Fig 1.
Generation and characterization of 6K mutants.
(A). The amino-acid sequence of the 6K region from WT SINV, Δ6K SINV, p7 SINV, M2 SINV, and Vpu SINV. (B). Plaque morphologies of WT and 6K mutant viruses. Viruses were collected from electroporated BHK-15 cells 24 h post-electroporation, and plaques were visualized by staining BHK-15 cells 48 hpi. (C). Mean plaque diameters of the virus plaques in BHK-15 cells measured on day 2 pi. Plaque size was determined by random selection of 10 plaques and values are expressed as the mean with standard error (SEM). Significance was determined using Dunnett’s multiple comparisons test as part of one-way ANOVA with a 95% confidence interval. p Values were considered significant when p < 0.05 (*), p < 0.01 (**), p < 0.001(***), and p < 0.0001(****). ns indicates “not significant”. (D). One-step growth curve analysis of WT and 6K mutant viruses from BHK-15 cells. BHK-15 cells were infected with WT or 6K mutant viruses at an MOI of 5, medium was harvested and replaced every hour for 12 h, and the rate of virus release (plaque-forming unit (pfu) per ml per hour) was determined using standard plaque assays. Data shown are from three independent experiments. Error bars indicate standard error of mean (SEM). (E). Quantification of the number of virus particles released into the medium for WT and 6K mutant viruses at 12 hpi. The total number of viral genomic RNA molecules was determined by qRT-PCR using a standard curve of a known amount of in vitro-transcribed SINV RNA molecules. The pfu in these samples were determined by standard plaque assays of the virus supernatant collected at 12 hpi from infected BHK-15 cells. Data shown are from three independent experiments. The error bars indicate the standard error of mean (SEM). (F) Specific infectivity of WT and chimeric viruses.
Fig 2.
Functional complementation of 6K by other viral ion channels.
(A) IF analysis of permeabilized BHK-15 cells infected with WT SINV, Δ6K SINV, M2 SINV, Vpu SINV, or P7 SINV at 6 or 12 hpi using antibodies against E2 (Green) and CP (Red). (B) IF analysis of glycoprotein trafficking to the plasma membrane under permeabilized or non-permeabilized conditions. BHK-15 cells were electroporated with RNA corresponding to WT or 6K mutant viruses and fixed at 12 h post-electroporation Anti-E2 monoclonal antibody was used to detect glycoproteins. Nuclei were stained with Hoechst stain. (C) Flow cytometry analysis of cells infected with WT or 6K mutant viruses at an MOI of 5. Cells were incubated with a monoclonal anti-E2 antibody followed by staining with FITC secondary antibody. Results are shown as a log10 of the geometric mean of the region of the curve measuring the amount of E2 fluorescence at the plasma membrane. Data shown are from two independent experiments. Error bars indicate standard error of mean (SEM). Significance was determined using Dunnett’s multiple comparisons test as part of two-way ANOVA with a 95% confidence interval. p Values were considered significant when p < 0.05 (*), p < 0.01 (**), p < 0.001(***), and p < 0.0001(****). ns indicates “not significant”. (D) Quantification of glycoprotein trafficking to the plasma membrane from (B). The number of cells expressing E2 (green) relative to the total number of cells (blue) was determined using the NIS Elements software. Cells from 10 independent frames per condition were counted. Error bars indicate standard error of mean (SEM). Significance was determined by multiple unpaired t-tests of data. p Values were considered significant when p < 0.05 (*), p < 0.01 (**), p < 0.001(***), and p < 0.0001(****). ns indicates “not significant”.
Fig 3.
6K is required for glycoprotein trafficking to the plasma membrane.
(A-B). Representative images of BHK-15 cells co-transfected with YFP-membrane (green) and E3-mCherry-E2-6K/TF-E1 (red) (A) or E3-mCherry-E2-E1 (red) (B). Images were collected at 12 h post-transfection. Images correspond to videos (S1 and S2 Videos). (C) Western blot analysis of cell lysates from BHK-15 cells infected with WT SINV or Δ6K SINV. The blot was probed with an anti-E2 primary antibody to detect the structural polyprotein-processing intermediates. (D) Images of BHK-15 cells transfected with YFP-membrane (green) and RNA corresponding to mCherry-E2 SINV (red) or Δ6K mCherry-E2 SINV (red). Images were collected at the indicated time points post-electroporation. Regions of interest are marked and represented as zoomed to the right in separate channels.
Fig 4.
Subcellular localization of 6K in infected cells.
(A) Schematic of the miniSOG-6K SINV construct. The sequence encoding the fluorescent protein miniSOG was cloned into WT toto64 after Thr2 of 6K as an N-terminal fusion. (B) Western blot analysis of the cell lysates from BHK-15 cells infected with WT SINV or miniSOG-6K SINV. The blot was probed with an anti-E2 primary antibody to detect the structural polyprotein processing intermediates. (C-E) Representative images of BHK-15 cells transfected with mCherry-SEC61B-C1 (red) (C), mTagBFP2-SiT-N-15 (blue) (D), or CP-E3-mCherry-E2-6K/TF-E1 (red) (E), and infected with miniSOG-6K SINV (green). Imaging was performed at 12 hpi. Regions of interest are marked and enlarged to the right as separate channels. Images correspond to videos (S3–S5 Videos) (F-G) Representative images of BHK-15 cells electroporated with RNA corresponding to the dual-labeled mCherry-E2/miniSOG SINV (mCherry-E2 is red and miniSOG-6K is green) (F) or ΔTF mCherry-E2/miniSOG SINV (mCherry-E2 is red and miniSOG-6K is green) (G) and imaged at 12 hpi Images correspond to videos (S6 and S7 Videos).
Fig 5.
Localization of 6K and the ion channel chimeras.
(A-D) IF analysis using anti-FLAG (red) and anti-Giantin (green) antibodies of permeabilized huh-7.5 cells infected with an MOI of 5 of (A) FLAG-6K SINV, (B) FLAG-Vpu SINV, (C) FLAG-M2 SINV, or (D) FLAG-P7 SINV. Cells were fixed at 12 hpi. Regions of interest are marked and enlarged to the right as separate channels. Fluorescent profiles of the lined areas are displayed at the bottom. Distance is in μm and intensity is in arbitrary fluorescence units (AFU).
Fig 6.
Ion channel activity of 6K is required for the biogenesis of CPV-IIs.
TEM analysis of BHK-15 cells. Cells were infected with WT SINV (A), Δ6K SINV (B), M2 SINV (C), Vpu SINV (D), or P7 SINV (E), and fixed at 12 hpi. Uninfected cells (F) were used as a control. Black arrowheads indicate CPV-Is, and white arrowheads indicate CPV-IIs. The scale bars are shown in each panel of the figure.
Fig 7.
Effect of HMA and amantadine on virus release from SINV and ion channel chimeras.
(A-B). Cytotoxicity of Amantadine (A) and HMA (B) was calculated using alamarBLUE reagent (C-D). BHK-15 cells were pretreated with the corresponding concentrations of HMA and Amantadine for 24 h and then infected with WT or 6K mutant viruses at an MOI of 0.1. Viral titers were determined using the supernatants collected at 12 hpi. Data shown are from three independent experiments. Error bars indicate standard error of mean (SEM). Significance was determined using Dunnett’s multiple comparisons test as part of one-way ANOVA with a 95% confidence interval. p Values were considered significant when p < 0.05 (*), p < 0.01 (**), p < 0.001(***), and p < 0.0001(****). ns indicates “not significant”.