Inhibition by Woodchuck Hepatitis Virus of Class I Major Histocompatibility Complex Presentation on Hepatocytes Is Mediated by Virus Envelope Pre-S2 Protein and Can Be Reversed by Treatment with Gamma Interferon

Hepatitis B virus (HBV) is a member of the hepadnavirus family, which includes, among several mammalian and avian viruses, woodchuck hepatitis virus (WHV), which naturally infects eastern North American woodchucks (Marmota monax). Woodchucks infected with WHV represent a highly valuable natural model of hepatitis B in which molecular, immunological, and histological sequelae of virus-induced liver inflammation closely resemble those encountered in humans, including development of chronic hepatitis (20, 32). It is now also evident that HBV and WHV invade and replicate not only in hepatic tissue but also in the lymphatic system, although under most circumstances, less vigorously than in the liver (24-26). The host cellular immune responses directed against hepadnavirus protein epitopes displayed on infected hepatocytes, particularly those mediated by cytotoxic T lymphocytes (CTL), are regarded to be crucial in the recovery from acute hepatitis as well as in the induction of hepatocellular damage in actively progressing infection (3). In contrast to acute hepatitis, which is characterized by a strong and specific CTL response directed towards multiple virus epitopes, chronic hepatitis B is characterized by a weak and narrowly focused CTL antiviral reactivity. It is assumed that this hindrance is one of the main factors underlying the development of chronic hepadnaviral infection and protracted liver damage. Because the triggering and strength of antiviral CTL responsiveness depend to a significant degree on the efficient presentation of viral peptides by class I major histocompatibility complex (MHC) molecules on the surfaces of infected cells, we have previously investigated characteristics of the class I MHC antigen display in woodchucks acutely and chronically infected with WHV (22). Among others, we have uncovered that chronic WHV hepatitis but not acute WHV hepatitis is associated with a profound suppression in class I antigen on the surface of infected hepatocytes. Thus, while acute hepatitis was accompanied by enhanced hepatocyte presentation of the class I antigen, inhibition of the antigen was consistently detected on these cells with chronic hepatitis. This occurred despite the fact that levels of intrahepatic expression of class I MHC-affiliated genes encoding woodchuck class I heavy and β2-microglobulin (β2m) chains and transporters associated with antigen processing (TAP1 and TAP2), as well as gamma interferon (IFN-γ), which is one of the most powerful stimulators of the class I antigen expression (1), were found to be augmented to the same degree in both acute and chronic hepatitis. Furthermore, the hepatocyte class I antigen suppression was not linked to the histological severity of hepatocellular injury, the extent of lymphocytic infiltrations or the hepatic load of WHV. It was concluded that the deficiency in hepatocyte class I antigen display is a uniform hallmark of chronic WHV hepatitis and is caused by a virus-dependent posttranscriptional interference. However, the basis of this interference, particularly which of the WHV genome translation products might be responsible for this impediment, was not investigated. The presentation of peptides by the class I MHC molecules on the cell surface accessible for CTL recognition is a multistep process involving the generation of peptides, their transport, loading onto class I heavy chain-β2m complexes in the endoplasmic reticulum (ER), and trafficking the complexes to the cell surface via the Golgi apparatus (30). The generation of short peptides in the cytosol is achieved by the ubiquitin-proteasome system. Briefly, the endogenously synthesized proteins are first conjugated with polyubiquitin by ubiquitin ligase (16). Then, the ubiquitin-tagged proteins are unfolded by the 19S cap of the proteasome, which is composed of proteasome activator 28 alpha (PA28α) and proteasome activator 28 beta (PA28β) and subsequently degraded into short peptides within the proteasome 20S core (18). The peptidase activity in the 20S proteasome resides in constitutive β subunits, termed X, Y, and Z, which can be readily substituted by IFN-γ-inducible β subunits, such as multicatalytic endopeptidase complex-like 1 (MECL-1), low-molecular-mass protein 2 (LMP2), and low-molecular-mass protein 7 (LMP7), which display catalytic activity. The generated peptides are either further degraded into amino acids (aa) for recycling by cytosolic peptidase or transported to the ER by TAP1/TAP2 heterodimer complexes (28). An interaction between the TAP molecules and class I MHC-β2m is required for the assembly of class I MHC-peptide complexes (29). Upon loading a peptide, the class I MHC complex is exported from the ER to the Golgi apparatus and finally transported to the cell surface, where it can be identified by the peptide-specific CTL. In the present study, to investigate the nature of the molecular hindrance responsible for hepadnavirus-induced inhibition of the class I MHC antigen on hepatocytes observed in chronic WHV (22), we established a panel of woodchuck hepatocyte transfectants transiently or stably expressing the whole genome of WHV, subgenomic fragments encoding individual virus proteins, or complete WHV sequences in which expression of individual translation products was disabled by premature stop codons introduced through site-specific mutagenesis. The display of class I MHC antigen on these hepatocytes was measured and compared to the control naive hepatocytes and those transfected with empty vector. It was uncovered that the expression of the complete WHV genome profoundly down-regulated presentation of the class I antigen on hepatocytes in vitro, and throughout the differential analysis of the created transfected hepatocyte lines, it was determined that the virus envelope pre-S2 protein was responsible for this effect. In parallel experiments, it was also found that the inhibited presentation of the class I antigen can be fully restored by exposure of hepatocytes to IFN-γ, implying that the virus-induced suppression was not tenacious but can be reversed by treatment with an appropriate exogenous agent.