Characterization of cell lines allowing tightly regulated expression of hepatitis C virus core protein.
A tetracycline-regulated system was used to generate cell lines allowing tightly controlled expression of a hepatitis C virus (HCV) cDNA comprising the 5' noncoding, the core, and part of the E1 regions. Production of 21-kDa processed nucleocapsid protein could be regulated over a broad range by the concentration of tetracycline present in the culture medium. Induction ratios of over 1,000-fold were found using an HCV core-luciferase fusion construct. Core protein had an intracellular half-life of 9 hr and corresponded to the product of 173 amino-terminal amino acids of the HCV open reading frame. Sequential immunofluorescence microscopy revealed the presence of core antigen first in a predominantly perinuclear fine-reticular staining pattern and subsequently also in cytoplasmic granules and vesicles. By immunoelectron microscopy core protein was found on the endoplasmic reticulum membrane and on the surface of cytoplasmic lipid droplets. Growth rate analyses and colony formation efficiency assays showed no major cytotoxic effect of HCV core protein expression per se. HCV gene expression could be inhibited by an antisense oligonucleotide targeting a region immediately downstream of the translation initiation codon. These cell lines represent important tools to investigate structural and functional properties of HCV core protein and may be useful to evaluate gene therapeutic strategies against HCV in a cellular system.
Moradpour
D
D
Molecular Hepatology Laboratory, Massachusetts General Hospital Cancer Center, Harvard Medical School, Charlestown 02129, USA.
Englert
C
C
Wakita
T
T
Wands
J R
JR
D002455:Cell Division; D002460:Cell Line / Q000648:ultrastructure / Q000821:virology*; D015967:Gene Expression Regulation, Viral / Q000187:drug effects; D006207:Half-Life; D016174:Hepacivirus / Q000235:genetics* / Q000648:ultrastructure; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D013347:Subcellular Fractions; D013752:Tetracycline / Q000494:pharmacology; D014758:Viral Core Proteins / Q000235:genetics* / Q000378:metabolism; D014759:Viral Envelope Proteins / Q000235:genetics*
C087035:E1 protein, Hepatitis C virus; D016376:Oligonucleotides, Antisense; D014758:Viral Core Proteins; D014759:Viral Envelope Proteins; C069130:nucleocapsid protein, Hepatitis C virus; D013752:Tetracycline