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.
Highlights
RNA viruses are the cause of numerous human diseases
To determine whether the bound proteins are specific to SL1, SL3d that is present in the 5Ј UTR of hepatitis C virus (HCV) RNA was used as a control (Fig. 1A and supplemental Fig. S1)
Because heterogeneous nuclear ribonucleoprotein K (hnRNP K) exhibited the strongest interactions to the HCV RNA, we further focused on hnRNP K study
Summary
RNA-binding Assays on the Human Proteome Microarrays—The human proteome microarrays, comprised of more than 17,000 fulllength human proteins with N-terminal GST-fusions, were constructed on the glass-based slides as previously reported [15]. The cover slips were removed and each microarray was washed with 500 ml DEPC-treated PBST (PBS, 0.05% tween-20) for 10 min at 37 °C, on a rotary shaker set at 10 rpm. SiRNA Knockdown Assays—siRNAs (20 nM per reaction) were transfected into Huh7.5 cells harboring the HCV replicon that expresses the Renilla luciferase (RLuc) [18]. The primers used were: HCV 5Ј UTR sense (5Ј-AGC CAT GGC GTT AGT ATG AGT GTC-3Ј) and HCV 5Ј UTR antisense (5Ј-ACA AGG CCT TTC GCG ACC CAA C-3Ј). The change in HCV RNA and -actin mRNA levels in siRNA-treated cells was compared with the mock-transfected control as previously reported [19]. HCV 5Ј-UTR sense (5Ј-AGC CAT GGC GTT AGT ATG AGT GTC-3Ј) and 5Ј-UTR antisense (5Ј- ACA AGG CCT TTC GCG ACC CAA C-3Ј) primers were used to amplify HCV 5Ј UTR-specific sequences. Quantification of the number of copies of the HCV RNA has been described previously [22]
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