Expression and self-assembly of HCV structural proteins into virus-like particles and their immunogenicity.
BACKGROUND
The synthesis of virus-like particles (VLPs) provides an important tool to determine the structural requirements for viral particle assembly and virus-host interactions. Our purpose was to express simultaneously all three structural proteins of hepatitis C virus (HCV) in insect cells to investigate the proteins assembly into VLPs and the immunogenicity of these particles.


METHODS
HCV gene sequences encoding the structural proteins C, E1, and E2 were amplified with PCR, and recombinant baculoviruses were constructed using recombinant DNA techniques. The expression of HCV structural proteins in insect cells was analyzed by immunofluorescence and SDS-PAGE. The interaction of expressed structural proteins was investigated by immunoprecipitation and immunoblotting. The VLPs in the insect cells were visualized by electron microscopy (EM). VLPs were then purified by sucrose gradient centrifugation and used to immunize BALB/c mice. Antibodies against HCV were tested for in mouse serum samples by an ELISA assay.


RESULTS
The recombinant baculoviruses reBV/C and reBV/E1-E2 were constructed successfully. Insect cells co-infected with reBV/C and reBV/E1-E2 expressed HCV C, E1, and E2 proteins with the expected molecular weights of 20kD, 35kD, and 66kD, respectively. The results of immunoprecipitation and immunoblotting assays revealed the coimmunoprecipitation of C, E1, and E2 proteins, indicating association of the three structural proteins. Electron microscopy of insect cells co-infected with reBV/C and reBV/E1-E2 demonstrated spherical particles (40 to 60 nm in diameter) similar to the HCV virions from serum samples or hepatic tissue samples of HCV infected humans. The VLPs were partially purified. Antibodies to HCV were detectable in the serum of mice immunized with VLPs.


CONCLUSIONS
HCV structural proteins simultaneously expressed in insect cells can interact with each other and assemble into HCV-like particles, which are shown to be immunogenic in mice.
Zhao
Wei
W
Laboratory of Vaccine Research, Institute of Medical Biology, Chinese Academy of Medical Sciences, Peking Union Medical College, Kunming 650118, China.
Liao
Guo-yang
GY
Jiang
Yan-jun
YJ
Jiang
Shu-de
SD
D000818:Animals; D005455:Fluorescent Antibody Technique; D016174:Hepacivirus / Q000276:immunology / Q000502:physiology*; D007114:Immunization; D047468:Immunoprecipitation; D051379:Mice; D008807:Mice, Inbred BALB C; D008854:Microscopy, Electron; D018411:Spodoptera; D015678:Viral Structural Proteins / Q000276:immunology / Q000502:physiology*; D014771:Virion / Q000502:physiology*; D019065:Virus Assembly
D015678:Viral Structural Proteins