A Particle Gel Assay for Detection of Intracellular Hepatitis B Virus Subviral Particles in Cultured Cells.

Chronic hepatitis B virus (HBV) infection is due to the failure of host immune system to resolve the viral infection. Accordingly, restoration or reconstitution of a functional antiviral immune response to HBV is essential to achieve durable control of HBV replication leading to a functional cure of chronic hepatitis B (CHB). Noninfectious subviral particles (SVPs), comprised of HBV surface antigen (HBsAg), are the predominant viral products secreted by HBV-infected hepatocytes. The high levels of SVPs in the circulation induce immune tolerance and contribute to the establishment of chronic HBV infection. The current standard-of-care medications for CHB efficiently suppress HBV replication but fail to reduce the levels of HBsAg in majority of treated patients. Further understanding the mechanisms underlying SVP morphogenesis, secretion and regulation by viral and host cellular factors are critical for the discovery of therapeutics that can inhibit SVP production and/or induce the degradation of HBV envelope proteins. We describe herein a protocol for intracellular SVP detection by a native agarose gel electrophoresis-based particle gel assy. The method is suitable for quantitative detection of intracellular HBV SVPs and can be applied in dissecting the molecular mechanism of SVP morphogenesis and the discovery of antiviral agents targeting SVP formation in hepatocytes.

Mechanism of interferon alpha therapy for chronic hepatitis B and potential approaches to improve its therapeutic efficacy.

Hepatitis B virus (HBV) chronically infects 296 million people worldwide and causes more than 820,000 deaths annually due to cirrhosis and hepatocellular carcinoma. Current standard-of-care medications for chronic hepatitis B (CHB) include nucleos(t)ide analogue (NA) viral DNA polymerase inhibitors and pegylated interferon alpha (PEG-IFN-α). NAs can efficiently suppress viral replication and improve liver pathology, but not eliminate or inactivate HBV covalently closed circular DNA (cccDNA). CCC DNA is the most stable HBV replication intermediate that exists as a minichromosome in the nucleus of infected hepatocyte to transcribe viral RNA and support viral protein translation and genome replication. Consequentially, a finite duration of NA therapy rarely achieves a sustained off-treatment suppression of viral replication and life-long NA treatment is most likely required. On the contrary, PEG-IFN-α has the benefit of finite treatment duration and achieves HBsAg seroclearance, the indication of durable immune control of HBV replication and functional cure of CHB, in approximately 5% of treated patients. However, the low antiviral efficacy and poor tolerability limit its use. Understanding how IFN-α suppresses HBV replication and regulates antiviral immune responses will help rational optimization of IFN therapy and development of novel immune modulators to improve the rate of functional cure. This review article highlights mechanistic insight on IFN control of HBV infection and recent progress in development of novel IFN regimens, small molecule IFN mimetics and combination therapy of PEG-IFN-α with new direct-acting antivirals and therapeutic vaccines to facilitate the functional cure of CHB.

Discovery of hepatitis B virus subviral particle biogenesis inhibitors from a bioactive compound library.

High levels of hepatitis B virus (HBV) surface antigen (HBsAg) in the blood of chronic HBV carriers are considered to drive the exhaustion of antigen-specific T and B lymphocytes and thus responsible for the persistence of infection. Accordingly, therapeutic elimination of HBsAg may facilitate the activation of adaptive antiviral immune responses against HBV and achieve a functional cure of chronic hepatitis B. We discovered recently that an amphipathic alpha helix spanning W156 to R169 of HBV small envelope (S) protein plays an essential role in the morphogenesis of subviral particles (SVPs) and metabolism of S protein. We thus hypothesized that pharmacological disruption of SVP morphogenesis may induce intracellular degradation of S protein and reduce HBsAg secretion. To identify inhibitors of SVP biogenesis, we screened 4417 bioactive compounds with a HepG2-derived cell line expressing HBV S protein and efficiently secreting small spherical SVPs. The screen identified 24 compounds that reduced intracellular SVPs and secreted HBsAg in a concentration-dependent manner. However, 18 of those compounds inhibited the secretion of HBsAg and HBeAg in HBV replicon transfected HepG2 cells at similar efficiency, suggesting each of those compounds may disrupt a common cellular function required for the synthesis and/or secretion of these viral proteins. Interestingly, lycorine more efficiently inhibited the secretion of HBsAg in HepG2 cells transfected with HBV replicons, HepG2.2.15 cells and HBV infected - HepG2 cells expressing sodium taurocholate cotransporting polypeptide (NTCP). The structure activity relationship and antiviral mechanism of lycorine against HBV have been determined.