The nucleocapsid core protein of hepatitis C virus (HCV) has been shown to
trans-act on several viral or cellular promoters. To get insight into the t
rans-action mechanism of HCV core protein, a yeast two-hybrid cloning syste
m was used for identification of core protein-interacting cellular protein.
One such cDNA clone encoding the DEAD box family of putative RNA helicase
was obtained. This cellular putative RNA helicase, designated CAP-Rf, exhib
its more than 95% amino acid sequence identity to other known RNA helicases
including human DBX and DBY, mouse mDEAD3, and PL10, a family of proteins
generally involved in translation, splicing, development, or cell growth. I
n vitro binding or in vivo coimmunoprecipitation studies demonstrated the d
irect interaction of the full-length/matured form and. C-terminally truncat
ed variants of HCV core protein with this targeted protein. Additionally, t
he protein's interaction domains were delineated at the N-terminal 40-amino
-acid segment of the HCV core protein and the C-terminal tail of CAP-Rf, wh
ich encompassed its RNA-binding and ATP hydrolysis domains. Immunoblotting
or indirect immunofluorescence analysis revealed that the endogenous CAP-Rf
was mainly localized in the nucleus and to a lesser extent in the cytoplas
m, and when fused with FLAG tag, it colocalized with the HCV core protein e
ither in the cytoplasm or in the nucleus. Similar to other RNA helicases, t
his cellular RNA helicase has nucleoside triphosphatase-deoxynucleoside tri
phosphatase activity, but this activity is inhibited by various forms of ho
mopolynucleotides and enhanced by the HCV core protein. Moreover, transient
expression of HCV core protein in human hepatoma HuH-7 cells significantly
potentiated the trans-activation effect of FLAG-tagged CAP-Rf or untagged
CAP-Rf on the luciferase reporter plasmid activity. All together, our resul
ts indicate that CAP-Rf is involved in regulation of gene expression and th
at HCV core protein promotes the trans-activation ability of CAP-Rf, likely
via the complex formation and the modulation of the ATPase-dATPase activit
y of CAP-Rf These findings provide evidence that HCV may have evolved a dis
tinct mechanism in alteration of host cellular gene expression regulation v
ia the interaction of its nucleocapsid core protein and cellular putative R
NA helicase known to participate in all aspects of cellular processes invol
ving RNA metabolism. This feature of core protein may impart pleiotropic ef
fects on host cells, which may partially account for its role in HCV pathog
enesis.