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. 2015 Apr 27:6:6985.
doi: 10.1038/ncomms7985.

Heparanase is a host enzyme required for herpes simplex virus-1 release from cells

Satvik R Hadigal  1 , Alex M Agelidis  2 , Ghadah A Karasneh  2 , Thessicar E Antoine  2 , Abraam M Yakoub  2 , Vishnu C Ramani  3 , Ali R Djalilian  1 , Ralph D Sanderson  3 , Deepak Shukla  2
Affiliations

Heparanase is a host enzyme required for herpes simplex virus-1 release from cells

Satvik R Hadigal et al. Nat Commun. .

Abstract

Herpesviruses exemplified by herpes simplex virus-1 (HSV-1) attach to cell surface heparan sulfate (HS) for entry into host cells. However, during a productive infection, the HS moieties on parent cells can trap newly exiting viral progenies and inhibit their release. Here we demonstrate that a HS-degrading enzyme of the host, heparanase (HPSE), is upregulated through NF-kB and translocated to the cell surface upon HSV-1 infection for the removal of HS to facilitate viral release. We also find a significant increase in HPSE release in vivo during infection of murine corneas and that knockdown of HPSE in vivo inhibits virus shedding. Overall, we propose that HPSE acts as a molecular switch for turning a virus-permissive 'attachment mode' of host cells to a virus-deterring 'detachment mode'. Since many human viruses use HS as an attachment receptor, the HPSE-HS interplay may delineate a common mechanism for virus release.

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Conflict of interest statement

Conflicts of Interest Statement:

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1. Loss of HS from cell surface after infection
a. Left- HS expression is decreased on HCE cell surface at different time points post-infection, measured by flow cytometry. Cells were infected with KOS-WT at MOI 0.1 and were stained for HS at 12, 24, and 36 hrs post-infection (hpi) with HS Antibody 10E4. Right- Fluorescence intensity measurements based on flow cytometry results. Integrated mean fluorescence intensity of the whole population was measured and fold change was normalized to uninfected mock samples for each time point. b. Left- Representative immunofluorescence microscopy images show decrease in HS expression on HCE cell surface of 24 and 36 hpi samples when compared to mock images. Cells were infected with KOS-WT at MOI 0.1 and were stained with HS Antibody 10E4. Green represents HS expression. Images are merged with brightfield to show cell borders. Scale bar 10 μm. Right- Images were quantified for HS intensity. Eighty cells were picked at random from each sample and intensity of HS was measured for each cell using IMAGE J. c. HS expression is decreased on HeLa cell surface after infection, measured by flow cytometry. Cells were infected with KOS-WT at MOIs of 1 and 10 for 24 hrs, and then stained for HS using HS antibody 10E4. d. ELISA measurement of HPSE activity indicates increased degradation of HS upon infection. Infected cells showed more HS degradation activity when compared to uninfected cells at each time point. Increased HPSE activity was observed in uninfected cells at 36 hrs due to expansion of the number of cells in culture; the assay provides a measurement of the total amount of HPSE produced rather than on a per cell basis. All data are presented as mean ± s.e.m. of three independent experiments (n=3). Asterisks denote a significant difference as determined by Student’s t-test; *P<0.05, **P<0.01, ****P<0.0001.
Figure 2
Figure 2. HPSE is upregulated after HSV-1 infection
a. Left- Western blot shows increased HPSE expression of latent (65kDa) and active (50kDa) after 24 and 36 hr infection of HCE cells at MOI 0.1. Right- Densitometry quantification of 50 kDa active HPSE normalized to GAPDH. Fold change of infected over uninfected cells is shown for each time point. b. Increase in HPSE transcripts in HCE cells after infection with KOS-WT at MOI 0.1. Results are shown as fold change of infected over uninfected cells at each time point, normalized to GAPDH. c. Increase in promoter activity of HPSE gene upon infection in HCE cells. KOS-WT was used to infect cells at MOI 0.1 for 12, 24, and 36 hrs. Shown is the average fold increase over uninfected control. Experimental values are normalized to those obtained with pGL3 as a control for transfection efficiency. d. Increase in HPSE transcripts when cells were infected with herpes simplex virus type-2 (HSV-2), cytomegalovirus (CMV), bovine herpes virus (BHV), and pseudorabies virus (PRV). HCE cells were used for HSV-2, BHV, and PRV infections whereas primary human foreskin fibroblasts (HFF) were used for CMV infections. HCE cells were infected for 24 hrs. HFF were infected with CMV for 7 days at indicated MOIs. Results are shown as fold change of infected over uninfected cells at each time point, normalized to GAPDH. e. Left- HPSE expression is increased on cell surface after infection, measured by flow cytometry. HCE cells were infected with KOS-WT at MOI 0.1 and were stained with HPSE antibody HP130. Right- Integrated mean fluorescence intensity of the whole population was measured and fold change was normalized to mock at each time point. f. Left- Immunofluorescence microscopy images show increased HPSE expression on surface of infected HCE cells. Red represents HPSE expression. Images are merged with brightfield to show cell borders. Scale bar 10 μm. Right- Images were quantified for HPSE intensity. Eighty cells were picked at random for each sample and intensity of HPSE was measured for each cell using IMAGE J. All data are presented as mean ± s.e.m. of three independent experiments (n=3). Asterisks denote a significant difference as determined by Student’s t-test; *P<0.05, **P<0.01, ****P<0.0001, ns, not significant. See Supplementary Figure 8 for full-length images of blots.
Figure 3
Figure 3. Mechanism of HPSE upregulation upon infection
a. Increase in NF-kB p65 transcript levels after infection with KOS-WT at MOI 0.1 at various time points. Results are normalized to GAPDH and reported as fold change of infected over uninfected cells. b. Nuclear translocation of NF-kB p65 upon HSV-1 infection, observed by immunofluorescence microscopy. HCE cells were infected with GFP-KOS at MOI 0.1 for 12 hrs. Translocation of NF-kB to the nucleus can be seen in GFP-positive cells (arrowheads). Scale bar 10 μm. c. Nuclear translocation of NF-kB p65 upon HSV-1 infection measured by western blot shows an increase in NF-kB translocation from cytoplasm to nucleus. Left- HCE cells were infected with KOS-WT at MOI 0.1 for 24 and 36 hrs, and nuclear/cytoplasmic extractions were performed. Right- Densitometry quantification of NF-kB p65 in cytoplasm and nucleus, normalized to GAPDH and Histone H3, respectively. d. Induction of NF-kB activation by Betulinic acid (BetA) increases HPSE mRNA. Left- HCE cells were treated with BetA at 10 μg/mL or DMSO vehicle for 24 hrs. NF-kB translocation into nucleus with BetA treatment is shown in HCE cells. Also shown is densitometry quantification of NF-kB p65 in cytoplasm and nucleus, normalized to GAPDH and Histone H3, respectively. Right- HPSE mRNA is increased with BetA treatment of HCE cells. e. Inhibition of NF-kB activation results in decreased HPSE promoter activity. HCE cells (left) and HeLa cells (right) were transfected with mutant IkBa incapable of degradation (S32A/S36A), thereby specifically inhibiting NF-kB activation and nuclear translocation. Cells were infected with KOS-WT at MOI 0.1 for 24 hrs. Cell lysates were isolated, and luciferase assay was performed. Results shown are normalized to empty pGL3 vector as a control for transfection efficiency. f. Inhibition of NF-kB activation results in decreased HPSE transcript levels upon infection. HCE cells were transfected with mutant IkBa as described above, and infected with KOS-WT at MOI 0.1 for 24 hrs. All data are presented as mean ± s.e.m. of three independent experiments (n=3). Asterisks denote a significant difference as determined by Student’s t-test; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, ns, not significant. See Supplementary Figure 8 for full-length images of blots.
Figure 4
Figure 4. Functional phenotype of HPSE on viral egress and attachment
a. Viral egress is increased in HPSE overexpressing cells. Left- HCE cells were infected with KOS-WT at MOI 0.1 24 hrs after transfection with HPSE or control vector. Supernatant was collected at indicated time points post-infection and virus was quantified by titering on Vero cells in a plaque assay. Right- HCE cells were transfected and infected as mentioned above and culture supernatant containing viruses was collected and quantified by PCR using primers against glycoprotein-D (gD). b. Viral egress is decreased in HPSE deficient cells. Stably transduced HCE cell lines were generated using lentiviral HPSE shRNA and scrambled shRNA constructs, and these were then infected with KOS-WT at MOI 0.1. As described above, supernatant was collected to perform plaque assay (left) and viral DNA was quantified by PCR for gD (right). c. HS is lost from the cell surface upon HPSE overexpression. HCE cells were transfected with HPSE plasmid or control vector, incubated at 37°C for 24 hrs. Cells were then stained for HS using Antibody 10E4 at 24 and 36 hpi and analyzed by flow cytometry. d. Decreased attachment of HSV-1 after HPSE overexpression. HCE cells were transfected with HPSE plasmid or control vector, and 24 hrs later incubated with GFP-KOS for 2 hrs at 4°C to allow viral binding. GFP fluorescence intensity from the virus was measured by flow cytometry. e. Decreased attachment of GFP-KOS after previous infection with KOS-WT at specified time points. HCE cells were infected with KOS-WT for 6, 24, or 36 hrs, then incubated with GFP-KOS for 2 hrs at 4°C to allow viral binding. GFP fluorescence intensity from the virus was measured by flow cytometry. Black curve represents 0 hr time point. f. Model of HSV egress, showing role of HPSE at the cell surface. This model shows the attachment and detachment modes of the HSV lifecycle. During attachment, virus is bound to HS. However, during detachment HPSE (red) is upregulated and translocated to the cell surface, cleaving HS during viral exit. All data are presented as mean ± s.e.m. of three independent experiments (n=3). Asterisks denote a significant difference as determined by Student’s t-test; *P<0.05, **P<0.01, ****P<0.0001.
Figure 5
Figure 5. Significance of HPSE and HSV egress in vivo
a. Knockdown of HPSE in mice eyes decreases viral spread through cornea. Mice corneas were injected with shRNA against HPSE or with scrambled shRNA and infected with GFP-KOS. Left- Fluorescence detection 72 hrs post-infection. Arrowhead indicates GFP-HSV. Right- Knockdown of HPSE in mouse cornea demonstrated by reverse transcriptase PCR. b. Left- Corneal eye swabs collected at 24 and 72 hpi for plaque assays show decreased HSV titer in HPSE knockdown corneas (n=5 for each group). Right- Decreased corneal tissue damage in HPSE knockdown mice corneas after HSV-1 infection. HPSE was knocked down in mice corneas as described above, and 7 days post-infection with KOS-WT, fluorescein was applied to mice eyes to highlight corneal damage. c. Left- Mice corneas were transfected with HPSE plasmid in corneal epithelium and infected with KOS-WT. Corneal swabs collected at 72 hpi for plaque assays show an increase in HSV titer in cornea transfected with HPSE plasmid (n=3 for each group). Right- Increased corneal tissue damage in HPSE overexpressing mouse corneas after HSV-1 infection. Fluorescein was applied to mice eyes to highlight tissue damage 7 days post-infection. d. Seven days post ocular infection, mouse corneas were examined for tissue damage by slit lamp biomicroscope and scored. Left- Decreased tissue damage in HPSE shRNA knockdown corneas when compared to scrambled shRNA controls. Right- Increased tissue damage in HPSE overexpressed corneas when compared to vector transfected controls (n=3 for each group). e. Decreased viral egress after heparin treatment, measured by plaque assay. Mice corneas were infected with KOS-WT at 105 PFU. After infection, 5 μL of 25 μg/μL heparin solution was applied directly to corneas at 24 and 48 hpi and corneal swabs for plaque assay were collected at 72 hpi. f. Human corneo-scleral buttons from the same subject were cultured and infected with KOS-WT at 105 pfu. The epithelium was extracted at 24 hpi and an increase in HSPE (50kDa) was observed. The experiment was repeated three times and representative western blot is shown. All data are presented as mean ± s.e.m. of three independent experiments (n=3). Asterisks denote a significant difference as determined by Student’s t-test; *P<0.05. See Supplementary Figure 8 for full-length images of gels and blots.
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