Prophylactic, therapeutic and neutralizing effects of zinc oxide tetrapod structures against herpes simplex virus type-2 infection

Abstract

The attachment of Herpes simplex virus type-2 (HSV-2) to a target cell requires ionic interactions between negatively charged cell surface co-receptor heparan sulfate (HS) and positively charged residues on viral envelop glycoproteins, gB and gC. Effective blocking of this first step of HSV-2 pathogenesis demonstrates significant prophylactic effects against the viral disease; any in vitro therapeutic effects of blocking this interaction, however, are not clear. Here, we provide new evidence that zinc oxide tetrapod micro-nanostructures synthesized by flame transport approach significantly block HSV-2 entry into target cells and, in addition, demonstrate the potential to stop the spread of the virus among already infected cells. The zinc oxide tetrapods (ZnOTs) also exhibit the ability to neutralize HSV-2 virions. Natural target cells such as human vaginal epithelial and HeLa cells showed highly reduced infectivity when infected with HSV-2 virions that were pre-incubated with the ZnOTs. The mechanism behind the ability of ZnOTs to prevent, neutralize or reduce HSV-2 infection relies on their ability to bind the HSV-2 virions. We used fluorescently labeled ZnOTs and GFP-expressing HSV-2 virions to demonstrate the binding of the ZnOTs with HSV-2. We also show that the binding and hence, the antiviral effects of ZnOTs can be enhanced by illuminating the ZnOTs with UV light. Our results provide new insights into the anti-HSV-2 effects of ZnOT and rationalize their development as a HSV-2 trapping agent for the prevention and/or treatment of infection. The observed results also demonstrate that blocking HSV-2 attachment can have prophylactic as well as therapeutic applications.

Figure 1. ZnOT Cytotoxicity

The viability of HeLa cells after ZnOT treatment was determined by MTS cytotoxicity assay at 24 hours post ZnO treatment. HeLa cells were seeded at a density of 2×10⁴ in a 96-well plate. UV ZnOT and NUV ZnOT were brought into suspension in PBS at concentrations of 1.5, 1, 0.8, 0.6, 0.4, 0.2, and 0.1 mg/mL. Cell viability was analyzed by a chromogenic kit (CellTiter Aqueous96; Promega, Madison, WI, USA). Colorimetric detection was measured by micro-pate reader (TECAN GENious Pro) at 492 nm. Results are representative of three independent experiments.

Figure 2. Zinc oxide nano-micro scale tetrapod structures synthesized by flame transport approach

(A) Glass bottle shows the large amount of ZnO tetrapod structures which were synthesized in just one run. (B–E) show the scanning electron microscopy images of different type of tetrapod structures from the ZnO powder shown in (A).

FIGURE 3. Neutralization, prophylaxis, or therapeutic treatment with ZnOT inhibits HSV-2 entry

A β-galactosidase-expressing reporter virus, HSV-2(333)gJ-was used for entry measurements. Viral entry was determined after each of the treatments indicated. UV treated (UV) or non-UV treated (NUV) ZnOTs were tested. (A) Neutralization. ZnOT was allowed to bind the virus and the virus/ZnOT mixture was then used to infect cells. (B) Prophylaxis treatment. Cells were pretreated with ZnOTs and then infected with HSV-2(333)gJ- virus. (C) Therapeutic treatment. Cells were first infected with HSV-2(333)gJ- and then treated with the ZnOTs.

FIGURE 4. Neutralization, prophylaxis or therapeutic treatment result in decreased internalization of HSV-2(333)

Western blot analysis of VP16 expression was performed to determine the effect of ZnOT on HSV-2 internalization. As indicated, VP16 protein expression was determined for neutralization, prophylaxis, therapeutic or mock treated cells infected with wild-type HSV-2(333). The cell lysates were prepared at 2 hours post infection and Western blots were performed. VP 16 expression and relative protein intensity are shown. (A) Neutralization (B) prophylactic and (C) therapeutic treatment. GAPDH was measured as a loading control. Results are representative of three independent experiments.

FIGURE 5. ZnOT Treatment inhibits virus free cell-to-cell fusion

A. The diagram shows the virus free cell-to-cell fusion assay used. Two populations of cells representing effector cells and target cells were generated. Effector cells express HSV-2 glycoproteins and T7 polymerase and the target cells express gD receptor nectin-1 and the firefly luciferase gene under T7 promoter. The luciferase activity can be detected when the cells fuse. (B) CHO-K1 effector cells were pretreated with UV and NUV ZnOT and mixed with target cells for 24 hours. As a negative control effector cells lacking gB were mixed with target cells. Results are representative of three independent experiments.

FIGURE 6. ZnOT reduces infectious cell cluster formation

HSV-2(333)GFP virus was used to determine the effect of ZnOT on viral replication and cell-to-cell spread. (A) Cellular expression of GFP and its distribution mark the ability of HSV-2 to form clusters of infected cells under various treatment conditions as indicated. Reduced clusters were noted in all conditions where ZnOT was present. (B). Relative sizes of infected cell cultures were determined by measurement of clusters under a fluorescent microscope. Images are representative of 3 independent experiments.

FIGURE 6. ZnOT reduces infectious cell cluster formation

HSV-2(333)GFP virus was used to determine the effect of ZnOT on viral replication and cell-to-cell spread. (A) Cellular expression of GFP and its distribution mark the ability of HSV-2 to form clusters of infected cells under various treatment conditions as indicated. Reduced clusters were noted in all conditions where ZnOT was present. (B). Relative sizes of infected cell cultures were determined by measurement of clusters under a fluorescent microscope. Images are representative of 3 independent experiments.

FIGURE 7. ZnOT reduces plaque formation

The effect of ZnOT treatment on viral spread and replication was determined by syncytial plaque assay. (A) Formation of plaques by HSV-2(333) infected cells under various treatment conditions as indicated. (B) Relative plaque sizes are shown. The data represents observations from three independent experiments.

FIGURE 7. ZnOT reduces plaque formation

The effect of ZnOT treatment on viral spread and replication was determined by syncytial plaque assay. (A) Formation of plaques by HSV-2(333) infected cells under various treatment conditions as indicated. (B) Relative plaque sizes are shown. The data represents observations from three independent experiments.

FIGURE 8. An illustration of red-blue syncytia assay used to understand the effect of ZnOT treatment

Effector cells expressing HSV-2 glycoproteins required for entry and NES-RFP signal were mixed with target cells expressing gD receptor nectin-1 and CFP-NLS signal. Effectors were pretreated with ZnOT prior to the mixing of target and effector cells. (B) Using a confocal microscope syncytia formation was recorded 24hours post mixing.

FIGURE 9. Fluorescently labeled ZnOT binds HSV-2(333)GFP virus

The direct interactions between ZnOT and HSV-2 virus were visualized through the incubation of fluorescently tagged ZnOT with GFP tagged HSV-2. Both the UV and NUV treated ZnOT show significant trapping ability. Images are representative of 2 independent experiments.

FIGURE 10. Model of ZnOT anti-HSV-2 action

A cartoon illustrates the infection process in the presence and the absence of ZnOT. (A)Non-UV treated ZnOT can trap the virus owing to the partial negative charge generated during synthesis. This partial negative charge attracts virus thus contributing to decreased entry and replication. (B) In the absence of ZnOT the virus is able to bind and interact with heparan sulfate and nectin-1 to mediate infection. (C) UV light treatment induces additional oxygen vacancies making ZnOT more partially negative.