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. 2013 Jun;12(6):1539-52.
doi: 10.1074/mcp.M112.017020. Epub 2013 Feb 21.

Affinity capture and identification of host cell factors associated with hepatitis C virus (+) strand subgenomic RNA

Alok Upadhyay  1 , Updesh Dixit, Dinesh Manvar, Nootan Chaturvedi, Virendra N Pandey
Affiliations

Affinity capture and identification of host cell factors associated with hepatitis C virus (+) strand subgenomic RNA

Alok Upadhyay et al. Mol Cell Proteomics. 2013 Jun.

Abstract

Hepatitis C virus (HCV) infection leading to chronic hepatitis is a major factor in the causation of liver cirrhosis, hepatocellular carcinoma, and liver failure. This process may involve the interplay of various host cell factors, as well as the interaction of these factors with viral RNA and proteins. We report a novel strategy using a sequence-specific biotinylated peptide nucleic acid (PNA)-neamine conjugate targeted to HCV RNA for the in situ capture of subgenomic HCV (+) RNA, along with cellular and viral factors associated with it in MH14 host cells. Using this affinity capture system in conjunction with LC/MS/MS, we have identified 83 cellular factors and three viral proteins (NS5B, NS5A, and NS3-4a protease-helicase) associated with the viral genome. The capture was highly specific. These proteins were not scored with cured MH14 cells devoid of HCV replicons because of the absence of the target sequence in cells for the PNA-neamine probe and also because, unlike oligomeric DNA, cellular proteins have no affinity for PNA. The identified cellular factors belong to different functional groups, including signaling, oncogenic, chaperonin, transcriptional regulators, and RNA helicases as well as DEAD box proteins, ribosomal proteins, translational regulators/factors, and metabolic enzymes, that represent a diverse set of cellular factors associated with the HCV RNA genome. Small interfering RNA-mediated silencing of a diverse class of selected proteins in an HCV replicon cell line either enhanced or inhibited HCV replication/translation, suggesting that these cellular factors have regulatory roles in HCV replication.

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Figures

Fig. 1.
Fig. 1.
A, structure of PNA. The bases (A, T, G, and C) are linked with polyamide backbone against a sugar phosphate backbone in DNA. B, neamine-PNA-biotin conjugate. PNA targeted to the HCV core coding region is conjugated with neamine at the N terminus and biotin at the C terminus. C, schematic representation of the MH14 HCV subgenomic replicon. The N-terminal HCV core sequence is shown downstream of 5′NTR within the reading frame of neomycin-resistant marker gene.
Fig. 2.
Fig. 2.
Specificity of PNA-neamine conjugate to its target sequence. A, 15-mer PNA-neamine conjugate complementary to the N-terminal HCV core of HCV RNA was incubated with 32P-labeled target RNA and analyzed by gel retardation assay. Lane 1, 3 nm of 32P-labeled 120-base-long RNA. Lanes 2–5 represent incubation of the labeled RNA with, respectively, 1.5, 2.0, 3, and 5 nm of PNA. B, 15-mer scrambled PNA-neamine conjugate incubated with 3 nm of labeled RNA. Lanes 1–5 represent the labeled RNA with, respectively, 0, 2, 5, 10, and 15 nm of scrambled PNA.
Fig. 3.
Fig. 3.
Binding affinity of cellular proteins to oligomeric PNA and DNA. The lysate from cured MH14 cells (HCV-negative) was incubated with 10 μm of biotinylated 15-mer PNA targeted to the HCV core coding region or biotinylated 15-mer oligo DNA containing an identical sequence. Biotin (10 μm) was included as control. After 30 min of incubation, the biotinylated PNA and DNA probes were captured on paramagnetic streptavidin beads and washed with buffer containing 0.5 m NaCl. Protein bands were visualized by staining the gel with Sypro Ruby. Lane 1, cellular proteins bound to oligomeric biotinylated DNA; lane 2, cellular proteins bound to oligomeric biotinylated PNA; lane 3, cellular protein bound to biotin.
Fig. 4.
Fig. 4.
Cellular uptake of PNA-neamine conjugates by Huh7 cells. MH14 cells grown in a monolayer were incubated at room temperature for 6 h with fluorescein-tagged 5 μm of naked PNA (A) or 2 μm of PNA-neamine (B) conjugate. The cells were then washed, detached, and resuspended at 2 × 106 cells/ml in PBS containing 2% FCS. Uptake was monitored by FACScan. Although PNA is neutral and has no charge, it is not taken up by the cell, although its neamine conjugate is efficiently internalized. C, localization of PNA-neamine conjugate in MH14 cells. Cells were incubated with 2 μm of fluorescein-tagged PNA-neamine conjugate. After 6 and 12 h of incubation, cells were washed and stained first with wheat germ agglutinin conjugated with rhodamine to label the membrane glycoproteins (red) to visualize the membrane boundary and then with DAPI to stain the nuclear DNA (blue). Uptake of PNA-neamine shows green fluorescence measured at 488 nm. Images were obtained Nikon A1R confocal microscope.
Fig. 5.
Fig. 5.
Affinity capture of HCV (+) RNA-protein complex from MH-14 cells (positive control) and cured MH14 cells (negative control). The biotinylated PNA-Nea-HCV-Core conjugate complementary to nucleotide sequence 342–356 of the HCV (+) strand RNA genome was incubated with MH14 cells (HCV-positive) (A) or cured MH14 cells (HCV-negative) (B). The conjugate that penetrated the cells and bound to its target sequence was captured from cell lysate on paramagnetic streptavidin beads. The beads were washed, suspended in SDS gel loading buffer, and heated at 90°C for 5 min. Following magnetic separation of beads, the eluate was resolved by SDS-PAGE and visualized by staining the gel with Sypro Ruby. Lane 1, cell lysate; lane 2, cell lysate supernatant flow-through following affinity capture; lane 3, affinity-captured proteins bound to biotinylated anti-HCV PNA-neamine conjugate; lanes 4 and 5, bead washes with 0.5 m NaCl in reticulocyte buffer.
Fig. 6.
Fig. 6.
Bioinformatics analysis of the identified proteins. The affinity-captured proteins identified by LC/MS/MS were matched against the Ingenuity pathway database of disease and disorder (A), molecular function (B), and canonical pathways (C) that were most significant to the set of identified proteins. The canonical pathway of the identified proteins with a p value for each pathway is indicated by the bar and is expressed as −1 times the log of the p value. The line indicated with the arrow represents the ratio of the number of genes in a given pathway that meet the cutoff criterion (p ≥ 0.05) divided by the total number of genes that make up that pathway due to chance alone. The percent of total protein matched in each category in the dataset of disease and disorder and molecular function is indicated. The gene symbol of each protein that matched against molecular function (B) and individual pathway (C) is also shown.
Fig. 7.
Fig. 7.
Modulation of HCV replication/translation by siRNA-mediated down-regulation of Stau1, ADAR1, DDX6, PA2G4, HSP60, and IGF2BP1. The control MH14 cells carrying replicating HCV replicon were grown for 24 h (lane 1) and then transfected with 20 nm of siRNA duplexes targeting individual cellular factors (lanes 4–6) or with control siRNA duplexes (lane 3). A mock transfection was also done using only transfection reagent (lane 2). Cells were grown for 72 h after transfection, and total protein and RNA were isolated. A shows controls (lanes 1–3) as well as siRNA-mediated down-regulation of targeted host cell proteins (lanes 4–6) as assessed by Western blotting, inhibition, or stimulation of HCV replication by RT-PCR (5′NTR region of the HCV genome) and HCV translation by Western blotting of HCV NS5A. B–D, respectively, show the protein bands of targeted cellular proteins, corresponding RT-PCR of HCV RNA, and expression of HCV NS5A quantified by Quantity One software (Bio-Rad).
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