Influence of hepatitis B virus surface protein variants associated with antiviral resistance on viral assembly and secretion of hepatitis B and hepatitis D viruses

摘要

The viral hepatitis is one of the five most important infectious diseases worldwide and is caused by the hepatitis B virus (HBV). The development of a chronic HBV infection raises the risk to generate liver fibrosis, cirrhosis, and hepatocellular carcinoma. Two different therapy-concepts to treat a chronic HBV infection are used: the treatment with IFN-α and the therapy with nucleos(t)ide analogs. Due to the high variability of the HBV genome and the missing proofreading function of the viral polymerase, several drug-resistant mutants arise during the long term treatment with nucleos(t)ide analogs in chronically infected patients. Because of the strong overlapping open reading structure of the HBV genome, mutations in the viral polymerase also affect the genes of the other viral proteins especially the gene for the HBV surface proteins.In the present work, a series of HBV surface protein variants were generated that can arise during the treatment with nucleos(t)ide analogs, bearing a stop mutation in the internal loop of the small hepatitis B surface antigen (SHBs) (sW172*, L176*, W182*, sW199*, W196*, L216*). These HBV variants show a strongly reduced secretion of HBV virions. The stop mutations in the SHBs of HBV probably lead to the loss of the internal loop between transmembrane domain III and IV resulting in a truncated SHBs variant. Surprisingly, a stop mutation at position 172 in the SHBs did not fully inhibit the secretion of HBV particles. Mutations in the HBV surface proteins can also have strong consequences for the satellite virus of HBV: the hepatitis delta virus (HDV). This virus is dependent on the surface proteins of its helper virus HBV for virion assembly, secretion, and infectivity. The antiviral treatment with nucleos(t)ide analogs may have consequences for the clinical prognosis and treatment of a patient chronically infected with HBV as well as for HBV/HDV co-infected patients. For analyzing the implications of HBV surface protein mutations on the viral life cycle of HDV in the future, an in vitro transfection system to generate recombinant HDV pseudo-particles in the hepatoma cell line Huh7 was established. With the help of these recombinant HDV-like particles, an infection system on primary hepatocytes, derived from the asian treeshrew Tupaia belangeri was then established. In this context, a full genomic HDV RNA standard was generated in vitro to be able to quantify the newly synthesized HDV RNA absolutely. The production of HDV pseudo-particles in Huh7 cells could show that the SHBs is crucial for the viral assembly and secretion, whereas the large hepatitis B virus surface antigen (LHBs) is required for a successful infection of primary Tupaia hepatocytes. Using these methods, HDV pseudo-particles were generated, covered with HBV surface protein variants possessing characteristic mutations in the SHBs (sW172*, sW172L, sW196*, sW196S, sW196L) that can arise during antiviral therapy. The present study could show that a stop mutation at position 172 and 196 as well as an amino acid change from tryptophan to serine or leucine at position 196 in the SHBs blocks the viral assembly and secretion of HDV pseudo-particles.