A Human Proteome Microarray Identifies that the Heterogeneous Nuclear Ribonucleoprotein K (hnRNP K) Recognizes the 5′ Terminal Sequence of the Hepatitis C Virus RNA

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 ϳ17,000 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-violetcrosslinking 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. Molecular & Cellular

Heterogeneous Ribonucleoprotein K (hnRNP K) Binds miR-122, a Mature Liver-Specific MicroRNA Required for Hepatitis C Virus Replication

“…Each reported value represents the results of a minimum of three independent experiments. SL1-hnRNPK Crosslinking Assay-HnRNPK binding to SL1 or variants was determined as previously described in Fan et al (15). Briefly, 100 ng of purified protein was mixed with 1 pmole of the RNA that was radiolabeled at its 5Ј terminus using polynucleotide kinase and [␥ -32 P]ATP.…”

“…Human Proteome Chip Assay-The human protein chip was screened as described previously (15). The protein chip was preblocked with a solution of 3% BSA and 0.1 mg/ml of salmon sperm DNA under a slide coverslip and then incubated with the probe RNA for 1 h at 37°C and 8 rpm using a BioMixer TM II (CapitalBio, Beijing, China).…”

“…Reagents-HnRNP K was expressed and purified from recombinant Sacchromyces cerevisae using a GST-tag as previously de-scribed (15) and stored in aliquots at Ϫ80°C until use. MiR-122 and variants, including those modified with fluorophores, were chemically synthesized (Bioneer, Alameda CA).…”

“…A version of the chip that contains ϳ17,000 GST-tagged human proteins has previously been used to screen for binding to specific RNAs (15). We used the chip to perform an unbiased screen of the human proteins that can bind to miR-122.…”

“…We previously reported that the HCV RNA sequence that anneals to miR-122 is recognized by the heterogeneous ribonucleoprotein K (hnRNP K), a multifunctional RNA-binding protein known to be involved in RNA processing, translation, and the replication of several RNA viruses (13)(14)(15). In an unbiased screen for proteins from human proteome chips containing over 17,000 proteins, we identified 40 proteins that bind mature miR-122, including hnRNP K. Recombinant hnRNP K recognizes short pyrimidine sequences in miR-122 in vitro and a similar sequence in the HCV 5Ј UTR.…”