Article Figures & Data
Figures
- FIG 1
Schematic diagrams of the genome structure of wild-type HSV-1(F) and the relevant domains of the recombinant viruses used in this study. Line 1, wild-type HSV-1(F) genome; line 2, domains encoding the Us3 to Us4 open reading frames; line 3, domains of Us3 and Us3.5; lines 4 to 11, domains in recombinant virus genomes with mutations in Us3.
- FIG 2
Immunoblots of electrophoretically separated lysates of Vero cells mock infected or infected with wild-type HSV-1(F), YK781 (Us3-chimera), YK783 (Us3-repair), or wild-type HSV-2 (186) at an MOI of 3. The infected cells were harvested at 18 h (A) and 12 h (B) postinfection and analyzed by immunoblotting with antibodies to Us3, VP5, and β-actin (A) or to Us2, Us4, and UL12 (B).
- FIG 3
Immunoblots of electrophoretically separated lysates of Vero cells mock infected or infected with wild-type HSV-1(F), YK823 (MEF-Us3), YK781 (Us3-chimera), or YK825 (MEF-Us3-chimera) at an MOI of 3. The infected cells were harvested at 18 h postinfection and analyzed by immunoblotting with antibodies to Myc tag, VP5, and β-actin.
- FIG 4
Growth curves and plaque sizes of the recombinant viruses generated in this study. Vero (A and B) and HaCaT (C and D) cells were infected at an MOI of 3 (A and C) or 0.01 (B and D) with wild-type HSV-1(F), YK511 (Us3K220M), YK513 (Us3K220M-repair), YK781 (Us3-chimera), or YK783 (Us3-repair). Total virus from the cell culture supernatants and infected cells was harvested at the indicated times and assayed on Vero cells. Vero (E) and HaCaT (F) cells were infected with wild-type HSV-1(F), YK511 (Us3K220M), YK513 (Us3K220M-repair), YK781 (Us3-chimera), or YK783 (Us3-repair) at an MOI of 0.0001 under plaque assay conditions. The diameters of 30 single plaques for each of the indicated viruses were measured 48 h postinfection. Each data point is the mean ± standard error of the measured plaque sizes. Statistical analysis was performed by one-way analysis of variance (ANOVA) with the Tukey test. Asterisks indicate statistically significant values (*, P < 0.001).
- FIG 5
Detection of phosphorylated proteins in Vero cells mock infected or infected with wild-type HSV-1(F), YK511 (Us3K220M), YK513 (Us3K220M-repair), YK781 (Us3-chimera), or YK783 (Us3-repair) at an MOI of 3. The infected cells were harvested at 18 h postinfection and analyzed by immunoblotting with anti-PKA substrate antibody 100G7.
- FIG 6
(A) Caspase 3/7 activity of infected SK-N-SH cells after induction of apoptosis by osmotic shock. SK-N-SH cells were infected with wild-type HSV-1(F), YK511 (Us3K220M), YK513 (Us3K220M-repair), YK781 (Us3-chimera), or YK783 (Us3-repair) at an MOI of 5. At 18 h postinfection, the infected cells were exposed to sorbitol for 2 h, incubated for an additional 5 h, harvested, and assayed for caspase 3/7 activity. Each value is the mean ± standard error of the results of triplicate experiments. Data are representative of three independent experiments. (B) Cell surface expression of gB in Vero cells infected with wild-type HSV-1(F), YK511 (Us3K220M), YK513 (Us3K220M-repair), YK781 (Us3-chimera), or YK783 (Us3-repair) at an MOI of 3. At 6 h postinfection, the infected cells were harvested and analyzed by flow cytometry. The relative mean fluorescence intensity for gB expression on the surface of cells infected with the indicated virus is shown as the fluorescence intensity of virus-infected cells relative to that of YK513 (Us3K220M-repair)-infected cells. Each value is the mean ± standard error of the results of three independent experiments. (C) Phosphorylation of gB at Thr-887 in Vero cells infected with wild-type HSV-1(F), YK511 (Us3K220M), YK513 (Us3K220M-repair), or YK781 (Us3-chimera) at an MOI of 3. At 18 h postinfection, the infected cells were harvested and analyzed by immunoblotting with the monoclonal antibody to gB-T887P and gB. (D) Localization of gB in Vero cells infected with wild-type HSV-1(F) or YK781 (Us3-chimera) at an MOI of 3. At 18 h postinfection, the infected cells were permeabilized, stained with anti-gB and anti-lamin B1 antibodies, and examined by confocal microscopy.
- FIG 7
Images of the CPE in Vero cells mock infected or infected at an MOI of 3 with wild-type HSV-1(F), YK511 (Us3K220M), YK513 (Us3K220M-repair), YK781 (Us3-chimera), or YK783 (Us3-repair). Live cells were examined at 24 h postinfection by confocal microscopy. Digital interference contrast images are shown.
- FIG 8
Effect of replacement of HSV-1 Us3 with HSV-2 Us3 on localization of UL31 and UL34 in infected Vero cells. Vero cells were infected with wild-type HSV-1(F), YK511 (Us3K220M), YK513 (Us3K220M-repair), YK781 (Us3-chimera), or YK783 (Us3-repair) at an MOI of 3, fixed at 12 h postinfection, permeabilized, stained with anti-UL34 and anti-UL31 antibodies, and examined by confocal microscopy. Each image in the far right column is the magnified image of the boxed area in the image to its left. Since two different patterns of UL31 and UL34 localization were observed in cells infected with YK781 (Us3-chimera), images of two of these infected cells are shown here as examples of UL31 and UL34 localization in these infected cells.
- FIG 9
Quantitation of infected Vero (A) and HaCaT (B) cells with aberrant punctate structures of UL31 and UL34 adjacent to the nuclear rim. The experiments were done as described for Fig. 8 and 10, and the percentage of cells with aberrant punctate structures at the nuclear rim was determined. Each value is the mean ± standard error of the results of three independent experiments. Statistical analysis was performed by one-way ANOVA with the Tukey test. Asterisks indicate statistically significant values (*, P < 0.001; **, P < 0.01). n.s., not statistically significant.
- FIG 10
Effect of replacement of HSV-1 Us3 with HSV-2 Us3 on localization of UL31 and UL34 in infected HaCaT cells. HaCaT cells were infected with wild-type HSV-1(F), YK511 (Us3K220M), YK513 (Us3K220M-repair), YK781 (Us3-chimera), or YK783 (Us3-repair) at an MOI of 3, fixed at 12 h postinfection, permeabilized, stained with anti-UL34 and anti-UL31 antibodies, and examined by confocal microscopy. Each image in the far right column is the magnified image of the boxed area in the image to its left. Since different patterns of UL31 and UL34 localization were observed in cells infected with HSV-1(F), YK513 (Us3K220M-repair), YK781 (Us3-chimera), and YK783 (Us3-repair), images of two of the HSV-1(F)-, YK513 (Us3K220M-repair)- and YK783 (Us3-repair)-infected cells and three of the YK781 (Us3-chimera)-infected cells are shown here as examples of UL31 and UL34 localization in these infected cells.
- FIG 11
Ultrastructural analysis of the effect of replacement of HSV-1 Us3 with HSV-2 Us3 on viral nuclear egress in Vero cells. Vero cells infected with wild-type HSV-1(F), YK511 (Us3K220M), YK513 (Us3K220M-repair), YK781 (Us3-chimera), or YK783 (Us3-repair) at an MOI of 3 were fixed at 18 h postinfection, embedded, sectioned, stained, and examined by transmission electron microscopy. Bars, 200 nm. N, nucleus.
- FIG 12
Ultrastructural analysis of the effect of replacement of HSV-1 Us3 with HSV-2 Us3 on viral nuclear egress in HaCaT cells. (A) HaCaT cells infected with wild-type HSV-1(F), YK511 (Us3K220M), YK513 (Us3K220M-repair), YK781 (Us3-chimera), or YK783 (Us3-repair) at an MOI of 3 were fixed at 18 h postinfection, embedded, sectioned, stained, and examined by transmission electron microscopy. (B) Area of some cells infected with wild-type HSV-1(F), YK513 (Us3K220M-repair), or YK783 (Us3-repair) showing the invagination structures. Bars, 200 nm. N, nucleus.
- FIG 13
Effect of replacement of HSV-1 Us3 with HSV-2 Us3 on expression of gB and gH. Vero cells mock infected or infected with wild-type HSV-1(F), YK781 (Us3-chimera), or YK783 (Us3-repair) at an MOI of 3 were harvested at 18 h postinfection and analyzed by immunoblotting with antibodies to gB, gH, and β-actin.
- FIG 14
Phosphorylation status of UL31, UL34, and vdUTPase in infected cells. Vero cells were mock infected or infected with wild-type HSV-1(F), YK511 (Us3K220M), YK513 (Us3K220M-repair), YK781 (Us3-chimera), or YK783 (Us3-repair) at an MOI of 3, harvested at 18 h postinfection, and analyzed on a Phos tag(+) SDS-PAGE gel (top) or Pho tag(−) SDS-PAGE gel (bottom) (A to E). The gels were immunoblotted with anti-UL31 (A and B), anti-UL34 (C and D), or anti-vdUTPase (E) antibody. The infected cell lysates were also either left untreated or treated with CIP (B and D). Asterisks indicates bands in which the amount of protein was affected in YK781 (Us3-chimera)-infected cells compared to that in cells infected with wild-type HSV-1(F) or YK783 (Us3-repair).
- FIG 15
Effect of replacement of HSV-1 Us3 with HSV-2 Us3 on viral pathogenesis and replication in mice following ocular infection. (A and B) Five-week-old female ICR mice were ocularly infected with YK781 (Us3-chimera) or YK783 (Us3-repair) and scored for HSK (A) and periocular skin disease (B) daily for 14 days (d). Each data point is the mean ± standard error of the scores. Asterisks indicate statistically significant values (*, P < 0.05) according to the two-tailed Student t test. (C) Tear films of infected mice at 1 and 5 days postinfection in the experiments shown in panels A and B were harvested, and virus titers were assayed. Each data point is the virus titer in the tear film of one mouse. The horizontal bars mark the means for the groups. P shows statistically significant values according to the two-tailed Student t test.
Tables
- TABLE 1
Electron microscopic localization of enveloped virions in infected Vero and HaCaT cells
Cells Virus No. of enveloped virions in compartment (mean ± SE)a Total no. of enveloped virions/cells Intranuclear invaginations Perinuclear space Cytoplasm Extracellular Vero HSV-1(F) 0.8 ± 0.4 (1.3) 16.4 ± 2.6 (27.2) 13.6 ± 1.7 (22.6) 29.3 ± 2.9 (48.6) 2,716/45 YK511 (Us3K220 M) 29.6 ± 2.4 (59.9) 1.9 ± 0.4 (3.9) 1.3 ± 0.6 (2.7) 11.5 ± 1.1 (23.2) 2,222/45 YK513 (Us3KM-repair) 1.7 ± 0.7 (4.1) 9.7 ± 2.1 (23.4) 7.0 ± 1.4 (16.9) 22.6 ± 1.9 (54.7) 1,861/45 YK781 (Us3-chimera) 9.7 ± 2.2 (16.9) 23.4 ± 3.3 (40.7) 6.9 ± 1.1 (12.1) 15.6 ± 1.7 (27.1) 2,586/45 YK783 (Us3-repair) 1.0 ± 0.5 (2.3) 14.6 ± 2.6 (33.8) 8.0 ± 1.3 (18.5) 19.3 ± 2.1 (44.8) 1,936/45 HaCaT HSV-1(F) 12.8 ± 2.2 (9.8) 10.0 ± 1.0 (7.6) 18.4 ± 3.1 (14.0) 87.8 ± 7.1 (67.1) 5,885/45 YK511 (Us3K220 M) 46.1 ± 3.5 (52.9) 1.5 ± 0.3 (1.7) 2.9 ± 0.6 (3.3) 28.7 ± 3.1 (33.0) 3,921/45 YK513 (Us3KM-repair) 12.6 ± 2.1 (9.5) 11.0 ± 1.0 (8.3) 22.5 ± 3.8 (17.0) 84.0 ± 7.1 (63.6) 5,947/45 YK781 (Us3-chimera) 42.1 ± 4.9 (35.7) 8.6 ± 1.0 (7.3) 7.2 ± 1.3 (6.1) 54.2 ± 5.1 (46.0) 5,305/45 YK783 (Us3-repair) 18.1 ± 5.0 (13.8) 11.6 ± 1.7 (8.8) 21.8 ± 4.2 (16.7) 77.6 ± 4.8 (59.3) 5,890/45 ↵a Numbers in parentheses are the percentages of enveloped virus particles in compartments.
