Expanding the role of 3-O sulfated heparan sulfate in herpes simplex virus type-1 entry

Abstract

Heparan sulfate (HS) proteoglycans are commonly exploited by multiple viruses for initial attachment to host cells. Herpes simplex virus-1 (HSV-1) is unique because it can use HS for both attachment and penetration, provided specific binding sites for HSV-1 envelope glycoprotein gD are present. The interaction with gD is mediated by specific HS moieties or 3-O sulfated HS (3-OS HS), which are generated by all but one of the seven isoforms of 3-O sulfotransferases (3-OSTs). Here we demonstrate that several common experimental cell lines express unique sets of 3-OST isoforms. While the isoforms 3-OST-3, -5 and -6 were most commonly expressed, isoforms 3-OST-2 and -4 were undetectable in the cell lines examined. Since most cell lines expressed multiple 3-OST isoforms, we addressed the significance of 3-OS HS in HSV-1 entry by down-regulating 2-O-sulfation, a prerequisite for 3-OS HS formation, by knocking down 2-OST expression by RNA interference (RNAi). 2-OST knockdown was verified by reverse-transcriptase PCR and Western blot analysis, while 3-OS HS knockdown was verified by immunofluorescence. Cells showed a significant decrease in viral entry, suggesting an important role for 3-OS HS. Implicating 3-OS HS further, cells knocked down for 2-OST expression also demonstrated decreased cell-cell fusion when cocultivated with effector cells transfected with HSV-1 glycoproteins. Our findings suggest that 3-OS HS may play an important role in HSV-1 entry into many different cell lines.

Fig. 1

Heparan sulfate modifications. Heparan sulfate chains are initially synthesized as repeating disaccharide units of N-acetylated glucosamine and glucuronic acid. HS can then be modified by a series of enzymatic reactions, including N-deacetylation and N-sulfation of N-acetylated glucosamine converting it to N-sulfo-glucosamine, C5 epimerization of glucuronic acid to iduronic acid, and O-sulfation at the 2-OH, 6-OH, and 3-OH positions. First is 2-O-sulfation of iduronic acid and glucuronic acid, followed by 6-O-sulfation of N-acetylated glucosamine and N-sulfo-glucosamine units, and finally 3-O-sulfation of glucosamine residues. 2-O (red) and 3-O (green) sulfations are highlighted.

Fig. 2

3-OST isoform expression in various cell lines. RT-PCR detection of isoforms 3-OST-3A (a), 3-OST-3B (b), 3-OST-5 (c), and 3-OST-6 (d) was performed in Vero, RPE, HeLa, and P19N cells. CHO-K1 cells transfected with 3-OST isoforms (-3A, -3B, -5, -6) served as a positive control. The cDNAs were produced from total RNA isolated from cells. Superscript II reverse transcriptase was used for RT-PCR. PCR products were separated by electrophoresis on an agarose gel and stained with ethidium bromide. Expected PCR product sizes were 604 bps (3-OST-3A), 442 bps (3-OST-3B), 777 bps (3-OST-5), and 570 bps (3-OST-6). β-actin mRNA (bottom panels) was used as a control with an expected PCR product size of 285 bps.

Fig. 3

RT-PCR to verify reduced 2-OST gene expression. RT-PCR analysis of 2-OST expression was performed with HeLa, RPE, and Vero cells (a), and CHO-K1 cells expressing 3-OST-3B, HVEM, or nectin-1 (b). Cells were mock treated (wt) or transfected with scrambled siRNA (+ negative siRNA) or 2-OST siRNA (+ siRNA). About 48 h after siRNA transfection, total RNA was isolated from each cell line. Superscript II reverse transcriptase was used for RT-PCR. PCR amplification of cDNA was done using specific 2-OST primers. Expected PCR product sizes were 792 bps (2-OST) and 285 bps (β-actin) (bottom panels).

Fig. 4

Western Blot analysis of 2-OST protein expression after siRNA down regulation. 2-OST protein expression was measured in a sample of CHO-K1 cells treated with 2-OST siRNA, scrambled siRNA, or mock treated cells (no siRNA). Protein expression was measured 48 h after siRNA transfection. β-actin protein expression was measured as a loading control. Protein bands were quantified using NIH ImageJ v1.41.

Fig. 5

Use of immunofluorescence to confirm reduced 3-OS HS expression. HeLa cells were incubated with the antibody HS4C3 for 1 h at 4 °C. Cells were then fixed for 20 min, and incubated with FITC conjugated anti-mouse IgG to label 3-OS HS surface expression. 3-OS HS surface expression was compared in HeLa cells that were mock treated (left panels) or transfected with scrambled siRNA (middle panels) or 2-OST siRNA (right panels). Imaging was performed using confocal microscopy at a 60x oil objective.

Fig. 6

HSV-1 entry is dependent on 2-OST and 3-OS HS expression. HSV-1 entry was analyzed in HeLa, Vero, RPE, and CHO-K1 cells expressing 3-OST-3B, nectin-1, or HVEM. Cells were mock treated (no siRNA) or transfected with scrambled siRNA or 2-OST siRNA. Cells were replated in a 96-well culture dishes and inoculated with β-galactosidase-expressing recombinant HSV-1(KOS) gL86 for 6 h. The soluble substrate ONPG was added, and enzymatic activity was measured with a microplate reader at 410 nm.

Fig. 7

2-OST downregulation affects HSV-1 binding. HSV-1 binding to the cell surface was measured in Vero, HeLa, RPE, and CHO-K1 cells expressing either 3-OST-3B or nectin-1 that were mock treated (no siRNA) or transfected with either scrambled siRNA or 2-OST siRNA. About 48 h after siRNA transfection, cells were infected with HSV-1 (K26GFP) (10 M.O.I.) for 1 h at 4 °C. Fluorescence readings were taken using a GENios Pro fluorescence reader.

Fig. 8

Cell-cell fusion is differentially affected by 2-OST downregulation in various cell lines. Target cells for Vero (a), HeLa (b), RPE (c), and CHO-K1 cells expressing 3-OST-3B (d) were mock treated (wild type), treated with scrambled siRNA (+negative siRNA), or treated with 2-OST siRNA (+siRNA). About 48 h after siRNA transfection, target and effector cells were mixed together in a 1:1 ratio and luciferase activity was measured after 24 h.