The Effect Of High Expression Of Beta2-glycoprotein Ⅰ In The Earliest Stages Of Hepatitis B Virus Infection

Human hepatitis B virus (HBV) infection is a global public health problem.Approximately2billion people are infected with HBV, and among them3.5-4million are chronic HBV carriers. Hepatitis B causes about6.5million deaths ofHBV-related cirrhosis and hepatocellular carcinoma annually. In China, HBV infection is ahighly endemic. The seroepideminological survey on HBV infection showed that HBsAgcarrier rate was10%in the overall population. Accordingly, there were an estimated93million HBV carriers, and35million die of cirrhosis and hepatocellular carcinoma each year.Current therapies include immune protection and sustained suppression of viral replication.However, inhibition of virus entry and the induction of hepatocyte proliferation are attractivetherapeutic strategies. Up to now, the early stages of HBV infection (i.e., attachment,receptor binding, and fusion) are not completely understood.Cellular entry of HBV is mediated by viral envelope proteins that aredesignated large (LHBs), middle (MHBs), and small (SHBs). The LHBsencompasses the PreS1domain, the PreS2domain and the S domain; the MHBsencompasses the PreS2and S domain and the SHBs consists of the S domain. Thepre-S1domain of the LHBs envelope protein is a primary determinant of binding toone or more receptors. Several proteins have been reported as viral receptors thatinteract with HBV surface proteins, including beta2-glycoprotein I (beta2-GPI),annexin V, annexin II (ANX2), asialoglycoprotein receptor (ASGPR), fibronectin,glycosaminoglycan (GAG), squamous cell carcinoma antige (SCCA),carboxypeptidase D and others; but no protein has been confirmed to support viralentry. Recently, it has been proposed that sodium taurocholate co-transportingpolypeptide (NTCP) is a functional receptor for human HBV and hepatitis D virusentry. Although the hepatotropism of HBV and its host specificity are believed to be associated with specific receptor binding, it remains unclear which pathway orpathways are essential for HBV entry.It has been shown that beta2-GPI, a human plasmatic protein primarilysynthesized in the liver, can bind to recombinant hepatitis B surface antigen(rHBsAg). However, beta2-GPI is known to act as a major autoantigen inantiphospholipid syndrome (APS), an autoantibody-mediated thrombotic disorder.Interestingly, beta2-GPI is a flexible molecule, and its conformation and functionalactivity depend on interactions with its surroundings. It consists of5homologousdomains (domains I-V), all of which are complement control protein repeats. Inplasma, beta2-GPI remains in the nonpathogenic circular form, with domains V and Iconnected. The protein opens into an S-shaped configuration when interacting withanionic surfaces via domain V. It was observed that glycosylation of beta2-GPI didnot change its high-affinity binding to rHBsAg, and rHBsAg and anionicphospholipids shared the same binding region in the fifth domain of beta2-GPI.Furthermore, the binding activity of beta2-GPI to HBsAg was higher in the serum ofpatients in the active virus replication phase. In addition, annexin II was reported tobe a possible receptor for beta2-GPI on the membrane of SMMC-7721hepatomacells, assisting in bridging HBV entry. Altogether, it can be concluded that beta2-GPImight be crucial for the initial stages of HBV infection.In this study, several cell lines were used to clarify the purpose of ourexperiment. Human hepatoma SMMC-7721cell line was used in our previous study.HepG2.2.15is a HBV-producing cell line, derived from human hepatoma cell lineHepG2. L02is an immortal human hepatic cell line. Human embryonickidney cell line, HEK-293T, has high transfection efficiency. Chinese HamsterOvary (CHO) cells represent a hugely popular research tool in the molecular biologycommunity. L02,293T and CHO cells were used as control in our study. Firstly,western blot and qRT-PCR analyses revealed that beta2-GPI expression wasupregulated in HepG2.2.15cells at both the mRNA and the protein level and wasalmost non-existent in293T and CHO cells. When rHBsAg (200ng mL-1) was pre-incubated with beta2-GPI (0-800ng mL-1) before contact with cells, ELISAanalyses demonstrated that beta2-GPI enhanced the ability of HBsAg to bind to cellsurfaces, and there was differential adhesion to L02, HepG2, HepG2.2.15, and293Tcells. However, there was no dose-dependent response for beta2-GPI. Thedifferences in the binding abilities of these cells might be attributable to differentexpression levels of annexin II. Then, western blot was performed to detect theexpression of annexin II in L02, SMMC-7721, HepG2, HepG2.2.15,293T and CHOcells. The result showed that annexin II was expressed at lower levels in HepG2.2.15cells compared to L02, HepG2, and SMMC-7721cells.Additionally, western blot and ELISA were then performed to assess the effectsof HBV and the HBsAg domain on beta2-GPI expression in co-transfected293Tcells, and determine the effects of HBV on annexin II expression in transfectedHepG2cells. This study revealed that HBV and the large HBV envelope proteinincreased beta2-GPI expression, and the middle and the small surface protein had noeffect on beta2-GPI expression. And beta2-GPI can inhibit the secretion of HBsAg inHBV life cycle. In addition, HBV can decrease annexin II expression in transfectedHepG2cells. Further investigation indicated that beta2-GPI colocalized with HBsAgin the cytosol of HepG2.2.15cells, with sodium taurocholate co-transportingpolypeptide (NTCP) on the cell membrane in NTCP-complemented HepG2cells,and with annexin II in the cytosol of HepG2and HepG2.2.15cells. And annexin IIcolocalized with HBsAg in the cytosol of HepG2.2.15cells.In conclusion, our findings demonstrated that high expression of beta2-GPIenhances HBsAg binding to cell surfaces, thus contributing to virus particle transfer to theNTCP receptor and interaction with annexin II for viral binding and membrane fusion. Theyprovide new insights into the route of human HBV entry into hepatocytes.