This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The aim of the project is to isolate and identify host proteins that interact with the HCV RNA structural element. The in vitro transcribed RNA element was first annealed to the biotinylated oligonucleotide, then was immobilized to the streptavidin magnetic beads. The soluble fraction (cytoplasmic) from Human Hepatocyte cell lysates was passed over with the RNA conjugated magnetic beads. After washing several times, the final pull-out was analyzed on SDS-PAGE gel, followed by MS and MS/MS analyses. We have performed comparisons between isolations using non-specific oligo, 320nt, or CRE and Specific 3'NTR, Cre-3"NTR, and Cre-3'NTR using Huh7.5 or Flneo cells. Competition with wild or mutant RNA helped to distinguish interacting proteins specific to the RNA. From the MS/MS analyses, we identified several RNA binding proteins. Follow-up with RNAi knock down analysis indicates that two identified proteins, LRP130 and DDX3, seems to be related with the HCV RNA replication. Further experiments will be followed to understand their roles in the HCV replication.