Screening Of Anti Hepatitis B Virus ISGs And Antiviral Research Of SAMD4A

Hepatitis B is an infectious disease caused by hepatitis B virus.In 2015,more than 800,000 people died of hepatitis B and its complications worldwide.Although the morbidity and mortality from the disease is declining through the years due to the vaccination and use of antivirals,hepatitis B remains a serious health threat to human.At present,there are two kinds of drugs for treating chronic hepatitis B,nucleoside analogues and interferons.Nucleoside analogues target various important enzymes in the life cycle of hepatitis B virus,such as Entecavir and tenofovir which are recommended by the World Health Organization for treating chronic hepatitis B.Interferons are with broad-spectrum antiviral activity and inhibit hepatitis B virus by activating a number of signaling pathways and the expression of interferon-stimulating genes(ISG).However interferons are also with side effects such as fever and pain.Unlike nucleoside analogs though,inferons are not prone to induce drug resistance.The treatment protocol for interferons are well established with a higher rate of serum HBeAg seroconversion,which correlates with a successful treatment.We constructed a model for screening anti-hepatitis B virus ISG using HepG2-2B1 cells expressing NTCP,a hepatitis B virus-specific receptor.After confirming the titer of infection and validating the screening model with IFN-α,the screening led to the identification and validation of four ISGs,MKX,SAMD4A,ZAP,ZBP1.Among these,SAMD4A was chosen for further studyAfter confirming that the expression of SAMD4A was inducible by interferon,we further confirmed that the inhibition or knockout of SAMD4A would lead to the increase of HBV titer by the techniques of RNAi and RISPR/Cas9.It was shown that different isoforms of SAMD4A were with different antiviral abilities and the SAM domain was required for SAMD4A to inhibit the replication of hepatitis B virus.SAMD4A had no effect on the expression of NTCP,but could inhibit the replication of hepatitis B virus at the protein level,RNA level and DNA level.Moreover,we found that SAMD4A expression completely eliminated ccc-DNA in cells.In summary,a model for screening anti-hepatitis B virus ISG was constructed,and SAMD4A was identified and selected for in-depth study as an anti-hepatitis B virus factor.The elimination of cccDNA by SMAD4A in HBV infected cells implicates a novel therapeutic strategy for the treatment of of chronic hepatitis B.