No requirement of HCV 5'NCR for HCV-like particles assembly in insect cells.
OBJECTIVE
To express all three HCV structural proteins in the presence or absence of HCV 5'NCR to investigate the requirement of 5'NCR for the assembly of HCV-like particles in insect cells.


METHODS
HCV structural protein encoding sequences CE1E2 and 5'NCR-CE1E2 were amplified with PCR. Recombinant baculovirus were constructed with recombinant DNA techniques. HCV structural proteins expressed in insect cells were analyzed by immunofluorescence and SDS-PAGE. Immunoprecipitation experiment of insect cell lysates with anti-E2 monoclonal antibody (MAb) was carried out and the immunoprecipitated proteins were subjected to SDS-PAGE and immunoblotting with anti-C, anti-E2 MAbs and HCV positive serum. The virus-like particles in insect cells were visualized by electron microscopy (EM). The HCV-like particles were purified by sucrose gradient centrifugation and identified by EM and immune aggregation EM.


RESULTS
The recombinant baculovirus reBV/CE1E2 containing HCV C, E1, E2 genes and reBV/CS containing the same structural protein genes plus 5'NCR were constructed. The insect cells infected with either reBV/CE1E2 or reBV/CS expressed HCV C, E1 and E2 proteins with a molecular weight of 20 kD, 35 kD and 66 kD respectively. The results of immunoprecipitation and the immunoblotting revealed the coimmunoprecipitation of C, E1, and E2 proteins, indicating the interaction of HCV structural proteins expressed in insect cells. Electron microscopy of insect cells infected with reBV/CE1E2 or reBV/CS demonstrated spherical particles (40 to 60 nm in diameter) similar to the HCV virions from sera or hepatic tissues of HCV infected humans. The HCV-like particles were partially purified by sucrose gradient centrifugation, and the purified VLPs showed immuno-reactivity with anti-HCV antibodies.


CONCLUSIONS
HCV 5'NCR is not required for the assembly of HCV-like particles in insect cells, HCV core and envelope proteins are sufficient for viral particle formation.
Zhao
Wei
W
Laboratory of Vaccine Research, Institute of Medical Biology, Chinese Academy of Medical Sciences, Peking Union Medical College, Kunming 650118, Yunnan Province, China.
Liao
Guo-Yang
GY
Jiang
Yan-Jun
YJ
Jiang
Shu-De
SD
D000818:Animals; D000911:Antibodies, Monoclonal; D016367:Baculoviridae / Q000235:genetics; D002478:Cells, Cultured; D005455:Fluorescent Antibody Technique; D015967:Gene Expression Regulation, Viral; D016174:Hepacivirus / Q000235:genetics* / Q000254:growth & development*; D006526:Hepatitis C / Q000821:virology*; D018936:Hepatitis C Antigens / Q000235:genetics / Q000276:immunology; D007313:Insecta; D008854:Microscopy, Electron; D011233:Precipitin Tests; D011994:Recombinant Proteins / Q000235:genetics; D014771:Virion / Q000254:growth & development* / Q000302:isolation & purification / Q000648:ultrastructure
D000911:Antibodies, Monoclonal; D018936:Hepatitis C Antigens; D011994:Recombinant Proteins