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1.
FIG. 3.

FIG. 3. From: Role for 3-O-Sulfated Heparan Sulfate as the Receptor for Herpes Simplex Virus Type 1 Entry into Primary Human Corneal Fibroblasts.

Expression of HVEM and 3-OST-3 mRNAs in cultured CF. (A) RT-PCR analysis of the expression of HVEM- and 3-OST-3 (as a surrogate marker for 3-OS HS)-specific messages in CF and HeLa cells (served as a control). The cDNAs were produced from total RNA isolated from the cells. The PCR products were separated by electrophoresis on an agarose gel and stained with ethidium bromide. Molecular size markers are indicated on the left (sizes are in kilobases). (B) RPIP-HPLC chromatograms of the disaccharide analysis of HS isolated from CF and CHO-K1 cells. 35S-HS was extracted from CHO-K1 cells (top) and CF (bottom) after they were incubated with Na235SO4. Extracted HS was then depolymerized by nitrous acid at pH 1.5, followed by sodium borohydride reduction. The resultant 35S-labeled disaccharides were resolved by RPIP-HPLC. The elution positions of disaccharides are indicated by arrows, where arrow 1 represents IdoUA2S-AnMan3S, arrow 2 represents IdoUA2S-AnMan6S, and arrow 3 represents IdoUA2S-AnMan3S6S.

Vaibhav Tiwari, et al. J Virol. 2006 Sep;80(18):8970-8980.
2.
FIG. 5.

FIG. 5. From: Role for 3-O-Sulfated Heparan Sulfate as the Receptor for Herpes Simplex Virus Type 1 Entry into Primary Human Corneal Fibroblasts.

Spinoculation of heparinase-treated cells recovers entry into Vero cells and nectin-1- or HVEM-expressing CHO-K1 cells but not cultured CF or 3-OST-5-expressing CHO-K1 cells. (A) CHO-K1 cells were transiently transfected with a nectin-1, HVEM, or 3-OST-5 plasmid. gD receptor-expressing cells, along with naturally susceptible Vero cells and cultured CF, were plated and treated with heparinases II and III (4 U/ml) or PBS buffer for 2 h and infected with HSV-1(KOS) gL86 at 35 PFU/cell for 2 h. During the 2-h infection, cells were either spun at 37°C or kept in an incubator. Infection was then continued for an additional 4 h, the plates were rinsed, and viral entry was measured by determining OD410. Results from heparinase-untreated and unspun cells (considered positive controls) are shown as black bars, while those from heparinase-treated and unspun or spun cells are shown as white and gray bars, respectively. Data represent the mean ± the standard deviation of results in triplicate wells in a representative experiment. The experiment was repeated three times with similar results. (B) Spinoculation of heparinase-treated CF recovers entry of CMV but not HSV-1. Cultured CF plated in a 96-well plate were treated with heparinases II and III (4 U/ml) or PBS buffer for 3 h and infected with reporter viruses HSV-1(KOS) gL86 and CMV at 35 PFU/cell for 2 h. During the 2-h infection, cells were spun at 37°C or kept in an incubator unspun. Infection was then continued for an additional 4 h, and viral entry was measured by determining OD410. Results from heparinase-untreated and unspun cells (considered positive controls) are shown as black bars, while those from heparinase-treated unspun or spun cells are shown as white and gray bars, respectively. Data represent the mean ± the standard deviation of results in triplicate wells in a representative experiment. The experiment was repeated three times with similar results.

Vaibhav Tiwari, et al. J Virol. 2006 Sep;80(18):8970-8980.
3.
FIG. 2.

FIG. 2. From: Role for 3-O-Sulfated Heparan Sulfate as the Receptor for Herpes Simplex Virus Type 1 Entry into Primary Human Corneal Fibroblasts.

Infectious yields of HSV-1 and involvement of gD receptors in viral infection. (A) HSV-1 replicates in cultured CF. Confluent monolayers of CF (○) and wild-type CHO-K1 cells (•) and CHO-K1 cells expressing 3-OS HS modified by 3-OST-3 (▾) were infected with HSV-1 at 0.01 PFU per cell for 90 min at 37°C. Inoculums were harvested at 12 to 48 h postinfection. The infectious-virus titer (PFU per milliliter) determined in triplicate in Vero cells by plaque assay indicates that the viral titer in cultured CF increased over time. Data represent the mean ± the standard deviation of results in triplicate wells in a representative experiment. Experiments were repeated three times, yielding similar results. (B) Receptors for gD are important for infection of CF. Cultured CF were transfected with an HSV-1 gD expression plasmid and infected either with HSV-1 (○) or with CMV (▿). In parallel, CF infected with the empty vector pCDNA3 were infected with either HSV-1 (•) or CMV (▾). Reporter enzyme activity, as a measurement of viral entry, was measured with a spectrophotometer at 410 nm.

Vaibhav Tiwari, et al. J Virol. 2006 Sep;80(18):8970-8980.
4.
FIG. 4.

FIG. 4. From: Role for 3-O-Sulfated Heparan Sulfate as the Receptor for Herpes Simplex Virus Type 1 Entry into Primary Human Corneal Fibroblasts.

Effect of heparinase treatment on HSV-1 entry and gD binding to CF. (A) Effect of heparinase treatment on HSV-1 entry. Cultured CF, HeLa cells, or control CHO-K1 cells expressing 3-OST-5 were treated with heparinases II and III (4 U/ml) in 96-well tissue culture dishes. A separate set of cultured cells were mock treated with PBS alone. The cells were then exposed to HSV-1(KOS) gL86 at 35 PFU/cell, and viral entry was quantitated 6 h later, as described in the legend to Fig. . Data represent the mean ± the standard deviation of results in triplicate wells in a representative experiment. The experiment was repeated three times with similar results. (B) Effect of heparinase treatment on gD binding. Cultured CF and control HeLa cells in 96-well dishes were treated with heparinases II and III (4 U/ml) or incubated with buffer. The cells were then incubated with gD-Fc for 30 min, followed by fixation and incubation with a secondary antibody and a horseradish peroxidase detection system. The values shown (means of triplicate determinations plus standard deviations) represent the amounts of binding product detected spectrophotometrically (OD650). (C) Confocal imaging of gD binding to heparinase-treated cells. Cultured HeLa cells and CF were plated in glass bottom culture dishes and grown overnight. The cells were treated with heparinases II and III (4 U/ml) or mock treated with buffer. After incubation with gD-Fc, the cells were washed, fixed with acetone, and incubated with FITC-conjugated anti-rabbit IgG. Micrographs of representative HeLa cells and CF are presented to show gD-Fc binding with or without heparinase treatment with a Leica confocal microscope.

Vaibhav Tiwari, et al. J Virol. 2006 Sep;80(18):8970-8980.
5.
FIG. 6.

FIG. 6. From: Role for 3-O-Sulfated Heparan Sulfate as the Receptor for Herpes Simplex Virus Type 1 Entry into Primary Human Corneal Fibroblasts.

Role of 3-OS HS in membrane fusion. (A) Enzymatic removal of cell surface HS blocks HSV-1-induced cell fusion in cultured CF. CHO-K1 cells expressing 3-OST-3 and naturally susceptible HeLa cells were used as controls. Target cells (CHO-K1 3-OST-3, CF, and HeLa) were treated with heparinase (black bars) or left untreated (white bars) prior to cocultivation with effector cells expressing four essential HSV-1 glycoproteins (gB, gD, gH, and gL). The luminometer readings (relative light units [RLU]) for untreated cells of each kind were determined three or more times, and the mean value was assigned a value of 100%. The number of relative light units obtained for untreated cells was then normalized accordingly to obtain the percentages shown. Significant difference compared to the heparinase-untreated controls, P < 0.01. (B) Enzymatic removal of cell surface HS does not block HHV-8 envelope glycoprotein-induced cell-to-cell fusion in CF. CHO-K1 cells, effector cells that express HHV-8 envelope glycoproteins (gB, gH, and gL) or control plasmid pCDNA3 were mixed at a 1:1 ratio with heparinase-treated or untreated CF. The percentage values were calculated as described above. (C) Soluble 3-OS HS generated by 3-OST-3 and 3-OST-5 but not 3-OST-1 or unmodified HS (UnHS) inhibits cell-to-cell fusion in cultured CF. Effector cells were treated with 1.0 μg of unmodified HS (UnHS) or purified 3-OSTs generated by specific enzymes (3-OST-1, 3-OST-3, and 3-OST-5) or mock treated as a positive control (white bar) prior to being mixed with target cells. Untreated effector cells mixed with cultured CF or 3-OST-3-expressing target cells served as a control. Luciferase activity was measured 24 h after cocultivating the effector and target cells. The number of relative light units shown is from one experiment performed in triplicate.

Vaibhav Tiwari, et al. J Virol. 2006 Sep;80(18):8970-8980.
6.
FIG. 1.

FIG. 1. From: Role for 3-O-Sulfated Heparan Sulfate as the Receptor for Herpes Simplex Virus Type 1 Entry into Primary Human Corneal Fibroblasts.

Entry of HSV-1 into cultured CF. (A) Dose-response analysis of HSV-1 entry into CF. Cultured CF (•), along with wild-type CHO-K1 cells (▪), were plated in 96-well plates and inoculated with twofold serial dilutions of β-galactosidase-expressing recombinant HSV-1(KOS) tk12 ranging from 1 to 180 PFU/cell. After 6 h, the cells were permeabilized and incubated with ONPG substrate for quantitation of β-galactosidase activity expressed from the input viral genome. Enzymatic activity was measured by determining OD410. Data represent the mean ± the standard deviation of results in triplicate wells in a representative experiment. The experiment was repeated three times with similar results. (B) Entry of HSV-1 into cultured CF, human LF cells, and CHO-K1 cells expressing either HVEM or 3-OS HS as a gD receptor. The cells were plated in 96-well dishes and then treated with serial dilutions of β-galactosidase-expressing recombinant HSV-1(KOS) tk12 as described for panel A. The values shown represent the amount of reaction product detected spectrophotometrically at a single input dose of 5 PFU/cell. Data represent the mean ± the standard deviation of results in triplicate wells in a representative experiment. The experiment was repeated three times with similar results. (C) Dark-field image of cultured CF infected with GFP-tagged HSV-1. (D) A section of the deconvolved stack showing GFP-tagged capsids near or at the periphery of DAPI-stained blue nuclei. The vertical line to the right indicates the YZ plane of z sections. The horizontal line at the bottom indicates the XZ plane of z sections. Images were collected on a Zeiss Axiovert 100 M microscope equipped with a 63× objective. (E) Three-dimensional rendering of the entire deconvolved stacks. (F) Optical plane of sections presented at 90°. (G) Optical plane of sections presented at 110°. (H) Plot of maximum light intensity due to GFP-tagged HSV-1 observed per stack going from the top to the bottom of a cell.

Vaibhav Tiwari, et al. J Virol. 2006 Sep;80(18):8970-8980.

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