determined the Fn/Fc ratio for these regions of interest in each immunofluorescence confocal microscopy image. (D) Image analysis results presented as the mean and SEM of the Person’s correlation coefficients (30 cells at each time point); *, P = 0.05; **, P = 0.001; ***, P = 0.0001; ****, P < 0.0001.
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