Exosome Delivery Of MiR-101 Inhibits The Replication Of Herpes Simplex Virus Type 2 And Varicella-zoster Virus

Both herpes simplex virus and varicella-zoster virus belong to the ? herpesvirus subfamily and can cause human disease.Herpes simplex virus type 2(HSV-2)mainly causes serious genital diseases,and is related to physiological diseases(such as genital ulcers and inflammation)and psychosocial problems.The currently available antiviral drugs have some limitations,and no available vaccines have been developed.VZV is the main pathogen that causes varicella zoster.The VZV virus can be latent in the nerves,and it can relapse when the host's immunity is low,and it will cause postherpetic neuralgia.For the treatment after infection with VZV virus,there is an urgent need to develop new treatment methods and find new antiviral mechanisms.MiRNA is a small non-coding RNA molecule that can participate in many cellular processes by targeting mRNA to regulate gene expression.Virus-infected host cells can regulate cellular miRNA expression and inhibit viral replication by directly binding to the viral genome or by regulating innate immune pathways and cytokine responses.For example,miR-101 can inhibit HSV-1 replication by targeting ATP5 B.MiRNA has great potential in the application of anti-viral infection,but due to the lack of an effective delivery system,the application of miRNA is hindered.Exosomes are lipid bilayer-coated nanovesicles with a diameter of about 30-150 nm.Exosomes contain proteins,lipids and nucleic acids,which can be transferred from the producing cells to the recipient cells to promote communication between cells.Recent studies have shown that exosomes are natural carriers of miRNA and can serve as gene delivery systems.Exosome gene delivery has the following advantages: good tolerance and low cytotoxicity.The purpose of this study was to investigate the inhibitory effect of exosome delivery of miR-101 into cells on the replication of HSV-2 viruses.In this paper,the effects of up-regulation or down-regulation of mir-101 expression on hsv-2 replication were detected by transfection of miR-101 mimics and inhibitors in cells.The results showed that miR-101 had inhibitory effects on HSV-2 by RT-PCR,Western Blotting and cellular immunofluorescence.Then Western Blotting and transmission electron microscopy(TEM)were used to identify exosomes purified by differential centrifugation,and the results showed that exosomes secreted by Hela cells were successfully prepared.RT-PCR was used to detect miR-101 in exosomes with different loading methods,and the modified calcium chloride was determined to be exosomeloaded with miR-101.Finally,RT-PCR,Western Blotting and cellular immunofluorescence were used to demonstrate that the exosome delivery of miR-101 mimics decreased replication of HSV-2 in cells.The same experimental method was then used to detect the effect of miR-101 on the replication of VZV in cells.The inhibition of miR-101 against VZV virus was demonstrated.Exosomes secreted by MRC5 cells were characterized by Western Blotting and TEM.Finally,RT-PCR,Western Blotting and cellular immunofluorescence were used to reduce the replication of VZV in the experimental group of miR-101 mimics delivered by exosomes.In summary,this paper proves that miR-101 has inhibitory effect on HSV-2 and VZV viruses.The exosome delivery system was successfully loaded with mir-101 mimics to inhibit the replication of HSV-2 and VZV viruses.This provides the technical method and experimental basis for the later antiviral delivery of miRNA by exosomes.