Zika Virus Protease Cleavage of Host Protein Septin-2 Mediates Mitotic Defects in Neural Progenitors

Abstract

Zika virus (ZIKV) targets neural progenitor cells in the brain, attenuates cell proliferation, and leads to cell death. Here, we describe a role for the ZIKV protease NS2B-NS3 heterodimer in mediating neurotoxicity through cleavage of a host protein required for neurogenesis. Similar to ZIKV infection, NS2B-NS3 expression led to cytokinesis defects and cell death in a protease activity-dependent fashion. Among binding partners, NS2B-NS3 cleaved Septin-2, a cytoskeletal factor involved in cytokinesis. Cleavage of Septin-2 occurred at residue 306 and forced expression of a non-cleavable Septin-2 restored cytokinesis, suggesting a direct mechanism of ZIKV-induced neural toxicity. VIDEO ABSTRACT.

Keywords: Zika; activated caspase; cytokinesis; microcephaly; protease; septin.

Figure 1.. Active, but Not Protease-Dead, ZIKV NS2B3 Is Sufficientto Reduce Cell Proliferation, MediateApoptosis, and Result in Multipolar Spindles

(A, C, and E) Cells stably expressing NS2B3 showed reduced incorporation of EdU (DNAsynthesis marker), reduced phospho-histone H3 (pH3) (mitotic marker), and increased caspase 3 (Cas3) (apoptotic marker) compared with vector and S135A (SA) enzymatic null from 293T cells. Scale bars represent 20 μm. (G) NS2B3 expression leads to accumulation of cells with multipolar spindle morphology (arrows) and supernumerary centrosomes, as evident by centrosome marker CEP63. Right-bottom corner boxes present enlargement of cells with mitotic spindle. Scale bars represent 20 μm. (B, D, F, and H) Distribution sample mean and SEM. N = 4 biological replicates. **p < 0.01.

Figure 2.. ZIKV Proteaseome Includes Septin Family Members

(A) Coomassie-stained gel comparing human neural precursor cell (hNPC) lysate, bacterial GST alone (black arrow), and GST-NS2B3 S135A (red arrow) prior to and after affinity purification. (B) Label-free-quantitation-based volcano plot representing quantitative proteomic analysis of NS2B3 S135A pull-down. p values (—log10) are plotted as a function oflog2 fold changes (NS2B3-GST versus GST). Proteins >4-fold changed are marked in red (Student’s t test; p<0.05). See Table S1 for details. Septin 2, Septin 7, and Septin 9 are marked in blue. N = 3 biological replicates. (C) Protein-protein interaction network analysis of ZIKV NS2B3-SA binding partners identified from MS/MS using String (https://string-db.org/; high confidence 0.700). Seven major networks with molecular functions were identified and labeled with different colors. Ball size represents the confidence level of each NS2B3-SA binging partner (—log p value). Line thickness represents the interaction strength (String combined score) between each protein. SEPT2, SEPT7, and SEPT9 are highlighted in red.

Figure 3.. ZIKV NS2B3 Protease Mediates Cleavage of Septin 2 at Residue R306

(A and B) Cells transfected with ZIKV NS2B3 or NS2B3-S135A assessed by western blotting for SEPT2 and SEPT7. The lower-molecular-weight cleavage product was evident only with wild-type ZIKV NS2B3 and correlated with reduced SEPT7 levels, quantified below from N = 3. (C and D) Cells infected with ZIKV showed the same SEPT2 cleavage band and reduced SEPT7 level, quantified below from N = 3 replicates. (E) In vitro cleavage assay with recombinant NS2B3-WT or NS2B3-SA at low or high concentration (0.15 or 1.5 μM) with SEPT2 for1 h, analyzed by western with N- or C-terminal SEPT2 antibody. Cleaved SEPT2 is marked with red arrow, suggesting a C-terminal cleavage event. (F) Schematic of SEPT2, GTPase, and coiled coil (CC) domains, with zoom in of residues 301–361 highlighting ZIKV NS2B3 polybasic motifs (tan). (G) Extracted areas of SEPT2 peptides obtained by the three different digests (trypsin only or propionic anhydride labeling followed by trypsin or Asp-N digestion) applied to intact SEPT2 (light gray and gray outlines, respectively) or C-terminal SEPT2 fragment (C-term) from (E) (orange and red, respectively) were summed per residue and plotted as a function of SEPT2 sequence, normalized to signal of the most C-terminal residues. The signal of the N-terminal-labeled Asp-N generated peptide that defined the very N-terminal of the SEPT2 C-terminal fragment above. (H) High-resolution/high-mass-accuracy tandem MS spectrum of the N-terminal and lysine propionylated (p)Asp-N peptide: pG³⁰⁷GRKpVENE(m/z = 500.75714 [1.5 ppm]; Mascot score of 37), consistent with R³⁰⁶ cleavage. B-, a-, and y-fragment ions are annotated. (I) In vitro cleavage assay with recombinant NS2B3-WT and SEPT2-WT or-R306A, blotted with N- or C-terminal SEPT2 antibody. SEPT2-R306Afailed to cleave in the presence of active ZIKV NS2B3.

Figure 4.. SEPT2 or SEPT7 Interference or ZIKV NS2B3 Expression Delays Cytokinesis, Partially Rescued by Non-cleavable SEPT2

(A–C) Knockdown of SEPT2 orSEPT7 showed reduced incorporation of EdU, reduced pH3, and increased cleaved Cas3 compared with scrambled short hairpin RNA (shRNA) from hNPCs. Bar represents 50 μm. From N = 4. (D and E) Time-lapse frames showed knockdown of SEPT2 or SEPT7 lengthened cytokinesis. For all images, arrows track to a single cell undergoing mitosis into two cells. Note the timescale is expanded in the frames to capture completion of telophase. Scale bars represent 15 μm. n = 15–20 cells in each group. (F and G) Transfection of NS2B3, but not NS2B3-SA, showed similar lengthening of cytokinesis. Bar represents 15 μm. n = 15–20 cells in each group. (H and I) Co-transfection with non-cleavable SEPT2-R306A partially rescued lengthened cytokinesis induced by NS2B3 transfection. Bar represents 15 μm. n = 12–18 cells in each group. *p < 0.05; **p < 0.01.