Fig 1.
Distribution of putative G4 motifs in the VZV genome.
(A) The locations for the predicted 148 G4 motifs (denoted as GQ1 to GQ148) in the Dumas strain are indicated as short vertical lines on the top and bottom sequences of the viral genome. The locations of the terminal repeat long (TRL), unique long (UL), internal repeat long (IRL), internal repeat short (IRS), unique short (US), terminal repeat short (TRS), oriS and reiteration sequences (R1, R2, R3, R4A, R4B, and R5) are also indicated. (B and C) The numbers of predicted G4-motifs in different categories (B) and in different reiteration sequence regions (C) in Dumas and pOka strains are summarized.
Fig 2.
Putative G4 motifs in R2 within ORF14.
(A) The position of the ORF14 coding sequence is indicated as an open arrow on the bottom strand of the VZV Dumas genome and the region containing putative G4 motifs in R2 is indicated as a black box on the top strand. The variations found in ORF14 of pOka, YC01, or YC02 compared with Dumas are indicated as black vertical arrows. (B) The G4 motifs (GQ12 to GQ41) found in R2 are indicated below the genome sequence of Dumas (from 20681 to 21017). The variations found in pOka, YC01, and YC02 are indicated above the Dumas sequences. All G4 motifs of R2 are predicted on the anti-sense strand of ORF14. Black arrows, GQs for long-looped G4; red arrows, GQs for bulged G4. Seven 42-bp elements and one 32-bp element in R2 are indicated as light and dark grey bars, respectively, above the sequences.
Fig 3.
CD spectroscopy analysis of ORF14 GQ ODNs from pOka.
CD spectra of wild-type and mutant ORF14 GQ ODNs (15 μM) from pOka annealed in 10 mM Tris-HCl pH 7.5 and 100 mM KCl are shown. Each spectrum was an average of three accumulations in the wavelength range between 230–320 nm. The spectra were blanked with buffer only. GQ15/16/18/19 and GQ23/24 are same sequences, but their mutant sequences are different. Similarly, GQ17/20 and GQ25 are the same sequences, but their mutant sequences are different. The data were normalized using the maximum ellipticity and smoothed using GraphPad Prism 5.
Fig 4.
Normalized TDS spectra of ORF14 GQ ODNs from pOka.
TDS spectra of wild-type (A) and mutant (B) ORF14 GQ ODNs (15 μM) from pOka annealed in 10 mM Tris-HCl pH 7.5 and 100 mM KCl are shown. Each spectrum was an average of three accumulations in the wavelength range between 220–320 nm. The spectra were blanked with buffer only before TDS calculation.
Fig 5.
Effect of PDS on thermal melting curves of ORF14 G4s.
(A) Chemical structure of PDS. (B) The CD melting curves of ORF14 GQ ODNs from pOka in the presence of DMSO and PDS are compared. GQ ODNs (15 μM) were annealed in the presence of 10 mM Tris-HCl (pH 7.5) and 100 mM KCl buffer with DMSO or 30 μM PDS (DNA to chemical ratio 1:2). The CD melting graph was calculated at 290 nm wavelength for all GQ ODNs. The melting curves obtained with mean values from triplicate results are shown. The data were normalized using the maximum ellipticity and smoothed using GraphPad Prism 5.
Table 1.
Effect of PDS on the Tm values of ORF14 G4s.
Fig 6.
Non-denaturing polyacrylamide gel electrophoresis (PAGE) analysis of GQ oligonucleotides.
Wild-type and mutant ORF14 GQ ODNs (1 μM) were subjected to native PAGE analyses after annealing in 100 mM K+ buffer (top) or denaturing PAGE with 7M urea (bottom). Scrambled (Sc DNA; 5′-TAACCGATGATATGAGTCAGATATAT-3′) and poly-T ODNs (Poly-T; 26mer) used as controls are shown. Double-stranded DNA (ds DNA) size makers are shown.
Fig 7.
Effect of G4 disruption in ORF14 on gC expression in transfected cells.
(A) 293T cells (1x106 cells per well) in six-well plates were transfected with 2 μg of empty vector (EV) or plasmids expressing pOka ORF14-HA or its G4-disrupted form (G4m). Total cell lysates were prepared in RIPA buffer at 72 h after transfection. The total cell lysates were subjected to immunoblotting with anti-HA antibody. Non-specific bands are indicated by the open circle. The levels of β-actin are shown as a loading control. (B) 293T cells were transfected as in (A) and treated with DW or increasing amounts (2.5 and 5 μM) of PDS at 16 h after transfection. At 72 h after transfection, total RNA was prepared and the ORF14 mRNA levels were measured by qRT-PCR and normalized with those of β-actin. Results are shown as mean values and standard errors of triplicates. (C) 293T cells (1x106 cells per well) in six-well plates were transfected with 2 μg of empty vector (EV) or plasmids expressing wild-type ORF14-HA (Wt) or its different G4-mutated forms (G4m-1, G4m-2, or G4m) and treated with DW (D) or 2.5 μM of PDS (P) at 16 h after transfection. At 72 h after transfection, total RNA was prepared and the ORF14 mRNA levels were measured by qRT-PCR as in (B). (D) 293T cells were transfected as in (A) and treated with actinomycin D for the indicated times. The gC mRNA levels were determined as in (B and C). (E) 293T cells were transfected with 2 μg of empty vector (EV) or plasmids expressing pOka ORF14 (Wt) or its G4-disrupted form (G4m). The gC mRNA levels were determined as in (B) and (C).
Fig 8.
Altered gC expression in the recombinant virus containing the G4-disrupted ORF14-HA gene.
(A) HF cells were infected with recombinant pOka viruses containing the wild-type ORF14 or ORF14-HA gene at an MOI of 0.01. Cells were harvested at 24, 48, 72 and 96 h after infection and luciferase activities in cell lysates were measured. Results are shown as mean values and standard errors of three independent experiments. (B) HF cells were infected with recombinant viruses (wild-type or ORF14-HA) at an MOI of 0.01 Total cell lysates were prepared at 96 h after infection and subjected to SDS-PAGE and immunoblotting with ant-HA and anti-gE (ORF68) antibodies. Levels of β-actin are shown as a loading control. (C) HF cells in 12-well plates were infected with recombinant pOka viruses [wild-type, G4-disrupted mutant (G4m), and its revertant (R)] at an MOI of 0.001. Cells were harvested at 24, 48, 72, 96, and 120 h after infection. The cells were trypsinized and the virus titers were determined by plaque assay. Results are shown as mean values and standard errors of three independent experiments. p-values <0.01 (**) are indicated when significance was observed both between G4m and Wt and between G4m and R. (D and E) HF cells in 12-well plates were infected with recombinant viruses as in (C). Total cell lysates were also prepared at 48, 72, 96, and 120 h after infection and the expression levels of gC-HA and gE were determined by immunoblotting with anti-HA and anti-gE antibodies, respectively. Levels of β-actin are shown as a loading control (panel D, left). The gC-HA and gE bands in three independent experiment were quantitated with ImageJ and shown as graphs (panel D, right). Total mRNA was also prepared at 72 and 96 h after infection and the mRNA levels of gC (ORF14) and gE (ORF68) were determined by qRT-PCR. Results are shown as mean values of viral mRNA normalized with β-actin and standard errors of three independent experiments (E). (F) HF cells in 12-well plates were infected with recombinant viruses as in (C). At 72 h after infection, cells were treated with PDS (5 μM) for 48 h before qRT-PCR assays. The gC and gE mRNA levels and the gC/gE ratio are shown as in (E).
Fig 9.
Comparison of plaque sizes of ORF14 recombinant viruses.
(A) MeWo cells in six-well plates were transfected with the VZV bacmids (1 μg) containing the ORF14-HA gene [wild-type, G4m, or revertant (R)]. At 8 days after transfection, images of GFP+ plaques were taken. A similar plaque size reduction pattern was found with the G4m virus in two independent experiments. Since the efficiency of plaque generation in bacmid-transfected cell is low, we combined all plaques (n = 11 for Wt, n = 17 for G4m, and n = 8 for G4m-R) in two experiments to make the graph. Plaque area size was measured with ImageJ and shown as a box and whisker graph. Box plots show the first and third quartiles as a box. Horizontal lines are samples median. X corresponds to the mean. Whiskers correspond to 1.5 times the inter-quartile distance (IQR) from the difference between first and third quartiles. Images showing plaques are shown in S7A Fig. (B) MeWo cells in six-well plates were infected with cell-associated recombinant viruses. At 5 days after infection, cells were stained with crystal violet and plaque size was measured (n = 20) and shown as a graph as in (A). The experiments were performed more than three times and showed similar results. The data from a representative experiment are shown. Plaques images are shown in S7B Fig.