Rosmarinic Acid inhibits Pseudorabies Virus (PRV) infection by activating the cGAS-STING signaling pathway

Pseudorabies virus (PRV), a swine alphaherpesvirus, is a double-stranded DNA virus. It may infect various animals, especially pigs. PRV infection in pigs leads to high mortality rates, and causes huge economic lose for swine industry. Currently, there are few effective antiviral treatments available. Rosmarinic acid (RA), a hydrophilic phenolic compound, shows potential for inhibiting herpes simplex virus. Given that PRV is a member of the Herpesviridae family, this study investigated the antiviral effects of RA against PRV infection through both in vitro and in vivo, as well as the underlying molecular mechanisms. PK-15 cells were used to assess the cytotoxicity of RA in vitro, followed by an investigation of its anti-PRV activity. The study then explored how RA regulates the cGAS-STING signaling pathway, along with inflammatory and apoptotic factors in PRV-infected cells. Molecular docking and dynamics simulations further elucidated the binding interactions between RA and cGAS-STING, providing insight into how RA activates the cGAS-STING pathway against PRV infection. In vivo, the antiviral efficacy of RA was evaluated in a PRV-infected mouse model by assessing tissue viral genome copies, the innate immune cGAS-STING signaling pathway activation, and inflammatory and apoptotic responses. The results showed that RA exhibited a half-maximal cytotoxic concentration (CC50) of 26.23 mu g/mL on PK-15 cells and a half-maximal inhibitory concentration (IC50) of 0.84 mu g/mL against PRV, resulting in a selectivity index (SI) of 31.22. These findings suggest that RA is a highly effective and low-toxicity compound. RA significantly inhibited PRV adsorption, penetration, and replication within cells. Additionally, while PRV infection suppresses the cGAS-STING signaling pathway, RA treatment activates the innate immune response, enhances downstream antiviral effector IFN-beta expression, and reduces inflammation and apoptosis in PRV-infected cells. Molecular docking results showed that the docking scores of cGAS_RA and STING_RA complexes were both less than - 5 kcal/mol, suggesting that RA binds well to cGAS and STING proteins. Molecular dynamics simulations, including RMSD, RMSF, and MM-GBSA analyses, confirmed the high binding stability of cGAS with RA, further validating the potential activity of RA as a cGAS agonist. In vivo studies revealed that RA dramatically lowered viral genome copies in various organs, activated the cGAS-STING signaling pathway, inhibited PRV-induced inflammation and apoptosis, alleviated clinical symptoms, and decreased mortality rate in PRV-infected mice. Overall, RA significantly inhibited PRV proliferation in vitro and in vivo, effectively reduced inflammation and apoptosis, and decreased the mortality rate in infected mice. The study supports the development of RA as an antiviral drug and emphasizes its potential as a candidate for PRV therapy.