Analysis of the subcellular localization of hepatitis C virus E2 glycoprotein in live cells using EGFP fusion proteins.
Hepatitis C virus (HCV) E1 and E2 glycoproteins assemble intracellularly to form a non-covalently linked heterodimer, which is retained in the endoplasmic reticulum (ER). To study the subcellular localization of E2 in live cells, the enhanced green fluorescent protein (EGFP) was fused to the N terminus of E2. Using fluorescence and confocal microscopy, we have confirmed that E2 is located in the ER, where budding of HCV virions is thought to occur. Immunoprecipitation experiments using a conformation-sensitive antibody and a GST pull-down assay showed that fusion of EGFP to E2 interferes neither with its heterodimeric assembly with E1, nor with proper folding of the ectodomain, nor with the capacity of E2 to interact with human CD81, indicating that the EGFP-E2 fusion protein is functional. As a tool to study binding of E2 to target cells, we also described the expression of an EGFP-E2 fusion protein at the cell surface.
Kien
François
F
INSERM U544, Institut de Virologie, 3 rue Koeberlé, 67000 Strasbourg, France.
Abraham
Jean-Daniel
JD
INSERM U544, Institut de Virologie, 3 rue Koeberlé, 67000 Strasbourg, France.
Schuster
Catherine
C
INSERM U544, Institut de Virologie, 3 rue Koeberlé, 67000 Strasbourg, France.
Kieny
Marie Paule
MP
INSERM U544, Institut de Virologie, 3 rue Koeberlé, 67000 Strasbourg, France.
D015703:Antigens, CD / Q000378:metabolism; D015153:Blotting, Western; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D049452:Green Fluorescent Proteins; D016174:Hepacivirus / Q000378:metabolism*; D006801:Humans; D007202:Indicators and Reagents; D008164:Luminescent Proteins / Q000032:analysis; D008565:Membrane Proteins / Q000378:metabolism; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000032:analysis; D060225:Tetraspanin 28; D014759:Viral Envelope Proteins / Q000032:analysis / Q000378:metabolism*
D015703:Antigens, CD; C554798:CD81 protein, human; D007202:Indicators and Reagents; D008164:Luminescent Proteins; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D060225:Tetraspanin 28; D014759:Viral Envelope Proteins; C114224:envelope protein E1, GB virus C; D049452:Green Fluorescent Proteins; C091015:glycoprotein E2, Hepatitis C virus