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. 2015 Nov;89(21):11116-28.
doi: 10.1128/JVI.01360-15. Epub 2015 Aug 26.

Genome-Wide Screen Reveals Valosin-Containing Protein Requirement for Coronavirus Exit from Endosomes

Hui Hui Wong  1 , Pankaj Kumar  2 , Felicia Pei Ling Tay  2 , Dimitri Moreau  2 , Ding Xiang Liu  3 , Frédéric Bard  4
Affiliations

Genome-Wide Screen Reveals Valosin-Containing Protein Requirement for Coronavirus Exit from Endosomes

Hui Hui Wong et al. J Virol. 2015 Nov.

Abstract

Coronaviruses are RNA viruses with a large zoonotic reservoir and propensity for host switching, representing a real threat for public health, as evidenced by severe acute respiratory syndrome (SARS) and the emerging Middle East respiratory syndrome (MERS). Cellular factors required for their replication are poorly understood. Using genome-wide small interfering RNA (siRNA) screening, we identified 83 novel genes supporting infectious bronchitis virus (IBV) replication in human cells. Thirty of these hits can be placed in a network of interactions with viral proteins and are involved in RNA splicing, membrane trafficking, and ubiquitin conjugation. In addition, our screen reveals an unexpected role for valosin-containing protein (VCP/p97) in early steps of infection. Loss of VCP inhibits a previously uncharacterized degradation of the nucleocapsid N protein. This inhibition derives from virus accumulation in early endosomes, suggesting a role for VCP in the maturation of virus-loaded endosomes. The several host factors identified in this study may provide avenues for targeted therapeutics.

Importance: Coronaviruses are RNA viruses representing a real threat for public health, as evidenced by SARS and the emerging MERS. However, cellular factors required for their replication are poorly understood. Using genome-wide siRNA screening, we identified novel genes supporting infectious bronchitis virus (IBV) replication in human cells. The several host factors identified in this study may provide directions for future research on targeted therapeutics.

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Figures

FIG 1
FIG 1
Genome-wide RNAi screen for cellular factors affecting IBV replication. (A) Assay overview. H1299-RL was infected with rIBV-Luc, and 16 h later, both firefly and renilla luciferases were measured. (B) Genome-wide RNAi screen. Average Z scores of renilla and firefly luciferase activities plotted on x and y axes, respectively. A cutoff of 2.5 SD from nontargeting control siRNAs defines hits. (C) Workflow of the validation process. (D) Gene ontology analysis was performed on validated hits. Graphs show statistically significant (P < 0.05) enrichment of cellular components, biological processes, and molecular functions. (E) Pie chart of enriched cellular components. ER, endoplasmic reticulum; ECM, extracellular space.
FIG 2
FIG 2
Common cellular processes important in coronavirus replication. (A) RNA splicing factors and spliceosomal components among hits. (B) RNA splicing genes and viral RNA (TRS, transcriptional regulatory sequence) interaction network. (C) Interaction network between CoV-interacting factors and hits. UTR, untranslated region. (D) Membrane-associated genes present in the screen. (E) Interactions between ubiquitin-proteasome pathway hits and coronavirus-interacting partners.
FIG 3
FIG 3
Depletion in VCP leads to decreased human coronavirus 229E (HCoV-229E) replication. (A) Huh7 cells transfected with nontargeting siRNA (siNT) or VCP siRNA (siVCP) were observed for cytopathic effect under a light microscope after 48 h of HCoV-229E infection (top panel). Cells were also then fixed, infected, and stained for dsRNA expression and nuclei by Hoechst staining (bottom panel). FITC, fluorescein isothiocyanate. (B) Viral genomic RNA (+gRNA) levels were then analyzed using quantitative real-time PCR. Error bars represent standard deviations obtained from two independent experiments performed in triplicate. (C) The level of nucleocapsid (N) expression in HCoV-229E-infected cells was probed using cross-reactive antibodies raised against IBV-N. (D) The titer of viral particles released into the respective culture supernatant was first determined with the method of TCID50 per milliliter and then confirmed via standard plaque assay.
FIG 4
FIG 4
Role of valosin-containing protein (VCP) in early virus replication. (A) Effect of VCP depletion on infectious bronchitis virus (IBV)-Luc replication at 10 or 16 hpi. Error bars represent SDs from average luciferase activity from two independent experiments with duplicates. (B) Effect of VCP depletion on dsRNA production, measured at 8 hpi with a specific monoclonal antibody. Cells were also stained with anti-IBV-S antibody, and nuclei were stained with Hoechst stain. DAPI, 4′,6-diamidino-2-phenylindole. (C) Effect of VCP knockdown on IBV-N expression at 8 and 16 hpi as measured by Western blotting. (D and E) Role of VCP in virion attachment. Attached viral particles were measured by counting the number of CFU by standard plaque assay (D) or via the detection of genomic RNA (+gRNA) levels by RT-PCR (E). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was measured as a loading control. (F and G) Role of VCP in virus entry. Internalized virus particles were quantified by plaque assay (F) as in panel D or by RT-PCR (G). NT, nontargeting.
FIG 5
FIG 5
Role of valosin-containing protein (VCP) in early-stage degradation of N protein. (A) Representative blot of IBV-N in VCP-depleted or control-treated (NT) cells infected with IBV for 4 or 8 h. Intensities of the bands were quantified and represented as an average of data from two independent experiments. (B) Comparison of effects of VCP and GBF1 depletion on IBV-N levels. (C) Effect of cycloheximide (CHX; 100 μg/ml) on IBV-N accumulation. β-Tubulin was used as a loading control. sgRNA, subgenomic RNA. (D) Effect of DMSO, MG132 (10 μM), brefeldin A (BFA; 5 μg/ml), Golgicide A (GCA; 10 μg/ml), or bafilomycin A1 (Baf-A1; 1 μM) applied 2 h prior to infection with IBV (MOI, 0.5). The cell lysate was probed for IBV-N as well as actin as a loading control.
FIG 6
FIG 6
Valosin-containing protein (VCP) depletion leads to accumulation of viral particles in early endosome vesicles. (A) H1299 cells transfected with nontargeting or anti-VCP siRNA (siNT or siVCP, respectively) were infected with recombinant IBV expressing Flag-tagged N protein (rIBV-FlagN) for 4 h, fixed, permeabilized, and stained for EEA1 (tetramethyl rhodamine isocyanate [TRITC], red) or anti-Flag (fluorescein isothiocyanate [FITC], green). A magnified view of colocalized particles is provided in the square inset. (B to D) Subcellular fractionation of IBV-N in VCP-silenced or siNT-treated cells using step sucrose gradient flotation. (B) Western blot analysis of whole-cell lysate (WCL). (C) RT-PCR against viral genomic RNA. (D) Western blot analysis of sucrose fractions against IBV-N, IBV-S, EEA1, and actin. Results are representative of three independent experiments. Tubulin and actin were used as loading controls.
FIG 7
FIG 7
Cellular map illustrating predominant subcellular localization and presumed functions of hits. We proposed that depletion of VCP prevents virus particles from exiting endosomes (1) and hence inhibits the subsequent uncoating and N protein degradation event (2).
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