Establishment And Early Invasion Of The Molecular Mechanisms Of HBV Infection Model

In China and worldwide, Hepatitis B virus (HBV) infection and its related diseases remains a big public health problem and causes about one million deaths annually. About2billion people have been infected with HBV, among whom35million people are suffered with HBV chronic infection and are at high risk of liver cirrhosis, hepatocyte carcinoma and liver failure due to the persisting HBV replication and the associated inflammation in the liver. More than50%of the hepatocyte carcinoma patients are attributable to HBV infection worldwide. Individuals co-infected with HBV and its satellite virus Hepatitis D virus (HDV) are at greater risk of more rapid progression and more severe outcome. Although a highly protective and effective vaccine against HBV and useful treatment for HBV infection are available, current preventive and therapeutic intervention for HBV infection is still inadequate, and there is no specific treatment clinically available for HDV infection. Studies on the molecular mechanisms of HBV infection, in particular the early entry processes, will not only greatly help to understand the viruses and its pathogenesis, but also help to develop new interventions for the treatment of the infection and its related diseases.In addition to the human and Chimpanzee, HBV can only infect a primate-like small animal termed treeshrew(Tupaia belangeri chinenesis). Susceptible cells for HBV infection in vitro are also quite limited, among which the tupaia primary hepatocyte (PTH) is very valuable for HBV infection studies. It is critically important to isolate the PTH with high viability and to establish a highly efficient HBV, HDV and Woolly Monkey HBV(WMHBV) in vitro infection system for studies on the molecular mechalisims of HBV early entry process. Such a system is also the key for evaluating the functionality of any candidate receptor and entry associate moleculars for HBV. After a lot of efforts on specifying and improving the two-step perfusion approach to isolate the primary hepatocytes from treeshrew liver, we worked out a highly reliable culture system for PTH, we also established very efficient and highly repeatable in vitro infection systems for HBV, HDV and WMHBV using PTH. During the optimization process of these systems, we also attempted to take a chemical-biology approach to explore the infection mechanism of HBV, in particular we tested a wide range of small molecular compounds with known functions and targets for their effect for HBV infection during different time windows of the infection processes. Considering that the genome database of Tupaia was incomplete and specific reagents such as antibodies for PTH were not available, but the compound activities are not likely to be limited in PTH, the compound experiments using the PTH infection system was a very useful and promising approach for us to investigate the entry processes of HBV. By screening a large set of compounds, we found that the Receptor Tyrosine kinase-phosphatidylinositol3kinase-Protein kinase B(RTK-PI3K-AKT)signaling pathway is involved in HBV early entry processes.We also explored a new strategy for tracing and isolating possible pre-SI/receptor complex. Using covalent cross linking followed by immunobloting to monitor the bait rather than the target protein, the putative functional receptor complex can be traced and be used for subsequent LC/MC/MC analysis to identify target proteins from the silver staining bands. This strategy has a key advantage because it was independent of the nature of the target, it was especially advantageous in HBV receptor studies as there were no specific antibodies and related reagents for treeshrew proteins. Our efforts of these explorations gave us lots of practical and theoretical implications, and those lots of worked or failed experiences greatly helped us in finally finding the functional receptor. For example, the covalent cross linking followed with immunobloting monitoring strategy was quite smart and efficient; it became clear to us that the quality of tupaia liver transcriptome at that time was not sufficient for the database searching, which itself is essential for MS identification; in addition, it might be needed to use multistep affinity purification in the denatured situation to trace and purify the membrane proteins. By taking a different approach, we also identified and partially verified that the membrane associated protein Myosin Heavy Chain9(myosin9) may be required for WMHBV binding and entry to the PTH.After sodium taurocholate cotransporting polypeptide (NTCP) was indentified as a common functional receptor for HBV, HDV and WMHBV in our lab, we propose that the early discovered RTK-PI3K-AKT signaling pathway may regulate the NTCP sub-cellular distribution and is involved in HBV early entry processes.Finally, to final prove that NTCP is a function receptor for the viruses in vivo, we have established the human NTCP transgenic mice lines in several months, then we validated the robust expression level of human NTCP in the transgenic mice hepatocyte according to series of approach such as western blot and immunofluoresence staining. The promising related functional study of virus infection is ongoing.