Syndecan-1 and syndecan-2 play key roles in herpes simplex virus type-1 infection

Abstract

Herpes simplex virus type 1 (HSV-1) is an important human pathogen and a leading cause of infectious blindness in the developed world. HSV-1 exploits heparan sulfate proteoglycans (HSPG) for attachment to cells. While the significance of heparan sulphate (HS) moieties in HSV-1 infection is well established, the role of specific proteoglycan core proteins in the infection process remains poorly understood. The objective of this study was to assess the roles of syndecan-1 and syndecan-2 core proteins in HSV-1 infection, both of which are expressed by many HSV-1 target cell types. Our results demonstrate that syndecan-1 and syndecan-2 gene silencing by RNA interference reduces HSV-1 entry, plaque formation and facilitates cell survival. Furthermore, HSV-1 infection increases syndecan-1 and syndecan-2 protein synthesis and a resultant increase in cell surface expression of HS. Our observations suggest that changes in syndecan-1 and syndecan-2 expression levels may be related to active viral infection. Taken together, our findings provide new insights into HSPG functions during HSV-1 entry and spread.

Fig. 1.

Western blot analysis of syndecan-1 and syndecan-2 protein expression after siRNA downregulation. Protein expression of syndecan-1 and syndecan-2 measured in a sample of HeLa cells mock treated (no transfection) or treated with scrambled siRNA, syndecan-1 siRNA or syndecan-2 siRNA for 48 h. Representative Western blots showed knockdown of syndecan-1 (a) or syndecan-2 (b) after siRNA downregulation. Densitometric analysis revealed 50 % reduction in the signal intensity of both syndecan-1 and syndecan-2. β-Actin protein expression was measured as the loading control. Representative blots from three independent experiments are shown. The results are expressed as means±sd values from three independent experiments.

Fig. 2.

Downregulation of syndecan-1 and syndecan-2 inhibits HSV-1 entry into HeLa cells. HSV-1 entry was analysed in HeLa cells mock treated (no transfection) or transfected with scrambled siRNA, syndecan-1 siRNA or syndecan-2 siRNA. After siRNA transfection (48 h), cells were inoculated with a serial dilution of β-galactosidase-expressing recombinant HSV-1 (KOS) gL86 virus for 6 h. The soluble substrate ONPG was added and enzymic activity was measured. Downregulation of both syndecan-1 (Fig. 2a) and syndecan-2 (Fig. 2b) inhibits HSV-1 (KOS) gL86 entry into HeLa cells although syndecan-2 siRNA has more significant effect. Per cent inhibition of HSV-1(KOS) gL86 entry by syndecan-1 siRNA or syndecan-2 siRNA treatment was calculated relative to mock-treated (no transfection) cells. Scrambled siRNA-transfected cells were used as negative control. Each value shown is the mean of six determinations (±sd) (*P<0.05, **P<0.0001).

Fig. 3.

Syndecan-1- or syndecan-2-specific antibody treatment blocked HSV-1(KOS) gL86 entry into HeLa cells. Cells were incubated with a serial dilution of syndecan-1-specific pAbs, syndecan-2-specific pAbs, or control anti-myc mAb for 30 min. Cells were then inoculated with a constant dose of β-galactosidase-expressing recombinant HSV-1(KOS) gL86 (m.o.i. of 10) for 2 h. The soluble substrate ONPG was added and enzymic activity was measured. Per cent inhibition of HSV-1(KOS) gL86 entry by syndecan-1 pAbs or syndecan-2 pAbs treatment was calculated relative to anti-myc mAb-treated cells. Each value shown is the mean of four determinations (±sd) (P<0.05).

Fig. 4.

Downregulation of syndecan-1 and syndecan-2 affects HSV-1 plaque formation, size and enhances cell survival in HeLa cells. Cells were either mock treated (no transfection), or treated with scrambled siRNA, syndecan-1 siRNA or syndecan-2 siRNA. Post-transfection cells (48 h) were infected with HSV-1(KOS) (m.o.i. of 0.01). (a) Post-infection infectivity at 72 h was measured by the number of p.f.u. Relative number of plaques was computed relative to mock-treated (no transfection) samples. Significant decreases of number of plaques were seen in both syndecan-1 siRNA- and syndecan-2 siRNA-transfected HeLa cells. Plaques that consisted of 15 or more nuclei were counted. Results are means±sd of four independent experiments conducted in triplicate (**P<0.0001). (b) Morphological appearance of Giemsa-stained HSV-1 (KOS) plaques 72 h post-infection. In syndecan-1 siRNA- and syndecan-2 siRNA-treated HeLa cells smaller plaques were observed compared with the plaques in mock-treated or scrambled siRNA-treated cells. Magnification, ×40. (c) Cytotoxicity was measured after 120 h HSV-1 (KOS) infection. Relative number of dead cells was calculated relative to mock-treated samples. Significant decreases of cytotoxicity were observed in both syndecan-1 siRNA- and syndecan-2 siRNA-treated cells; however, only the effect of syndecan-2 siRNA treatment was statistically significant. Results are means±sd of four independent experiments conducted in triplicate (*P<0.05).

Fig. 5.

HSV-1 infection of host cell enhances syndecan-1 and syndecan-2 cell surface expression in HeLa cells. Cells were infected with a constant dose of HSV-1 (KOS) (m.o.i. of 10) for 2, 4 or 6 h. Syndecan-1 and syndecan-2 cell surface expression was then detected by FACS analysis. Enhanced syndecan-1 (a) and syndecan-2 (b) cell surface expression was detected when HeLa cells were infected with HSV-1. Mock-infected FITC stained cells were used as background control. Results are representative of three independent experiments.

Fig. 6.

Western blot analysis of syndecan-1 and syndecan-2 protein expression after HSV-1 (KOS) infection. HeLa cells were infected with a constant dose of HSV-1 (KOS) (m.o.i. of 10) for 2 and 6 h. Cell lysates were then blotted against syndecan-1 and syndecan-2. According to the densitometric analysis syndecan-1 (a) and syndecan-2 (b) were more strongly expressed at 2 and 6 h post-infection compared with the uninfected cells in HeLa cells. Relative intensity of HSV-1(KOS) virus-infected bands expressed as a ratio relative to the mock-infected sample is shown. β-Actin protein expression was measured as loading control. The results are expressed as means±sd values from three independent experiments.

Fig. 7.

Flow cytometry analysis of HS expression on HeLa cells infected with HSV-1. HeLa cells were infected with HSV-1(KOS) (m.o.i. of 10) at 37 °C for 2, 4 or 6 h. HS cell surface expression was then detected by FACS analysis. In HSV-1-infected cells, HS cell surface expression was enhanced compared with uninfected cells. Untreated FITC stained HeLa cells were used as background control.