Identification Of The Subdomains In Hepatitis B Virus Envelope Proteins Involved In Viral Entry

Hepatitis B virus (HBV), the prototype for the family Hepadnaviridae, is known as the smallest DNA virus that infects human beings. HBV infection causes acute and chronic necroinflammatory liver disease. Infected individuals are at high risk of developing liver cirrhosis and, eventually, hepatocellular carcinoma. After discovery of the virus in 1960s, replication of HBV has been extensively studied in transfected hepatoma cells, yet the mechanisms by which HBV infect hepatocytes are still not well understood by the lack of easily accessible in vitro infection models.HBV is an envelope virus and its envelope proteins play a key role during viral entry. So it is very important to identify the subdomains in HBV envelope proteins involved in HBV infection. These studies would help us to understand the HBV entry mechanism, promote the discovery of HBV receptor(s) and develop new kinds of anti-HBV drugs.HBV and its satellite virus hepatitis delta virus (HDV) are widely used in HBV entry research. To screen the subdomains of HBV envelope proteins involved in viral entry, HBV and HDV have their own limitations. For example, the subdomains involved in HBV/HDV production couldn't be investigated. Recently, John Taylor's lab reported that HBV pseudotype particles (HBVpp) could infect primary human hepatocytes as efficiently and specifically as HBV and could be used as a new liver-targeted delivery vehicle. In this study, we tried to use HBVpp and primary tupaia hepatocytes (PTH) to identify the roles of"preS2 domain, Transmembrane domain I (TMD-I), Transmembrane domain II (TMD-II), Cys-107 in S domain, C-terminal domain of L protein and S protein"during HBV entry.First, a simple PTH isolation method was established based on two-step in situ collagenase perfusion. With this method, 1×108 cells could be prepared from each animal. The cell viability was around 90% and the attachment percentage was above 70% on average. The PTH cryopreservation method was also optimized and after thawing the cell viability and attachment percentage was about 80% and 40% respectively.Next, HBVpp was produced by cotransfection 293T cells with HBV envelope protein expression plasmids and lentiviral packaging system. HBVpp species and tissue specific infection ability was analyzed using PTH, primary rat hepatocytes (PRH), HepG2 and 293T cells. It was found that HBVpp could infect PTH specifically in vitro and its infectivity could be neutralized by preS1 antibody. These characteristics were similar to HBV. The HBVpp-PTH in vitro infection model was also used to assess the roles of M protein, the myristoylation and cytosolic domain I of L during HBVpp entry. It was indicated that these domains'roles for HBVpp were the same to HBV. So it was concluded that the HBVpp-PTH in vitro infection model could be used to identify the subdomains of HBV envelope proteins for HBV entry.Then, a series of plasmids expressing HBV recombinant envelope proteins with some subdomain's deletion or substitution were constructed. WB and ELISA methods were employed to demonstrate their expression and secretion. It was indicated that all of the recombinant envelope proteins with preS1 were intracellular expressed. For S protein, deletion or substitution of TMD-I, Cys-107 and C-terminal amino acid would severely reduce or abolish S protein secretion. So HBV or HDV couldn't be used in this study.The HBVpp production only required HBV recombinant envelope proteins expressing on 293T cell membrane. The flow cytometry results showed that most of HBV recombinant envelope proteins could be detected on the surface of transfected 293T cells. Various HBVpps were produced and their species and tissue specific infection abilities were analyzed with PTH, PRH, hepG2 and 293T cells. All of thses HBVpps couldn't infect PRH, hepG2 and 293T cells, and some of them could infect PTH specifically. From these results, it was concluded that:1. Deletion of preS2 completely from L protein wouldn't abolish HBVpp entry2. TMD-I was important for HBVpp entry, but it could be substituted with other transmembrane domains which were with no fusion activities.3. TMD-II played a key role for the structures of HBV envelope proteins and no conclusion was got for its function in HBVpp entry.4. Cys-107 of S domain was dispensable for HBVpp entry5. The C-termianl 21 amino acids of HBV envelope proteins was not required for HBVpp entry6. S protein was not necessary for HBVpp entry, but it could enhance HBVpp infection ability when coexpressd in 293T cells with L protein. The S domain and preS1 of L protein should be in one peptide chain to enable HBVpp entry. Some of infectious HBVpps were chosen for HBVpps infection inhibition experiments. HBVpps copy numbers were first quantitated by one step real-time RT-PCR. The inhibitor Myr-HBVpreS/2-48XaGST which was known by its HBV entry suppression activities was expressed and purified. The infection abilities of all the HBVpps we chose were sensitive to the inhibitor Myr-HBVpreS/2-48XaGST.On the whole, it was concluded that HBVpp and HBV should employ similar mechanisms to entry primary hepatocytes and the six conclusions above for the roles of the subdomains in HBV envelope proteins during HBVpps entry would also apply to HBV.