Figure 1.
(A–B) Cloning of Zebrafish (ZF) encoded 3-OST-4 isoform into pCDNA3.1.
Zebrafish encoding 3-OST-4 plasmid was constructed by inserting the open reading frame of 3-OST-4 into pcDNA3.1 and the construct was designated pcDNA3.1-ZF-3-OST-4. The inserted sequence of 1278 bp of 3-OST-4 was verified after digestion using BamH1 and Xho1. (C–D). RT-PCR analysis for ZF encoded 3-OST-2 and 3-OST-4 isoforms expression in CHO-K1 cells. The housekeeping gene GAPDH was used as a normalization control.
Figure 2.
(A–C) Wild type Chinese hamster ovary (CHO-K1) cells expressing ZF 3-OST-4 are susceptible to HSV-1 entry.
(A). Dose response curve of HSV-1 entry into ZF expressing 3-OST-4 CHO-K1 cells. Resistant wild-type CHO-K1 cells were transfected with ZF-3-OST-4 at 2.5 µg DNA resulted HSV-1 gL86 entry, similar to human 3-OST-3 expression. Cells transfected with empty vector pcDNA3.1 at 2.5 µg DNA was used as a negative control. Cultured cells were plated in 96-well plates and inoculated with two-fold serial dilutions of β-galactosidase-expressing recombinant virus HSV-1 (KOS) gL86 at the plaque forming units (PFU) indicated. After 6 hr, the cells were washed, permeabilized and incubated with ONPG substrate (3.0 mg/ml) for quantitation of β-galactosidase activity expressed from the input viral genome. The enzymatic activity was measured at an optical density of 410 nm (OD 410). (B). HSV-1 entry into ZF 3-OST-4 expressing CHO-K1 cells was further confirmed by X-gal staining. Cells grown (4×106 cells) in six well dishes were challenged with β-galactosidase-expressing recombinant HSV-1 (gL86) at 20 pfu/cell. Wild-type CHO-K1 cells transfected with empty vector (pcDNA3.1) were also infected in parallel as negative control. After 6 h of infection at 37°C, cells were washed with PBS, fixed and permeabilized, and incubated with X-gal (5 bromo-4 chloro-3-indoyl- β-D- galactosidase) at 1.0 mg/ml, which yields an insoluble blue product upon hydrolysis by β-galactosidase. Blue cells (representing viral entry) were seen as shown. Microscopy was performed using a 20 × objective of Zeiss Axiovert 100. C. Co-expression of ZF encoded 3-OST-2 and 3-OST-4 resulted significant increase in HSV-1 infection. CHO-K1 cells cultured in 6 well dishes were transiently transfected with plasmids expressing human 3-OST-2, ZF 3-OST-2, ZF 3-OST-4 and co-expressing ZF 3-OST-2 and 3-OST-4. CHO-K1 cells expressing pcDNA3.1 was used as negative control. Thirty six hr. post transfection cells were challenged with HSV-1 gL86 reporter virus. CHO-K1 cells expressing both 3-OST-2 and 3-OST-4 showed increase in HSV-1 entry. β-galactosidase based viral assay were performed using a soluble substrate o-nitrophenyl-β-D-galactopyranoside (ONPG; ImmunoPure, Pierce) using plate reader at 405 nm.
Figure 3.
Enzymatic removal of cell surface heparan sulfate (HS) by heparinase treatment in ZF 3-OST-4 expressing CHO-K1 cells reduces HSV-1 infection.
Three groups of cultured CHO-K1 cells expressing empty vector pcDNA3.1 or ZF 3-OST-4 or human 3-OST-3 were treated with heparinase II/III (1.5 U/ml; grey bar) or mock treated (black bar) followed by exposing cells to HSV-1 (KOS) gL86 at 20 PFU/cell and viral entry was quantitated 6 hr later by ONPG assay (panel A) and fluorescent microscopy and quantification (panel B, and panel C). In the latter case HSV-1 capsid-tagged to RFP (HSV-1K26RFP) virus was used. The heparinase treated ZF-3-OST-4 and human 3-OST-3 cells had significantly lesser number of viral entry (panel A) compared to mock treated ZF-3-OST-4 or 3-OST-3 cells. Similarly confocal visualization (panel B) and quantification (panel C) resulted less red punctate of HSV-1K26RFP on heparinase treated ZF 3-OST-4 or human 3-OST-3 cells compared to mock treated cells.
Figure 4.
Effect of heparinase enzyme on Zebrafish (ZF) encoded 3-OST-4 mediated cell to cell fusion with HSV-1 glycoprotein expressing cells (A–C).
A. ZF 3-OST-4-expressing target CHO-K1 cells gain the ability to fuse with effector cells co-expressing HSV-1 glycoproteins gB, gD, gH, and gL while heparinase treatment significantly blocks ZF 3-OST-4 mediated fusion. The target CHO-K1 cells were transfected with plasmids expressing ZF 3-OST-4 and luciferase reporter gene. The effector CHO-K1 cells were transfected with HSV-1 glycoproteins gB, gD, gH, and gL, and T7 RNA polymerase. CHO-K1 effector cells expressing control plasmid without HSV-1 glycoproteins were used as a negative control. In addition, target CHO-K1 cells expressing ZF 3-OST-4 were treated with heparinase II/III (1.5 U/ml) (blur bar) or left untreated (red bar) for 1 hr prior to co-cultivation with effector CHO-K1 cells expressing four HSV-1 essential glycoproteins (gB, gD, gH-gL; 0.5 µg DNA each glycoprotein). A luciferase reporter assay was performed 24 h after the two cell populations were mixed together. Cell fusion was measured in relative luciferase units (RLUs) using a Sirius luminometer (Berthold Detection System). Similarly visual observation resulted multinucleated giant cells (panel B; subpanels a and b) with CHO-K1 cells expressing ZF 3-OST-4 mixed with effector cells expressing HSV-1 glycoproteins. However hepainase treatment to ZF 3-OST-4 cells resulted significant decrease in giant cell formation. Panel c represent cartoon indicating cell fusion in mock treated ZF 3-OST-4 cells (subpanel a) vs. heparinase treated ZF 3-OST-4 cells inhibiting HSV-1 glycoprotein mediated cell fusion (subpanel b).
Figure 5.
Anti-3OS HS (G2) peptide block HSV-1 entry into ZF 3-OST-4 cells.
CHO-K1 cells expressing ZF 3-OST-4 and human 3-OST-3 were pretreated for 60 min with 0.2 mM concentrations of G2, or control peptide (Cp) peptides. Pretreated cells were infected with a β galactosidase-expressing recombinant virus HSV-1(KOS) gL86 (20 pfu/cell) for 6 h. Viral entry was measured via microplate reader at 405 nm.
Figure 6.
Proposed model for ZF 3-OST-4 mediated HSV-1 entry.
Cell expressing unmodified heparan sulfate (HS) do allow viral binding but not viral entry (panel a), while cells expressing HS modifying enzymes 3-O sulfotransferases (3-OST) such as ZF encoded 3-OST-2 or 3-OST-4 mediates both viral binding and entry (panel b). Pre-treatment of CHO-K1 cells expressing ZF encoded 3-OST-4 with anti-3-OS HS (G2) peptide generated against human 3-OST-3 binds to the sites on the modified HS used by HSV-1 and thereby prevents both viral binding as well as viral entry (panel c).