ABSTRACT
Background/Aims@#The major histocompatibility class II (MHC II) transactivator, known as CIITA, is induced by Interferon gamma (IFN-γ) and plays a well-established role in regulating the expression of class II MHC molecules in antigen-presenting cells. @*Methods@#Primary human hepatocytes (PHH) were isolated via therapeutic hepatectomy from two donors. The hepatocellular carcinoma (HCC) cell lines HepG2 and Huh7 were used for the mechanistic study, and HBV infection was performed in HepG2-NTCP cells. HBV DNA replication intermediates and secreted antigen levels were measured using Southern blotting and ELISA, respectively. @*Results@#We identified a non-canonical function of CIITA in the inhibition of hepatitis B virus (HBV) replication in both HCC cells and patient-derived PHH. Notably, in vivo experiments demonstrated that HBV DNA and secreted antigen levels were significantly decreased in mice injected with the CIITA construct. Mechanistically, CIITA inhibited HBV transcription and replication by suppressing the activity of HBV-specific enhancers/promoters. Indeed, CIITA exerts antiviral activity in hepatocytes through ERK1/2-mediated down-regulation of the expression of hepatocyte nuclear factor 1α (HNF1α) and HNF4α, which are essential factors for virus replication. In addition, silencing of CIITA significantly abolished the IFN-γ-mediated anti-HBV activity, suggesting that CIITA mediates the anti-HBV activity of IFN-γ to some extent. HBV X protein (HBx) counteracts the antiviral activity of CIITA via direct binding and impairing its function. @*Conclusions@#Our findings reveal a novel antiviral mechanism of CIITA that involves the modulation of the ERK pathway to restrict HBV transcription. Additionally, our results suggest the possibility of a new immune avoidance mechanism involving HBx.
ABSTRACT
BACKGROUND/AIMS: Direct sequencing is the gold standard for the detection of drug-resistance mutations in hepatitis B virus (HBV); however, this procedure is time-consuming, labor-intensive, and difficult to adapt to high-throughput screening. In this study, we aimed to develop a dendron-modified DNA microarray for the detection of genotypic resistance mutations and evaluate its efficiency. METHODS: The specificity, sensitivity, and selectivity of dendron-modified slides for the detection of representative drug-resistance mutations were evaluated and compared to those of conventional slides. The diagnostic accuracy was validated using sera obtained from 13 patients who developed viral breakthrough during lamivudine, adefovir, or entecavir therapy and compared with the accuracy of restriction fragment mass polymorphism and direct sequencing data. RESULTS: The dendron-modified slides significantly outperformed the conventional microarray slides and were able to detect HBV DNA at a very low level (1 copy/μL). Notably, HBV mutants could be detected in the chronic hepatitis B patient sera without virus purification. The validation of our data revealed that this technique is fully compatible with sequencing data of drug-resistant HBV. CONCLUSIONS: We developed a novel diagnostic technique for the simultaneous detection of several drug-resistance mutations using a dendron-modified DNA microarray. This technique can be directly applied to sera from chronic hepatitis B patients who show resistance to several nucleos(t)ide analogues.