Antiviral Effects And Mechanisms Of ABMA And DABMA Against Influenza Virus And Varicella-zoster Virus

Influenza is an acute upper respiratory disease caused by influenza viruses,characterized by high contagious and spreads rapidly.Influenza A virus(IAV),especially H1N1 and H3N2 subtypes,are associated with seasonal influenza,causing millions of cases worldwide each year.Small molecule drugs are the effective approach for treating influenza,including M2 ion channel blockers,neuraminidase(NA)inhibitors,and RNA polymerase inhibitors.However,the emergence of drug-resistant virus strains and side effects of drugs limited the development of these antivirals.Varicella-zoster virus(VZV)is a highly infectious neurotropic virus.The primary infection causes varicella.VZV is latent in the dorsal root ganglia and cranial root ganglia.Reactivation of the virus causes herpes zoster,which leads to postherpetic neuralgia(PHN)and severe neurological complications.In the United States,roughly 1 million cases of shingles are diagnosed each year,with economic costs of$1.1 billion.Antivirals of VZV can significantly reduce the morbidity and mortality of varicella and herpes zoster,especially in immunodeficient populations.Nucleoside analogs are the effective antivirals for treating VZV infection including acyclovir,famciclovir,and valacyclovir.However,low bioavailability and drug resistance limited the use of these antivirals in the clinic.ABMA was identified by a cell-based high-throughput screening against ricin toxin.It demonstrated that ABMA and its optimized analog DABMA can protect cells against multiple toxins,including Leishmania,ricin,diphtheria toxin,lethal toxin from Bacillus anthracis(LT),Clostridium difficile toxin B(Tcd B),Clostridium sordellii lethal toxin(Tcs L),Pseudomonas Exotoxin A(PE),Rabies virus,dengue virus,herpes simplex virus type 2 and Ebola virus.The mechanism of action involves the delay of virus and toxin trafficking through late endosomes by inhibiting the fusion of late endosomes with lysosomes and reducing the autophagic flux.Autophagy is a physiological process unique to eukaryotic cells that involve lysosomes to degrade intracellular components to maintain the structural stability and function of cells.Autophagy is involved in IAV and VZV infections.Influenza virus M2 hijacks autophagic vesicles by interacting with LC3;autophagy plays a role in the early stages of the H5N1 life cycle;IAV can increase autophagic flux through M2binding to Beclin1 and upregulating the expression of autophagy-related genes;VZV can hijack the LC3 envelope as their second envelope;VZV infection of Hela cells causes autophagosome aggregation;lipidated LC3 can also be found in purified VZV particles,while VZV g E partially co-localizes with LC3 in the cytoplasm.This study consists of two main parts.Part I:Antiviral effects and mechanisms of ABMA and DABMA against influenza viruses.(Chapter 2,3)We first evaluated the toxicity of ABMA and DABMA in MDCK and A549 cells.Results showed that the CC₅₀s of ABMA and DABMA were 72.30?M and 42.71?M in MDCK cells and were 83.77?M and 47.42?M in A549 cells,respectively.We then explored the anti-influenza virus activities of the drugs.ABMA and DABMA showed antiviral activities against multiple strains of influenza viruses with the EC₅₀s ranging from 2.83?7.36?M(ABMA)and 1.82?6.73?M(DABMA).Amantadine as the positive control drug showed antiviral activities against amantadine-sensitive influenza viruses(S31),but not against amantadine-resistant influenza viruses(S31N)and influenza B virus.Meanwhile,ABMA and DABMA inhibited H1N1 genomic RNA replication and protein synthesis.Moreover,we evaluated the protective effect of ABMA and DABMA in vivo.The body weight of mice in the administered group recovered significantly compared to the placebo group.ABMA and DABMA(5mg/kg)increased the survival rate of mice by 67%.In addition,lung viral load and titer,lung index,and the lesions induced by IAV were significantly reduced in the drug-treated groups compared to the placebo group.To investigate the role of ABMA and DABMA in the H1N1 life cycle,time of drug addition experiments were performed.Results showed that ABMA and DABMA impair H1N1 infection at the early stages of the virus life cycle.Furthermore,molecular docking results showed that the relative binding affinities(-CDocker energy)of ABMA and DABMA with the influenza M2 channel(PDB:6BKK)were much lower than that of amantadine,suggesting that ABMA and DABMA do not act as M2 ion channel blockers.Moreover,labeling of acidic vesicles by lysotracker and endosomal marker proteins revealed that ABMA caused the accumulation of late endosomes in A549 and MDCK cells.In addition,co-localization of influenza virus NP proteins and late endosomes revealed that virions accumulated in the late endosomes.Autophagy converges with the endolysosomal pathway through the fusion of autophagosomes with late endosomes to give amphisomes that fuse with lysosomes to form the autolysosomes.In the autolysosomes,the sequestered proteins and dysfunctional cellular components are digested by lysosomal hydrolases.A549 cells treated with ABMA significantly increased the expression of LC3-II and p62(two marker proteins of autophagy)in a dose-and time-dependent manner,suggesting a defective autophagic flux.Localization of p62 and Rab7,marker proteins of autophagosomes and late endosomes in A549 cells,revealed that ABMA caused the accumulation of amphisomes.In addition,3-MA and Baf A1 inhibited the replication of the influenza virus,demonstrating that autophagy is essential for H1N1 infection.Part II:Antiviral effect and mechanism of ABMA and DABMA against varicella-zoster virus(Chapter 4)We first verified the toxicities of ABMA and DABMA on VZV-susceptible MRC-5 cells and Me Wo cells.The CC₅₀s of ABMA and DABMA were 63.36?M and 43.37?M for MRC-5 cells;55.95?M and 41.80?M for Me Wo cells.ABMA and DABMA were found to inhibit the VZV-induced MRC-5(EC₅₀=1.89?M and 1.05?M,SI=33.52 and 41.30)and Me Wo(EC₅₀=1.47?M and 1.34?M,SI=38.06 and31.19)CPE with a dose-dependent effect.In addition,ABMA and DABMA effectively inhibited VZV DNA replication and g E protein synthesisand and the titers of progeny virus.Time of drug addition assay revealed that ABMA mainly inhibited the entry and release stage of VZV lifecycle.ABMA increased the expression levels of LC3-II and p62 with dose-and time-dependent effects,suggesting that ABMA inhibited the late phase of autophagy in Me Wo cells.Further localized p62 and Rab7 revealed that ABMA caused the accumulation of amphisomes in Me Wo cells.Moreover,autophagy inhibitors 3-MA,Baf A1,and chloroquine inhibited VZV replication.In contrast,the rapamycin(RAPA,autophagy activator)increased the relative expression of VZV DNA.This demonstrated that autophagy plays an important role during VZV infection in Me Wo cells.In addition,co-localized VZV g E protein and LC3 indicated that VZV can hijack autophagic vesicle.In conclusion,this study demonstrated the antiviral activities of ABMA and DABMA against influenza virus and VZV in vitro and H1N1 in vivo and elucidated the mechanism of action of ABMA.These results expand the antiviral landscape of ABMA and DABMA,providing lead compounds for the development of antivirals against influenza virus and herpesvirus.In addition,ABMA can also use as probes for regulating intracellular vesicle transport.