Hypericin blocks the function of HSV-1 alkaline nuclease and suppresses viral replication

Ethnopharmacological relevance: Hypericum perforatum L. has a long history in many countries of being used as a herbal medicine. It is also widely used in Chinese herbal medicine for the treatment of infections. Hypericin, a main component extracted from Hypericum perforatum L., has attracted the attention of many researchers for its remarkable antiviral, antitumor and antidepressant effects.

Aim of the study: To find plant molecules that inhibit the alkaline nuclease (AN) of herpes simplex virus type 1 (HSV-1) and suppress viral replication.

Materials and methods: Bioinformatics methods were used to determine which compounds from a variety of natural compounds in our laboratory interact with AN. By this means we predicted that hypericin may interact with AN and suppress HSV-1 replication. Experiments were then carried out to verify whether hypericin inhibits the bioactivity of AN. The Pichia pastoris expression system was used to obtain recombinant AN. The exonuclease and endonuclease activity of AN treated with hypericin were tested by electrophoresis. Immunohistochemical staining of the HSV-1 nucleocapsids was used to find out whether hypericin inhibits the intracellular function of AN. Real-time PCR and western blotting analysis were performed to test viral gene expression and viral protein synthesis. The extent of viral replication inhibited by hypericin was determined by a plaque assay and a time of addition assay.

Results: Recombinant AN was obtained by Pichia pastoris expression system. The exonuclease and endonuclease activity of recombinant AN were inhibited by hypericin in the electrophoresis assay. Hypericin showed no inhibitory effect on BeyoZonase™ Super Nuclease or DNase I. T5 Exonuclease activity was inhibited partially by10 μM hypericin, and was completely suppressed by 50 μM hypericin. Hind Ⅲ was inhibited by hypericin at concentrations greater than 100 μM, but EcoR I, BamH I, and Sal I were not inhibited by hypericin. HSV-1 nucleocapsids gathered in the nucleus when the viruses were treated with hypericin. Plaque formation was significantly reduced by hypericin (EC₅₀ against HSV-1 F is 2.59 ± 0.08 μM and EC₅₀ against HSV-1 SM44 is 2.94 ± 0.10 μM). UL12, ICP27, ICP8, gD, and UL53 gene expression (P < 0.01, 4.0 μM hypericin treated group vs control group) and ICP4 (P < 0.05, 6.0 μM hypericin treated group vs control group), ICP8 and gD (P < 0.05, 2.0 μM hypericin treated group vs control group) protein synthesis were inhibited by hypericin. In the time of addition assay, HSV-1 was suppressed by hypericin in the early stages of viral replication. Hypericin exhibits potent virucidal activity against HSV-1 and inhibits the adsorption and penetration of HSV-1.

Conclusion: Hypericin inhibits the bioactivity of AN and suppresses HSV-1 replication. The data revealed a novel mechanism of the antiherpetic effect of hypericin.