The high level of chronicity resulting from HBV infection likely results from the inability to produce interferon gamma, tumor necrosis factor alpha (TNF) and interferon alpha which inhibit HBV replication without killing infected hepatocytes (noncytopathic inhibition). Studies have shown that TNF blocks HBV replication through the loss of viral cytoplasmic capsids, structures in which HBV must replicate. This application is directed to understanding the molecular mechanism by which TNF inhibits HBV replication by eliminating viral capsids. Since TNF pathways intersect with those acted on by the HBV regulatory protein HBx, which could influence TNF activities, studies will also investigate the effect of HBx expression on TNF suppression of HBV.
Aim 1 will characterize the mechanism by which TNF inhibits HBV replication through loss of cytoplasmic capsids. Studies will determine whether downregulation of capsids by TNF activity is due to destabilization of previously formed capsids, prevention of new capsid formation, or both. Studies will also determine whether capsids are lost by TNF-meditated capsid destabilizing phosphorylation of core protein, redistribution of core protein, inducible anti-capsid chaperone activity or other potential mechanisms.
Aim 2 will identify cellular genes and activities that mediate TNF and NF-kB inhibition of HBV capsid abundance and potential involvement of HBx expression in altering this response. Studies will identify the cellular genes that mediate TNF inhibition of HBV replication through reduced capsid integrity. Candidate genes will be identified by characterizing an in vitro system we developed in which TNF destabilizes HBV capsids, and attempt to identify the components involved. Other studies will use differential TNF-inducible gene expression.
Aim 3 will examine the mechanism and function of TNF-mediated downregulation of HBV replication by loss of capsids in a mouse model hepatocyte system. Studies will characterize an HBV replication system in primary mouse hepatocytes that closely reproduces many of the features of natural infection. Studies will utilize hepatocytes from knockout mice containing specific lesions in the TNF pathway. Primary hepatocyte growth in an extracellular matrix permits analysis of HBV replication, anti-capsid effects of TNF, and dissection of the pathway for capsid destabilization in a system that significantly reproduces a state of semi-chronic infection that more closely parallels authentic infection.
