An Experimental Study On Intracellular Movement Of HBV Core Protein In Living Cells Using Biarsenical Fluorescent Labeling Method

Hepatitis B virus (HBV) represents a serious threat to public health since its infection is a significant cause of liver disease in the world today, ranging from acute liver failure to chronic active hepatitis, liver cirrhosis, and hepatocellular carcinoma (HCC). HBV belongs to a family of DNA virus called Hepadnaviridae. The HBV core protein (HBc), also named HBV core antigen (HBcAg), is the capsid protein of the virus and is also an important serological marker for diagnosis of HBV infection. During HBV infection, multiple copies of core protein assemble in the cytosol to form icosahedral capsid particles packaging the viral polymerase and pregenomic RNA. Besides capsid-building, HBc has been shown to play a multifunctional role in viral life cycle, including the synthesis of double-stranded DNA as a cofactor of the viral reverse transcriptase, interacting with viral envelope proteins to form infectious virions which later will be secreted into extracellular space. Although some important information about HBc's multifunctionality have been deciphered, many questions concerning the transport of core protein from systhesis sites to assembly sites, as well as the intracellular trafficking of nucleocapsid remain poorly understood. Studies to provide an understanding of these questions will be facilitated by biarsenical fluorescent labeling, which enables researchers to observe the movement of targeted protein in living cell.【Objective】To investigate the effects of tetracysteine peptide insertion on protein expression and viral particle formation. To preliminarily discuss the feasibility of applying biarsenical labeling method to visualizing HBc in living cells, and at the same time to provide some experimental basis to HBV viral particle visualization using the same method.【Methods】Utilizing PCR and molecular cloning techniques, a small synthetic oligonucleotides encoding tetracysteine peptide Cys-Cys-Pro-Gly-Cys-Cys was introduced into C-ORF of the parental plasmid pTHBV1.3, and the new recombinant plasmid produced was designated as pTHBV1.3-TC. Both pTHBV1.3 and pTHBV1.3-TC were transfected into HepG2 cells respectively by Lipofectamine2000. The expression and intracellular localization of wild-type HBV core protein (WT-HBc) and tetracysteine-tagged HBV core protein (TC-HBc) were analyzed by Western Blot and Immunofluorescence assays. Transimmision electron microscope was used to detect the existence of intracellular and extracellular HBV viral particles produced by transient transfected HepG2. For TC-HBc imaging in living cells, HepG2 cells were transfected with pTHBV1.3-TC. After 36 h, cells were washed with HBSS and labeled with ReAsH for 10 min. Information on mobile fraction of TC-HBc protein was obtained through FRAP analysis performed on an Olympus FV-500 laser scanning confocal microscope.【Results】The plasmid pTHBV1.3-TC was successfully constructed as originally designed, identified by restriction enzyme digestion and sequencing. Results of Western Blot confirmed the expression of TC-HBc protein in pTHBV1.3-TC-transfected HepG2 cells and no significant difference in expression amount was observed when comparing WT-HBc expressing cells with TC-HBc expressing cells (t=0.157, P=0.88). Immunofluorescence analysis showed that in the pTHBV1.3-transfected cell, WT-HBc was localized primarily in or near nucleus, while the TC-HBc-expressing cells showed a few fluorescent signals in the cytoplasma, in addition to the strong staining in nucleus. Under the transmission electron microscope, viral particles with diameter about 40nm were found both in the cytoplasm of pTHBV1.3-TC- transfected HepG2 cells and in its culture supernatants. Besides, 20nm particles were also found in the supernatants. In biarsenical labeling experiment, red fluorescent signals were found in the cytoplasm and nucleus. The movement of TC-HBc was recorded and the mobile fraction (Mf) of TC-HBc was estimated to be 66.9% by FRAP analysis.【Conclusion】The TC tag inserted into HBc protein was close to the c/e1 site in MIR (major immunodominant region). However, it had no significant impact on HBc expression and HBV viron formation. This confirmed that the Cys-Cys-Pro-Gly-Cys-Cys insertion was small enough and did not affect the secondary structure of important functional domains of HBc. Biarsenical compound ReAsH is membrane-permeable and can bind the TC tag, which was exposed on the surface of the capsid, with high specificity and high affinity. Thus, ReAsH could be used to detect TC-HBc in living cells. In the FRAP experiment, HBc protein showed an Mf of 66.9%, indicating that most of the HBc was mobile in living cells. This high mobile fraction potentially allows hundreds of HBc molecules to come together to form viron on a short time scale. The dynamic imaging observed in this study demonstrated that this method could be improved to visualize HBV viral particles in living cells, which will benefit a lot the study on infection steps of HBV.