Green fluorescent protein - Tagged HCV non-enveloped capsid like particles: development of a new tool for tracking HCV core uptake.
Circulating 'free' non-enveloped Hepatitis C virus (HCV) core protein has been demonstrated in HCV-infected patients, and HCV subgenomes with deletions of the envelope proteins have been previously identified. Initial studies from our laboratory, previously published, indicated that expression of HCV core in insect cells can direct the formation of capsid-like particles lacking the envelope glycoproteins. These protein nanospheres, morphologically similar to natural capsids, were shown to be taken up by human hepatic cells and to produce cell-signalling events. To follow the intracellular fate of these particles we fused the core protein to eGFP. We demonstrate that the chimeric proteins core(173)-eGFP, eGFP-core(191) and eGFP-core(173) can be efficiently expressed, self-assembled, and form fluorescent non-enveloped capsid-like particles. By using confocal microscopy and FACS analysis, we provide evidence that the fluorescent nanospheres can not only enter human hepatic cells - the main target of HCV - but also human immune cells such as T and B lymphocytes, as well as human myeloid leukaemia cells differentiated along the monocyte/macrophage-like pathway. The fluorescent particles might thus be used to trace the intracellular trafficking of naked HCV capsids as showed by live microscopy and to further understand their biological significance.
Katsarou
Konstantina
K
Molecular Virology Laboratory, Hellenic Pasteur Institute, 11521 Athens, Greece.
Serti
Elisavet
E
Tsitoura
Panagiota
P
Lavdas
Alexandros A
AA
Varaklioti
Agoritsa
A
Pickl-Herk
Angela-Maria
AM
Blaas
Dieter
D
Oz-Arslan
Devrim
D
Zhu
Rong
R
Hinterdorfer
Peter
P
Mavromara
Penelope
P
Georgopoulou
Urania
U
D001402:B-Lymphocytes / Q000821:virology; D002213:Capsid / Q000502:physiology* / Q000648:ultrastructure; D045744:Cell Line, Tumor; D049452:Green Fluorescent Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D016174:Hepacivirus / Q000502:physiology* / Q000648:ultrastructure; D022781:Hepatocytes / Q000821:virology*; D006801:Humans; D009000:Monocytes / Q000821:virology; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D013601:T-Lymphocytes / Q000821:virology; D014758:Viral Core Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D053586:Virus Internalization
D011993:Recombinant Fusion Proteins; D014758:Viral Core Proteins; C485184:enhanced green fluorescent protein; C069130:nucleocapsid protein, Hepatitis C virus; D049452:Green Fluorescent Proteins