A Semliki Forest virus expression system as a model for investigating the nuclear import and export of hepatitis B virus nucleocapsid protein

Abstract

Summary. -HBV core protein (HBc), the major component of nucleocapsid is known to have an essential role in the virus life cycle as the transporter of virus genome to the host nucleus; however, molecular details of the intracellular transport of HBc remain unknown. In this study, we investigated the intracellular distribution of the HBc protein resulting from Semliki Forest virus (SFV)-driven HBc gene and HBV RNA pregenome (pgRNA) expression in BHK-21 cells. Fluorescent confocal microscopy revealed extensive HBc protein synthesis in the cytoplasm 12 hr post infection (p.i.) with recombinant pSFV1/HBc or pSFV1/HBVpgRNA viruses. Twenty-four hr p.i., the HBc protein showed asymmetric, predominantly nuclear localization in most cells. However, 42 hr p.i., the number of cells containing intranuclear HBc protein, dramatically decreased to ~ 5%, while cytoplasmic HBc protein was detected in all cells. Remarkably, 24 hr p.i. with pSFV1/HBVpgRNA virus, cytoplasmic HBc protein colocalized with HBs protein. We conclude that a HBs-HBc interaction partially prevents the transport of HBc from the cytoplasm to nucleus. The SFV-driven system can be used for identification of new factors involved in the HBV nuclear import and export.

Design and Synthesis of Hepatitis B Virus (HBV) Capsid Assembly Modulators and Evaluation of Their Activity in Mammalian Cell Model

“…Previously, we developed and optimized the use of the recombinant alphavirus expression vector for HBc synthesis within 24 h [ 30 , 53 ], which cannot be achieved in HBV-producing cell cultures [ 32 ]. According to our protocol, there is a potential for the production of recombinant alphaviruses containing different HBV genotypes and HBc mutant variants, thus providing an opportunity to study the differences in the effects of capsid self-assembly inhibitors on different HBV genotypes and clinical mutants, allowing, to a large extent, individualized drug treatment testing.…”

“…The BHK-21 cells transduced with recombinant SFV/HBc virus express the HBc protein which is assembled into capsids [ 30 ]. Incubation with a potential capsid assembly inhibitor in cell growth medium followed by native agarose gel electrophoresis of cell lysates and subsequent immunoblot analysis allowed observation of the large-molecule HBc assembly products.…”

“…The cell medium containing pSFV1/HBc virus particles was collected and frozen next day after electroporation. The titre of the recombinant pSFV1/HBc virus particles was assessed by immunostaining for the HBc protein with anti-HBc antibodies in infected BHK-21 cells [ 30 ]. For infection, BHK-21 cells were incubated with a range of dilutions of recombinant pSFV1/HBc alphavirus particles for one hour in PBS with magnesium and calcium salts (PBS+/+, Gibco).…”

“…Cells were incubated with primary anti-HBc antibody mouse monoclonal antibody C1-5 [54] in blocking solution for 1 h at 37 • C. After incubation, the cells were washed three times with PBS and the secondary Alexa Fluor-488 labelled anti-mouse antibody in blocking solution was added followed by incubation for 30 min at 37 • C. Then, the cells were washed three times with PBS, incubated with the DNA stain reagent DAPI (1 mg/mL, dilution 1:10,000 in PBS) for 10 min at room temperature. Cells were mounted into Mowiol mounting medium with 2.5% DABCO (Sigma Aldrich, Darmstadt, Germany), left at room temperature in the dark overnight and examined for DAPI and HBc staining by confocal fluorescence microscopy (Leica TCS Sp2 SE) as previously described [30].…”

“…We previously developed and optimized the use of the recombinant alphavirus expression vector for effective target protein synthesis within 24 h [ 30 , 31 ], which cannot be achieved in standard HBV-producing mammalian cell cultures [ 32 ]. Here, we applied the alphavirus-based expression of full-length HBc gene and performed the assessment of HBV capsid assembly in a natural mammalian cell environment, allowing the direct selection of capsid modulators.…”