NEDD4-binding protein 1 suppresses HBV replication by degrading pgRNA

Abstract

AbstractChronic infection with hepatitis B virus (HBV) places patients at increased risk for liver cirrhosis and hepatocellular carcinoma. Although nucleos(t)ide analogs are mainly used for the treatment of HBV, they require long-term administration and may lead to the emergence of drug resistant mutants. Therefore, to identify targets for the development of novel anti-HBV drugs, we screened for HBV-suppressive host factors using a plasmid expression library of RNA-binding proteins (RBPs). We screened 132 RBPs using an expression plasmid library by measuring HBV relaxed circular DNA (rcDNA) levels in hepatocellular carcinoma. After we identified one RBP which suppressed rcDNA, the domain-deficient mutants were generated to determine the region contributing to the anti-HBV effect. Also, we measured pregenomic RNA (pgRNA) and covalently closed circular DNA (cccDNA) level in the RBP transfected cells and confirmed the degradation of pgRNA by Northern blotting. Our screen identified NEDD4-binding protein 1 (N4BP1) having an anti-HBV effect. In hepatocellular carcinoma cell lines transfected or infected with HBV, overexpression of N4BP1 decreased rcDNA levels while knockdown or knockout of N4BP1 expression rescued rcDNA levels. We found that both the KH-like and RNase domains of N4BP1 were required for the protein’s anti-HBV effect. N4BP1 suppressed HBV replication by promoting pgRNA, PreS1 and PreS2/S degradation. In summary, we found that N4BP1 is a newly identified host factor able to counteract HBV production by reducing HBV RNA levels.Author summaryThere is still a large number of HBV-infected people in the world today because of no curative treatment for HBV infection. In this study, we focused on and screened RNA-binding proteins to identify new host factors which inhibit HBV replication. As a result, we found that NEDD4-binding protein 1 (N4BP1) expression suppresses rcDNA production by promoting the degradation of pregenomic RNA, 2.4kb and 2.1kb HBV RNA. Furthermore, KH-like domain or RNase domain of N4BP1 were involved in this anti-HBV effect. In addition, the N4BP1 levels were lower in HCC resection samples of exacerbated patients, suggesting that individual N4BP1 levels might be related to HCC progression. This novel factor can potentially become a key to new HBV treatments.