Fig 1.
Mutational analysis of ZIKVC α3.
(A) Micrographs showing fluorescent clusters of Vero E6 cells infected with wild-type ZIKVvenus or ZIKVvenus containing mutant C protein I66A(ZIKVvenus-CI66A), N67A(ZIKVvenus-CN67A), R68A(ZIKVvenus-CR68A), S71A(ZIKVvenus-CS71A), and combination mutant INRWSG/AAAWAG (ZIKVvenus-Cm), showing virus spread. Images were acquired at 5 d.p.i. (B) The crystal structure of ZIKV C dimer (PDB:5YGH) was generated using UCSF Chimera software. Chain A is green, and chain B is purple with α1–4 labeled on the structure. Residues selected for mutational analysis are depicted as ball and stick. (C) Virus titers of wild-type and mutant ZIKVvenus (passage 0) as determined by plaque assay in Vero E6 cells. Column labels correspond to viruses in (A). The graph represents average titers (n = 3) with error bars representing the standard error of the mean (SEM). Statistical significance was calculated by Ordinary one-way ANOVA using GraphPad PRISM 7 software at a 95% confidence interval. Relative significance is indicated by asterisks (p<0.0001 = ****). (D) Sequence alignment of flavivirus C α3 helices with the residue of interest N67 denoted by an arrow above. Sequence accession numbers [ZIKV MR766: AMR39835.1, ZIKV P6740: AVK43549.1, ZIKV PRV: AMC13911.1, ZIKV PF13: ARB08112.1, ZIKV Senegal: AMR39832.1, DENV 1: ADK37471.1, DENV 2: NP_056776.2, DENV 3: ABW82020.1, DENV 4: ARM59249.1, WNV: Q9Q6P4.2, JEV: NP_059434.1, YFV: NP_041726.1, SLEV: YP_001008348.1, MVEV: NP_051124.1, DTV: AAL32169.1, POWV: NP_620099.1, TBEV: ABI 31771.1, LGTV: QBR53298.1].
Fig 2.
Biochemical characterization of WT and N67A mutant C proteins.
Wild-type (MBP-CWT) and N67A mutant (MBP-CN67A) C were expressed and purified as MBP fusion proteins. (A) SDS-PAGE gel showing the purified MBP-CWT and MBP-CN67A. (B) EMSA showing binding of MBP-CWT (top panel), MBP-CN67A (middle panel), or MBP alone (bottom panel) to RNA representing 5’UTR and C gene of ZIKV. Relative molar ratios of RNA: protein are indicated by the triangle above, from left to right: 1:0, 1:1.5, 1:3, 1:6, 1:9. (C) SDS-PAGE of samples from glutaraldehyde (GA) crosslinking assay. Samples from MBP-CWT and MBP-CN67A after 20 min crosslinking reaction. Relative GA concentrations are indicated by triangles above, from left to right: 0 mM, 0.25 mM, 0.5 mM, 1.0 mM. The size of the C-MBP monomer, dimer, and possible tetramer is indicated on the right.
Fig 3.
Colocalization of WT and N67A mutant C protein to lipid droplets and nucleolus.
N-terminal mCherry tagged WT (mCherry-CWT) and N67A mutant (mCherry-CN67A) C proteins were expressed in JEG-3 cells, and their colocalization to LD and nucleolus were determined by confocal microscopy. (A-B) Fluorescent micrographs of JEG-3 cells transfected with mCherry-CWT (A) or mCherry-CN67A (B) and stained with MDH for LD (blue). (C-D) Fluorescent micrographs of JEG-3 cells co-expressing mCherry-CWT (C) or mCherry-CN67A (D) with nucleolar maker GFP-nucleolin. Regions of interest (ROI) are marked and represented as zoomed images to the right in separate channels. Line graphs to the right of each image show the relative intensity of red and blue (A-B) or red and green (C-D) channels corresponding to the white arrow inside the ROI. All images were acquired at 24 h.p.t.
Fig 4.
Effect of C N67A mutation on ZIKV assembly and replication.
JEG-3 cells were transfected with full-length cDNA clones of WT ZIKVvenus (A, D) ZIKVvenus-CN67A (B, E) or C assembly negative control mutant K85A/K86A (ZIKVvenus-CK85A/K86A) (C, F). Permeabilized cells were probed with antibodies against C (red) and Golgi marker Giantin (magenta) to determine the localization of C protein to the Golgi apparatus (A-C) or anti-dsRNA antibody (magenta) to detect replicating viral RNA (D-F). All cells were stained with Hoechst (blue) to detect the nucleus. The green color represents venus-tagged NS2A translated from the viral RNA. ROIs are outlined in cyan and represented as zoomed images to the right in separate channels. (G) Pearson’s correlation coefficients were calculated for the colocalization of C protein and Golgi using Nikon NIS Elements software. ROIs containing Golgi were selected from the confocal micrographs for each virus for calculation. Data points represent Pearson’s correlation coefficient (n = 9), with error bars representing mean and standard deviation (SD). Statistical significance was calculated by Ordinary one-way ANOVA using PRISM software at a 95% confidence interval. Relative significance is indicated by asterisks (p<0.0001 = ****). The heatmap indicates the colocalization range, and the data points are colored accordingly. (H-I) Detecting release of assembled virus in culture supernatants by qRT-PCR (H) and Western blot (I) 48 h.p.i. (H) Number of RNA molecules per ml of cell culture supernatant estimated by qRT-PCR. The number of RNA molecules/ml was calculated based on the Ct standard curve generated from ZIKV genomic RNA of a known quantity. The graph represents average RNA molecules/ml (n = 3) with error bars representing SEM. Statistical significance was calculated by Ordinary one-way ANOVA using GraphPad PRISM 7 software at a 95% confidence interval. Relative significance is indicated by asterisks (p<0.0001 = ****). Graph normalized to mock infected samples. (I) Western blot represents virus pellets obtained after ultracentrifugation of the cell culture supernatants probed with anti-ZIKV C antibody. Label C on the right indicates a band corresponding to the released ZIKV C protein.
Fig 5.
Amino acid substitution of C residue 67.
(A) Micrographs showing fluorescent clusters of Vero E6 cells infected with WT ZIKVvenus or C mutant viruses ZIKVvenus-CN67A, N67M(ZIKVvenus-CN67M), N67G(ZIKVvenus-CN67G), N67L(ZIKVvenus-CN67L), N67R(ZIKVvenus-CR67R), N67K(ZIKVvenus-CR67K), and negative control mutant ZIKVvenus-CK85A/K86A, demonstrating relative virus spread. Images were acquired at 5 d.p.i. (B) Virus titers of wild-type and mutant ZIKVvenus as determined by plaque assay in Vero E6 cells. The graph represents average titers (n = 3) with error bars representing SEM. Statistical significance was calculated by Ordinary one-way ANOVA using GraphPad PRISM 7 software at a 95% confidence interval. Relative significance is indicated by asterisks (p<0.0001 = ****).
Fig 6.
Identification and characterization of CN67A+MF37L revertant.
(A) Micrographs showing fluorescent clusters of Vero E6 cells infected with ZIKVvenus-CN67A at passage 0 (P0) and passage 5 (P5). (B) Sanger sequencing results analyzed using FinchTV software showing the region of mutation in M protein in the P5 revertant virus. A single nucleotide T→C mutation (highlighted in blue) created an F→L amino acid mutation in the M protein resulting in a ZIKVvenus-CN67A++MF37L double mutant. (C) Virus titers of ZIKVvenus-CN67A mutant and ZIKVvenus-CN67A++MF37L revertant compared to wild-type ZIKVvenus as determined by plaque assay on Vero E6 cells. The graph represents average titers (n = 3) with error bars representing SEM. Statistical significance was calculated by Ordinary one-way ANOVA using GraphPad PRISM 7 software at a 95% confidence interval. Relative significance is indicated by asterisks (p<0.01 = **). (D) Growth curve analysis of ZIKVWT and ZIKVWT-CN67A++MF37L. The graph represents average titers (n = 3) at 24-, 48-, 72-, and 96 h.p.i with error bars representing SEM. (E) Western blot of virus pellets obtained after ultracentrifugation of the cell culture supernatants probed with anti-ZIKV C antibody. Label -C on the right indicates a band corresponding to the released ZIKV C protein.
Fig 7.
Mutational analysis of ZIKV M residue 37.
(A) Cryo-EM structure of mature ZIKV M/E heterodimers (PDB:6CO8) showing the position of MF37 residue in magenta and marked with black arrows. E protein chains are shown with standard domain coloring (domain I: red, domain II: yellow, domain III: blue), and M protein chains are represented in cyan. The lipid bilayer is indicated by curved gray lines. (B) Multiple sequence alignment of flavivirus M perimembrane helix (M-H1) annotated in Jalview with Zappo coloring. The residue of interest F37 is denoted by an arrow above. Sequence accessions numbers [ZIKV MR766: AMR39835.1, ZIKV P6740: AVK43549.1, ZIKV PRV: AMC13911.1, ZIKV PF13: ARB08112.1, ZIKV Senegal: AMR39832.1, DENV 1: ADK37471.1, DENV 2: NP_056776.2, DENV 3: ABW82020.1, DENV 4: ARM59249.1, WNV: Q9Q6P4.2, JEV: NP_059434.1, YFV: NP_041726.1, SLEV: YP_001008348.1, MVEV: NP_051124.1, DTV: AAL32169.1, POWV: NP_620099.1, TBEV: ABI 31771.1, LGTV: QBR53298.1] (C) Virus titers (passage 0) calculated from plaque assay of ZIKVvenus, and M37 substitution mutant viruses F37A(ZIKVvenus-MF37A), F37I(ZIKVvenus-MF37I), F37W(ZIKVvenus-MF37W), and F37L(ZIKVvenus-MF37L). The graph represents average titers (n = 3) with error bars representing SEM. Statistical significance was calculated by Ordinary one-way ANOVA using GraphPad PRISM 7 software at a 95% confidence interval. Relative significance is indicated by asterisks (p<0.01 = **, p>0.05 considered not significant (ns)). (D) Growth curve analysis of ZIKVvenus, ZIKVvenus-MF37I, and ZIKVvenus-MF37L viruses. The graph represents average titers (n = 3) at 24-, 48-, 72-, and 96 h.p.i with error bars representing SEM.
Fig 8.
Mutational analysis of DENV C α3 and ML37.
(A) NMR structure of DENV C (PDB:1R6R) generated using UCSF Chimera software with α3 residues selected for mutational analysis shown as ball and stick and colored in red. Chain A is shown in beige, chain B is shown in blue, and helices α1–4 are labeled on the structure. (B) Plaque morphologies of DENVWT and plaque-forming C mutants L66A(DENV-CL66A), K67A(DENV-CK67A), and T71A(DENV-CT71A), as well as plaque-forming M mutants L37A(DENV-ML37A), and L37F(DENV-ML37F) in BHK-15 cells fixed at 7 d.p.i. and stained with crystal violet. (C) The graph shows plaque diameters measured from DENVWT (black circles) or mutant DENV (orange circles) plaque assays shown in (B). The graph represents average plaque diameters (n = 5) with error bars representing SD. Statistical significance was calculated by Ordinary one-way ANOVA using GraphPad PRISM 7 software at a 95% confidence interval. Relative significance indicated by asterisks (p<0.01 = **, p<0.001 = ***, values with p>0.05 considered not significant (ns)). (D) Virus titers (passage 0) calculated from plaque assay in BHK-15 cells of wild-type and mutant DENV. DENV-Cm represents a combination mutant (C-62TAGILKRWGT71/62AAGAAARWGA71), DENV-Dm-1 represents a double mutant one (C-66LK67/66AA67), DENV-Dm-2 represents a double mutant two (C-67KRWGT71/67ARWGA71), and DENV-Sw represents helix swap mutant with ZIKV C α3 in place of DENV C α3 (C-62TAGILKRWGT71/62SLGLINRWGSVI73). The graph represents average titers (n = 3) with error bars representing SEM. Statistical significance was calculated by Ordinary one-way ANOVA using GraphPad PRISM 7 software at a 95% confidence interval. Relative significance is indicated by asterisks (p<0.001 = ***). (E) Growth curve analysis of WT DENV, DENV-CK67A, DENV-CT71A, and DENV-MF37L viruses (passage 0) in BHK-15 cells infected at MOI = 0.01. The graph represents average titers (n = 3) at 24-, 48-, 72-, and 96 h.p.i. with error bars representing SEM.
Fig 9.
Proposed impacts of CN67A and CN67A+MF37L mutations on flavivirus assembly.
(A) In the wild-type virus, capsid protein (purple) and prM/E heterodimers (green and yellow) assemble along with viral RNA (red) and the lipid bilayer to generate immature viral particles (left). Mutation CN67A (red star) prevents virus assembly, leading to a lack of immature particle budding. In CN67A-MF37L revertant (magenta star), assembly is restored (right). (B) Potential core formation defects caused by CN67A mutation. (C) Potential mechanisms of MF37L rescue of CN67A mutant. Icons for flavivirus images were generated manually using PowerPoint and InkScape drawing tools with published cryo-EM structures of mature (PDB: 6CO8) and immature (PDB:5U4W) ZIKV structures generated using ChimeraX as templates. All icons in A are drawn using Microsoft PowerPoint. Icons representing protein structures in B and C were generated using ChimeraX https://www.rbvi.ucsf.edu/chimerax.