Expression studies of the core+1 protein of the hepatitis C virus 1a in mammalian cells. The influence of the core protein and proteasomes on the intracellular levels of core+1.
Recent studies have suggested the existence of a novel protein of hepatitis C virus (HCV) encoded by an ORF overlapping the core gene in the +1 frame (core+1 ORF). Two alternative translation mechanisms have been proposed for expression of the core+1 ORF of HCV-1a in cultured cells; a frameshift mechanism within codons 8-11, yielding a protein known as core+1/F, and/or translation initiation from internal codons in the core+1 ORF, yielding a shorter protein known as core+1/S. To date, the main evidence for the expression of this protein in vivo has been the specific humoral and cellular immune responses against the protein in HCV-infected patients, inasmuch as its detection in biopsies or the HCV infectious system remains elusive. In this study, we characterized the expression properties of the HCV-1a core+1 protein in mammalian cells in order to identify conditions that facilitate its detection. We showed that core+1/S is a very unstable protein, and that expression of the core protein in addition to proteosome activity can downregulate its intracellular levels. Also, we showed that in the Huh-7/T7 cytoplasmic expression system the core+1 ORF from the HCV-1 isolate supports the synthesis of both the core+1/S and core+1/F proteins. Finally, immunofluorescence and subcellular fractionation analyses indicated that core+1/S and core+1/F are cytoplasmic proteins with partial endoplasmic reticulum distribution in interphase cells, whereas in dividing cells they also localize to the microtubules of the mitotic spindle.
Vassilaki
Niki
N
Molecular Virology Laboratory, Hellenic Pasteur Institute, Athens, Greece.
Boleti
Haralabia
H
Mavromara
Penelope
P
D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D015870:Gene Expression; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D007424:Intracellular Fluid / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D046988:Proteasome Endopeptidase Complex / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D014758:Viral Core Proteins / Q000235:genetics / Q000378:metabolism*
D011993:Recombinant Fusion Proteins; D014758:Viral Core Proteins; C433031:hepatitis C protein F, Hepatitis C virus; D049452:Green Fluorescent Proteins; D046988:Proteasome Endopeptidase Complex