Immunology and Microbiology An Investigative Peptide-Acyclovir Combination to Control Herpes Simplex Virus Type 1 Ocular Infection

PURPOSE. To investigate the efficacy of a combination treatment composed of the cationic, membrane-penetrating peptide G2, and acyclovir (ACV) in both in vitro and ex vivo models of herpes simplex virus 1 (HSV-1) ocular infection. METHODS. The antiviral activity of a combined G2 peptide and ACV therapy (G2-ACV) was assessed in various treatment models. Viral entry, spread, and plaque assays were performed in vitro to assess the prophylactic efficacy of G2, G2-ACV, and ACV treatments. In the ex vivo model of HSV-1 infection, the level of viral inhibition was also compared among the three treatment groups via Western blot analysis and immunohistochemistry. The potential change in expression of the target receptor for G2 was also assessed using immunohistochemistry and RT-PCR. RESULTS. Statistically significant effects against HSV-1 infection were seen in all treatment groups in the viral entry, spread, and plaque assays. The greatest effects against HSV-1 infection in vitro were seen in the G2-ACV group. In the ex vivo model, statistically significant anti-HSV-1 effects were also noted in all control groups. At 24 hours, the greatest inhibitory effect against HSV-1 infection was seen in the ACV group. At 48 hours, however, the G2-ACVtreated group demonstrated the greatest antiviral activity. Syndecan-1, a target of G2, was found to be upregulated at 12-hours postinfection. CONCLUSIONS. This study shows that G2-ACV may be an effective antiviral against HSV-1 (KOS) strain when applied as single prophylactic applications with or without continuous doses postinfection. Keywords: herpes simplex keratitis, heparan sulfate, acyclovir H erpes simplex virus is an enveloped, double-stranded DNA virus and a member of Alphaherpesvirinae, a subfamily of Herpesviridae. Of the three members of this subfamily, which include herpes simplex virus 1 (HSV-1), herpes simplex virus 2 (HSV-2), and varicella zoster virus (VZV), HSV-1 has the greatest association with ocular infection. 1 With approximately 8.4 to 13.2 new cases per 100,000 people per year, HSV-1 is actually the main cause of infectious blindness in developed countries. 2 In the United States, approximately 500,000 individuals are afflicted with HSV ocular infection, with treatment costs rising to US$ 17.7 million annually for initial onset and recurring cases. 2,3 Ocular manifestations of HSV-1 include iridocyclitis, acute retinal necrosis, conjunctivitis, and keratitis. 1 Current mainstay treatment options against ocular HSV-1 infection include a dual regimen consisting of topical antivirals and topical corticosteroids. Prior work in our laboratory has yielded two 12-mer peptides with antiviral activity against HSV-1. The mechanism of action of these peptides rely on binding specifically to heparan sulfate (HS) and a modified form of HS, 3-O-sulfated HS (3-OS HS), both of which serve as entry receptors for HSV-1 on many different cell lines, including human corneal epithelial (HCE) cells. The peptides G1 (LRSRTKIIRIRH) and G2 (MPRRRRIRRRQK) both have high positive charge densities, and specific arginine and lysine residues are necessary to inhibit virus-cell binding and virus-induced membrane fusion. 7 In addition to inhibitory effects on HSV-1 entry into primary cultures of human corneal fibroblasts, both peptides have been demonstrated to effectively serve as prophylactic eye drops in an in vivo murine corneal model

Herpes Simplex Virus Infectivity And The Development Of Therapeutics Against Viral Invasion

Herpes Simplex Virus is one of the most commonly transmitted viral infections worldwide. HSV Type-1 and type -2 infections are known to cause painful, ulcerative lesions and blisters in the genital and oral mucosa and in severe cases can lead to blindness, encephalitis, and death. After infection HSV permanently resides within the host in a quiescent state of latency which is established in the ganglions of nerves. Once latency is established complete clearance of the virus cannot be achieved. For the past 3 decades, physicians have utilized suppressive therapy to treat HSV positive individuals. Unfortunately, suppressive treatments such as acyclovir (ACV) and other nucleoside analogs are only effective at reducing recurrent infections as they cannot clear latent infections or prevent asymptomatic shedding. The frequent emergence of acyclovir resistant HSV strains and high toxicity associated with long term usage of ACV and other nucleoside analogs regimens highlight a major limitation of the current treatment approach. To provide an alternative to suppressive therapeutics, we investigated the stages of viral entry that could be targeted to provide protection against viral invasion. As HSV pathogenesis begins with viral entry, our goal was to target this step of the viral lifecycle. By utilizing the protein kinase inhibitors ML-7, ML-9, and Blebbistatin plus the sequence specific G2 peptide and Zinc Oxide tetrapod nanoparticles, we significantly reduced viral attachment, internalization and intracellular trafficking of virus particles. In addition, cell-to-cell transmission of virus particles, the formation of multiple nucleated cells, viral glycoprotein mediated cellular fusion, and the development of disease associated with HSV infection were diminished. This study reveals that antiviral agents specifically designed to target or disrupt interactions of the virus with its receptors during the entry phase of infection can effectively serve as a new therapeutic approach for HSV treatment

Zebrafish 3-<i>O</i>-Sulfotransferase-4 Generated Heparan Sulfate Mediates HSV-1 Entry and Spread

<div><p>Rare modification of heparan sulfate (HS) by glucosaminyl 3-<i>O</i> sulfotransferase (3-<i>O</i>ST) isforms generates an entry receptor for herpes simplex virus type-1 (HSV-1). In the zebrafish (ZF) model multiple 3-<i>O</i>ST isoforms are differentially expressed. One such isoform is 3-<i>O</i>ST-4 which is widely expressed in the central nervous system of ZF. In this report we characterize the role of ZF encoded 3-<i>O</i>ST-4 isoform for HSV-1 entry. Expression of ZF 3-<i>O</i>ST-4 into resistant Chinese hamster ovary (CHO-K1) cells promoted susceptibility to HSV-1 infection. This entry was 3-<i>O</i> sulfated HS (3-<i>O</i>S HS) dependent as pre-treatment of ZF 3-<i>O</i>ST-4 cells with enzyme HS lyases (heparinase II/III) significantly reduced HSV-1 entry. Interestingly, co-expression of ZF 3-<i>O</i>ST-4 along with ZF 3-<i>O</i>ST-2 which is also expressed in brain rendered cells more susceptible to HSV-1 than 3-<i>O</i>ST-4 alone. The role of ZF-3-<i>O</i>ST-4 in the spread of HSV-1 was also evaluated as CHO-K1 cells that expressed HSV-1 glycoproteins fused with ZF 3-<i>O</i>ST-4 expressing effector CHO-K1 cells. Finally, adding further evidence ZF 3-<i>O</i>ST-4 mediated HSV-1 entry was inhibited by anti-3<i>O</i> HS G2 peptide. Taken together our results demonstrate a role for ZF 3-<i>O</i>ST-4 in HSV-1 pathogenesis and support the use of ZF as a model to study it.</p></div

Enzymatic removal of cell surface heparan sulfate (HS) by heparinase treatment in ZF 3-<i>O</i>ST-4 expressing CHO-K1 cells reduces HSV-1 infection.

<p>Three groups of cultured CHO-K1 cells expressing empty vector pcDNA3.1 or ZF 3-<i>O</i>ST-4 or human 3-<i>O</i>ST-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 (<b>panel A</b>) 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-<i>O</i>ST-4 and human 3-<i>O</i>ST-3 cells had significantly lesser number of viral entry (<b>panel A</b>) compared to mock treated ZF-3-<i>O</i>ST-4 or 3-<i>O</i>ST-3 cells. Similarly confocal visualization (<b>panel B</b>) and quantification (<b>panel C</b>) resulted less red punctate of HSV-1K26RFP on heparinase treated ZF 3-<i>O</i>ST-4 or human 3-<i>O</i>ST-3 cells compared to mock treated cells.</p

(A–C) Wild type Chinese hamster ovary (CHO-K1) cells expressing ZF 3-<i>O</i>ST-4 are susceptible to HSV-1 entry.

<p>(<b>A</b>). Dose response curve of HSV-1 entry into ZF expressing 3-<i>O</i>ST-4 CHO-K1 cells. Resistant wild-type CHO-K1 cells were transfected with ZF-3-<i>O</i>ST-4 at 2.5 µg DNA resulted HSV-1 gL86 entry, similar to human 3-<i>O</i>ST-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>B</b>). HSV-1 entry into ZF 3-<i>O</i>ST-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. <b>C.</b> Co-expression of ZF encoded 3-<i>O</i>ST-2 and 3-<i>O</i>ST-4 resulted significant increase in HSV-1 infection. CHO-K1 cells cultured in 6 well dishes were transiently transfected with plasmids expressing human 3-<i>O</i>ST-2, ZF 3-<i>O</i>ST-2, ZF 3-<i>O</i>ST-4 and co-expressing ZF 3-<i>O</i>ST-2 and 3-<i>O</i>ST-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-<i>O</i>ST-2 and 3-<i>O</i>ST-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.</p

Anti-3OS HS (G2) peptide block HSV-1 entry into ZF 3-<i>O</i>ST-4 cells.

<p>CHO-K1 cells expressing ZF 3-<i>O</i>ST-4 and human 3-<i>O</i>ST-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.</p

Effect of heparinase enzyme on Zebrafish (ZF) encoded 3-<i>O</i>ST-4 mediated cell to cell fusion with HSV-1 glycoprotein expressing cells (A–C).

<p><b>A.</b> ZF 3-<i>O</i>ST-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-<i>O</i>ST-4 mediated fusion. The target CHO-K1 cells were transfected with plasmids expressing ZF 3-<i>O</i>ST-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-<i>O</i>ST-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 (<b>panel B</b>; subpanels a and b) with CHO-K1 cells expressing ZF 3-<i>O</i>ST-4 mixed with effector cells expressing HSV-1 glycoproteins. However hepainase treatment to ZF 3-<i>O</i>ST-4 cells resulted significant decrease in giant cell formation. <b>Panel c</b> represent cartoon indicating cell fusion in mock treated ZF 3-<i>O</i>ST-4 cells (subpanel a) vs. heparinase treated ZF 3-<i>O</i>ST-4 cells inhibiting HSV-1 glycoprotein mediated cell fusion (subpanel b).</p

(A–B) Cloning of Zebrafish (ZF) encoded 3-<i>O</i>ST-4 isoform into pCDNA3.1.

<p>Zebrafish encoding 3-<i>O</i>ST-4 plasmid was constructed by inserting the open reading frame of 3-<i>O</i>ST-4 into pcDNA3.1 and the construct was designated pcDNA3.1-ZF-3-<i>O</i>ST-4. The inserted sequence of 1278 bp of 3-<i>O</i>ST-4 was verified after digestion using BamH1 and Xho1. (<b>C–D</b>). RT-PCR analysis for ZF encoded 3-<i>O</i>ST-2 and 3-<i>O</i>ST-4 isoforms expression in CHO-K1 cells. The housekeeping gene GAPDH was used as a normalization control.</p

Proposed model for ZF 3-<i>O</i>ST-4 mediated HSV-1 entry.

<p>Cell expressing unmodified heparan sulfate (HS) do allow viral binding but not viral entry (panel a), while cells expressing HS modifying enzymes 3-<i>O</i> sulfotransferases (3-<i>O</i>ST) such as ZF encoded 3-<i>O</i>ST-2 or 3-<i>O</i>ST-4 mediates both viral binding and entry (panel b). Pre-treatment of CHO-K1 cells expressing ZF encoded 3-<i>O</i>ST-4 with anti-3-<i>O</i>S HS (G2) peptide generated against human 3-<i>O</i>ST-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).</p

SnO₂ treatment reduces glycoprotein mediated cell-to-cell fusion.

<p>Two populations of cells were generated to determine the effect of SnO₂ treatment on cell fusion. Effector cells were transfected with plasmids gB, gD, gH, gL and T7. Target cells were transfected with gD, receptor Nectin-1 and a luciferase expressing plasmid under the control of a T7 promoter. Target and Effector cells were mixed together at a 1∶1 ratio. Luciferase activity was determined in the presence of firefly luciferase, allowing the measurement of relative light units (RLU). CHO-K1 cells were either mock treated or treated with SnO₂. As a negative control, effector cells lacking gB were mixed with the target cells.</p