| T-cells and the cytokines they secrete are critical components of the immune response to Hepatitis B virus (HBV). Tumor necrosis factor (TNF) and interferon-gamma (IFN-gamma) are known central mediators involved in inhibiting HBV replication during infection. TNF has been shown to reduce the levels of the viral nucleocapsid through decreased capsid stability, thereby impairing viral DNA replication. The molecular mechanism by which IFN-gamma reduces HBV replication, however, has remained elusive. Here we present evidence describing the effect of IFN-gamma on HBV replication in a hepatocyte culture-based system. We show that IFN-gamma reduces the levels of HBV DNA without decreasing host cell viability (non-cytopathic inhibition). Further, IFN-gamma likely has no effect on virus particle production, as IFN-gamma had no observable effect on the key steps involved in particle production. Surprisingly, IFN-gamma was found to increase the levels of extra-cellular viral particles that contain HBV DNA replicative intermediates. In addition, the increase in extra-cellular particles was found to be specific to HBV DNA containing particles, as IFN-gamma treatment did not affect overall cellular protein secretion nor extracellular HBsAg sub-viral (immature) particle levels. We show that IFN-gamma increases the extracellular levels of viral particles that lack the HBV envelope proteins (HBsAg deficient particles). This is intriguing, as particles that lack HBsAg should be non-infectious. Increasing the levels of non-infectious particles represents a novel anti-viral mechanism for IFN-gamma. Finally, we show that IFN-gamma requires Nedd4, but not Vps4A, to alter intracellular and extracellular HBV DNA levels. This modulation of the MVB pathway is likely specific as IFN-gamma does not alter the total protein levels of the EGFR, a protein known to be modulated by the MVB pathway.;The signal transduction pathways activated by and important for IFN-gamma mediated reduction of HBV DNA were also studied to gain a better understanding of the molecular mechanism IFN-gamma utilizes to reduce intracellular HBV replication. We show that the transcription factors Stat1 and IRF-1 are active for an extended period of time in IFN-gamma treated hepatocytic cells. Further, Stat1, but not NF-kappaB, was found to be required for IFN-gamma mediated reduction of intracellular HBV DNA replicative intermediates. TNF has been shown to require NF-kappaB mediated gene transcription to reduce HBV DNA replication. These data indicate that IFN-gamma and TNF therefore use distinct pathways and mechanisms to reduce HBV replication. Further, we show here that IFN-gamma and TNF additively reduce intracellular HBV DNA replicative intermediates.;Together, these studies demonstrate that IFN-gamma has a profound effect on HBV DNA replication and secreted particle levels. Further, IFN-gamma functions through a novel mechanism of action that is distinct from that utilized by TNF, but together these cytokines play a critical role controlling the HBV infection. |
