This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.
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 streptavidinmagnetic 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 3NTR, Cre-3 NTR, and Cre-3NTR 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.
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