A human proteome microarray identifies that the heterogeneous nuclear ribonucleoprotein K (hnRNP K) recognizes the 5' terminal sequence of the hepatitis C virus RNA

Mol Cell Proteomics. 2014 Jan;13(1):84-92. doi: 10.1074/mcp.M113.031682. Epub 2013 Oct 10.

Abstract

Stem-loop I (SL1) located in the 5' untranslated region of the hepatitis C virus (HCV) genome initiates binding to miR-122, a microRNA required for hepatitis HCV replication. However, proteins that bind SL1 remain elusive. In this study, we employed a human proteome microarray, comprised of ∼17,000 individually purified human proteins in full-length, and identified 313 proteins that recognize HCV SL1. Eighty-three of the identified proteins were annotated as liver-expressing proteins, and twelve of which were known to be associated with hepatitis virus. siRNA-induced silencing of eight out of 12 candidate genes led to at least 25% decrease in HCV replication efficiency. In particular, knockdown of heterogeneous nuclear ribonucleoprotein K (hnRNP K) reduced HCV replication in a concentration-dependent manner. Ultra-violet-crosslinking assay also showed that hnRNP K, which functions in pre-mRNA processing and transport, showed the strongest binding to the HCV SL1. We observed that hnRNP K, a nuclear protein, is relocated in the cytoplasm in HCV-expressing cells. Immunoprecipitation of the hnRNP K from Huh7.5 cells stably expressing HCV replicon resulted in the co-immunoprecipitation of SL1. This work identifies a cellular protein that could have an important role in the regulation of HCV RNA gene expression and metabolism.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Gene Expression Regulation, Viral
  • Hepacivirus / genetics*
  • Hepacivirus / pathogenicity
  • Hepatitis / genetics
  • Hepatitis / virology*
  • Heterogeneous-Nuclear Ribonucleoprotein K / genetics*
  • Humans
  • MicroRNAs / genetics
  • MicroRNAs / metabolism
  • Protein Array Analysis
  • Proteome
  • RNA, Viral / genetics*
  • RNA-Binding Proteins / genetics
  • Virus Replication / genetics

Substances

  • Heterogeneous-Nuclear Ribonucleoprotein K
  • MIRN122 microRNA, human
  • MicroRNAs
  • Proteome
  • RNA, Viral
  • RNA-Binding Proteins