PPM1F Inhibitor Prevents HSV-1/2 Infection By Inhibiting PPM1F-Mediated TBK1/IRF3 Dephosphorylation

BACKGROUND:Infection with herpes simplex virus,commonly known as herpes,can be due to either herpes simplex virus type 1(HSV-1)or herpes simplex virus type 2(HSV-2).HSV-1 is mainly transmitted through oral-oral and oral-genital contact.Most people are first infected in childhood and remain infected for every day.HSV-2 is spread almost exclusively through sex or genital contact during childbirth and HSV-2 can greatly increase the risk of HIV infection.Herpes simplex virus infection is mostly asymptomatic.The onset of herpes simplex virus often produces vesicular lesions in or around tissues,ranging from mild to severe,which can cause neuralgia and encephalitis.In acute symptoms,HSV has been studied as a contributing factor to cognitive decline,contributing to the development of Alzheimer,Parkinson,fibromyalgia,encephalitis,and other neurological disorders.Approximately 6 billion people worldwide carry HSV-1 virus(approximately 66%of the world of population carries the HSV-1 virus,13%carries the HSV-2 virus).According to the Herpes Cure Advocacy,approximately 40 to 80 million people suffer from recurrent painful genital lesions of HSV-2.Approximately 420,000 HIV infections occur each year as a result of genital herpes HSV-2.About 14,000 new baby infections a year.In the United States,about 1,000 babies die each year from neonatal herpes.10 million people suffer from herpetic eye disease.At present,HSV vaccine is still in the early stage of development,and it is possible to induce the activation of virus latent reservoir.Therefore,there is a huge and urgent need for antiviral therapy.At present,in addition to some supportive treatments,the clinical use of nucleoside anti-HSV drugs such as acyclovir and valaciclovir is mainly used to control symptoms and recurrence,but the adverse reactions of such drugs are relatively large,and cross-resistance is increasing year by year.According to the statistics of drug registration information in clinical trials at home and abroad,no new drug has been successfully launched in the field of anti-HSV in the past 20 years.Now,patients with HSV have been in a dilemma of single drugs for treatment,many adverse reactions and increasing viral resistance every time.METHODS:By analyzing and comparing the samples infected with various microorganisms in the GEO database,we found that the expression levels of PPM1F gene in different samples were different.The PPM1F gene was knocked out by CRISPR-CAS9 in Hela cells.At the same time,we explored the direct interaction between PPM1F and the protein through Co-IP test,to reveal the mechanism of PPM1F in antiviral immunity.We repeatedly verified the anti-HSV-1/2 effect of PPM1F Inhibitor by observing cytopathic effect,qRT-PCR,western blotting and plaque assay.The antiviral action pattern of PPM1F Inhibitor was explored by constructing different virus-drug incubation models.Since PPM1F Inhibitor and acyclovir have different antiviral mechanisms,we also tested whether there is a synergistic effect between PPM1F Inhibitor and acyclovir through combination drug experiment.To evaluate the antiviral effect of PPM1F Inhibitor in vivo,we constructed a herpes simplex virus type 1(HSV-1)infected mouse model in IFNAR KO C57B1/6 mice.RESULTS:Our study found that the mRNA expression level of PPM1F was significantly increased in Hela cells infected with HSV-1/2.Knockout of PPM1F by CRISPR-Cas9 can inhibit HSV-1/2 infection,while overexpression of PPM1F can promote viral infection.In addition,we found that PPM IF can directly interact with TBK1 and dephosphorylate TBK1.The PPM1F Inhibitor we found had a good anti-HSV-1/2 effect.It was found that PPM1F Inhibitor restored the phosphorylation level of TBK1 by inhibiting PPM1F,and then restored the production of IFN-βdownstream,playing a certain antiviral effect.At the same time,we found that PPM1F Inhibitor can also play an antiviral role by influencing the process of virus entry.PPM1F Inhibitor had a synergistic effect with acyclovir Inhibitor.PPM1F Inhibitor also has an anti-HSV-1 infection effect in vivo.CONCLUSIONS:Our study for the first time found that PPM1F as a novel target plays an important role in the anti-HSV-1/2 process.There is a direct interaction between PPM1F and TBK1,the PPM1F dephosphorylate TBK1/IRF3 pathway to inhibit antiviral response,in turn promoting viral infection.Meanwhile,we identified inhibitors of PPM1F and evaluated its antiviral effect in vitro and in vivo.We found that PPM1F Inhibitor has a strong antiviral effect in co-incubation with HSV-1/2 and confirmed that it exerts antiviral effect mainly by blocking the entry process of HSV-1/2.