Quantitative proteomic analysis of host-virus interactions reveals a role for Golgi brefeldin A resistance factor 1 (GBF1) in dengue infection

Abstract

Dengue virus is considered to be the most important mosquito-borne virus worldwide and poses formidable economic and health care burdens on many tropical and subtropical countries. Dengue infection induces drastic rearrangement of host endoplasmic reticulum membranes into complex membranous structures housing replication complexes; the contribution(s) of host proteins and pathways to this process is poorly understood but is likely to be mediated by protein-protein interactions. We have developed an approach for obtaining high confidence protein-protein interaction data by employing affinity tags and quantitative proteomics, in the context of viral infection, followed by robust statistical analysis. Using this approach, we identified high confidence interactors of NS5, the viral polymerase, and NS3, the helicase/protease. Quantitative proteomics allowed us to exclude a large number of presumably nonspecific interactors from our data sets and imparted a high level of confidence to our resulting data sets. We identified 53 host proteins reproducibly associated with NS5 and 41 with NS3, with 13 of these candidates present in both data sets. The host factors identified have diverse functions, including retrograde Golgi-to-endoplasmic reticulum transport, biosynthesis of long-chain fatty-acyl-coenzyme As, and in the unfolded protein response. We selected GBF1, a guanine nucleotide exchange factor responsible for ARF activation, from the NS5 data set for follow up and functional validation. We show that GBF1 plays a critical role early in dengue infection that is independent of its role in the maintenance of Golgi structure. Importantly, the approach described here can be applied to virtually any organism/system as a tool for better understanding its molecular interactions.

Fig. 1.

Workflow for identifying high confidence interactors of DENV NS5 and NS3 and typical I-DIRT immunoaffinity purification (IP). A, Schematic diagram of workflow, including incorporation of the I-DIRT technique. Metabolically heavy cells were generated by passaging in medium containing exclusively heavy lysine and arginine (Lys8 and Arg10) until incorporation was verified to be >95%. In parallel, metabolically light cells were maintained in identical medium, with the exception that light lysine and arginine were substituted for their isotopically heavy counterparts. For I-DIRT IPs, only light cells were transfected with constructs driving the expression of GFP-tagged viral baits, or the GFP tag alone as an additional control. In parallel, heavy cells were mock transfected. As soon as transfection mixtures could be removed, all cells (heavy and light) were infected with DENV. At 24 h post-transfection (19 h postinfection), cell lysates were made and their protein contents quantitated. For the I-DIRT IPs, equal protein amounts of heavy and light lysates were thoroughly mixed, and an aliquot was reserved for determination of protein ratio inputs. Then, rapid IPs of tagged protein complexes were performed using anti-GFP antibodies, cross-linked to Protein G Dynabeads, and the heavy/light lysate mixtures. After washing the beads, proteins associated with the GFP-tagged viral baits were eluted and resolved by SDS-PAGE. In-gel trypsin digestion and C18 clean-up were then performed in preparation for LC-MS/MS. After database searching and analysis, the cumulative distributions of the light to heavy ratios in the mixed lysate inputs were used to identify the cutoff threshold ratio that included 95% of the input proteins. In our analysis, high confidence interactors were considered to be proteins that passed this cutoff in each of three independent experiments. B, Typical I-DIRT immunoaffinity purification. Mixed lysate inputs and proteins associated with GFP-tagged protein complexes, obtained in a typical I-DIRT IP, were resolved by SDS-PAGE and stained with Coomassie dye. Lanes 1–3, 0.1% of mixed lysate inputs: lane 1, GFP input; lane 2, NS5-GFP input; lane 3, NS3-GFP input. Lanes 4–6, 67% of eluates: lane 4, GFP IP; lane 5, NS5-GFP IP; and lane 6, NS3-GFP IP. Asterisks designate GFP and the GFP-tagged viral baits.

Fig. 2.

High confidence data sets of NS5-GFP and NS3-GFP interactors. A, NS5-GFP interactors. The background distributions of the light to heavy ratios of proteins in the mixed lysate input of each NS5-GFP IP are shown in green triangles. Each biological replicate is shown in a different shade of green. Associated host proteins (53 in total) whose light to heavy ratio was higher than that of the appropriate cutoff (supplemental Fig. S3) in each of three individual experiments are shown in orange circles with purple borders (average ratios and intensities of three biological replicates). The NS5-GFP bait is also shown. B, NS3-GFP interactors. The background distributions of the light to heavy ratios of proteins in the mixed lysate input of each NS3-GFP IP are shown in blue triangles. Each biological replicate is shown in a different shade of blue. Associated host proteins (41 in total) whose light to heavy ratio was higher than that of the appropriate cutoff (supplemental Fig. S3) in each of three biological replicates are shown in yellow circles with purple borders (average ratios and intensities of three biological replicates). The NS3-GFP bait is also shown. x axis, light to heavy protein ratio; y axis, intensity (the sum of the extracted ion currents of all isotopic clusters assigned to each particular amino acid sequence). To obtain the average light to heavy ratios of the GFP-tagged baits, the light to heavy ratios of GFP and either NS5 or NS3 from each experiment were averaged. Average intensities were obtained similarly.

Fig. 3.

Reciprocal co-immunoprecipitation validation of a panel of high confidence NS5 and NS3 interactors. A, Validation of 5/5 high confidence NS5 interactors. Cell lysates were prepared from DENV-infected HepG2 cells, and immunoprecipitations with antibodies cross-linked to Protein G-Dynabeads (25 μl beads/immunoprecipitation; anti-GBF1, anti-SLIRP, anti-XPOT, anti-CAD, anti-HSP90α, and rabbit IgG control) were performed as in Fig. 1. After extensive washing, bound proteins were eluted in loading sample buffer, resolved by SDS-PAGE, and transferred to nitrocellulose membranes. Membranes were then immunoblotted with the designated antibodies. Left panel: Amounts of NS5 present in immunoprecipitation inputs and amounts of NS5 associated with immunoprecipitated endogenous proteins or rabbit IgG controls. Right panel: Efficiencies of antibodies in immunoprecipitations their target antigens. Left lanes: immunoprecipitation inputs; middle lanes: rabbit IgG controls; right lanes: immunoprecipitations with the designated antibodies. Results shown are representative of at least three independent experiments for each antibody. B, Validation of 6/7 high confidence NS3 interactors. As in A, except the following antibodies were used: anti-IPO4, anti-HSP27, anti-SERPINH1, anti-ZW10, anti-HSP90β, anti-HSP70, and rabbit or mouse IgG control. Left panel: Amounts of NS3 present in immunoprecipitation inputs and amounts of NS3 associated with immunoprecipitated endogenous proteins or IgG controls. Right panel: Efficiencies of antibodies in immunoprecipitating their target antigens. Left lanes: immunoprecipitation inputs; middle lanes: IgG controls; right lanes: immunoprecipitations with the designated antibodies. All inputs and eluates shown in a horizontal row are from the same film exposure. Results shown are representative of at least three independent experiments for each antibody. C, Efficiency of DENV infection in a panel of KD cell lines. A panel of 8 KD cell lines, each of which inducibly expressed shRNA against a high confidence NS5 and/or NS3 interactor, was generated by lentiviral transduction. Cells were incubated in doxycycline (2 μg/ml) for 96 h to induce shRNA expression and then infected with DENV (moi = 1). Intracellular RNA was harvested at 24 h postinfection and quantified by qPCR. Gray bars designate the levels of DENV replication (NS2A levels) in each KD cell line. Results are expressed as means ± S.D. (n = 3) of relative quantification values, normalized to the internal control RPL13A. The level of DENV RNA in control cells expressing nonsilencing shRNA was set to 1. D, Efficiency of host gene silencing in the panel of KD cell lines. The extent to which the appropriate host target was silenced in each KD cell line shown in C, was determined by qPCR as above. Each colored bar designates a different host target. Results are expressed as in C, with the level of target RNA in control cells expressing nonsilencing shRNA set to 1.

Fig. 4.

Interaction networks for NS5-GFP and NS3-GFP. The 53 high confidence NS5-GFP interactors (A) and the 41 high confidence NS3-GFP interactors (B) were entered into GeneMANIA to generate interaction networks, using the settings described in the Experimental Procedures section. Significantly enriched GO annotations of interest, and their corresponding proteins, are labeled. Note that occasionally a protein belongs to two or more significantly enriched annotations; in these cases, the interactor is assigned only one color for clarity. Proteins not belonging to any of the selected enriched annotations are represented by black circles. Pink lines designate physical interactions identified by GeneMANIA. The complete list of all significantly enriched GO annotations for each interaction network is shown in supplemental Table S4.

Fig. 5.

GBF1 plays an essential and specific role early in DENV infection. A, Pharmacological inhibition of GBF1 inhibits DENV infection. HepG2 or HEK293 cells were infected with DENV for 1 h (moi = 2–5) and then treated with DMSO, BFA (5 μg/ml), or GCA (10 μm). After 16 h, the levels of DENV NS2A were quantified by qPCR and normalized to the internal control RPL13A. The amount of DENV RNA in DMSO-treated cells was set to 1. Data are expressed as means ± S.D. of relative quantification values of NS2A (n = 3 for HepG2; n = 4 for HEK293). B, GCA-mediated inhibition of DENV infection is rescued on the RNA level by overexpression of GBF1_WT and a drug-insensitive version thereof (GBF1_M832L). HEK 293-T cells were transfected with pCI, pCI-GBF1_WT, or pCI-GBF1_M832L. At 24 h post-transfection, cells were reseeded into 6-well plates. The following day, cells in representative wells were counted to ensure that infections were set up at the same moi. After infection with DENV (moi = 1–2), cells were incubated in medium with either DMSO or GCA (10 μm) for 16 h. DENV RNA was then quantified as in A. For each condition (pCI, pCI-GBF1_WT, or pCI-GBF1_M832L), the relative quantification value of DENV NS2A in DMSO-treated cells was set to 1. Results are expressed as means ± S.D. of relative quantification values of NS2A (n = 4). * = p < 0.05. C, GCA-mediated inhibition of DENV infection is rescued on the protein level by overexpression of GBF1_WT and a drug-insensitive version thereof (GBF1_M832L). Experiments were performed as in B, except that equal amounts of protein in cell lysates were resolved by SDS-PAGE and transferred to nitrocellulose membranes. Membranes were then probed with anti-GBF1, anti-NS5, and anti-tubulin antibodies. D, NS5 interacts with GBF1, but not BIG1. HepG2 cells were infected with DENV (moi = 2) for 1 h, and cell lysates were prepared at 24 h postinfection. Immunoprecipitations were performed with the following antibodies: rabbit IgG (control), rabbit polyclonal anti-BIG1, and rabbit polyclonal anti-GBF1. Coimmunoprecipitated proteins were analyzed by immunoblotting the eluates with anti-NS5, anti-BIG1, and anti-GBF1 antibodies. Inputs and eluates shown in the same horizontal row are from the same film exposure. A representative result from three independent experiments is shown. E, Golgicide A does not inhibit DENV internalization. HepG2 cells were prechilled at 4 °C and then infected with DENV (moi = 3) for 1 h at 4 °C to allow virion adsorption. After 1 h, viral inoculum was removed and replaced with prewarmed medium with either DMSO or GCA (10 μm). After a 30 min incubation to allow virus internalization, bound (i.e. uninternalized) virus was removed via several washes with PBS and a 2 min incubation with a high-salt, high-pH solution at 0 °C. Intracellular DENV RNA was then quantified as in A. Data are expressed as means ± S.D. (n = 3). F, DENV replication is most sensitive to GCA at early time points in infection. HepG2 cells were prechilled at 4 °C and then infected with DENV (moi = 5–10). Virus was adsorbed for 1 h at 4 °C, and cells were washed to remove virus input. Inoculum was replaced with prewarmed medium (24, 36, and 48 h samples) or medium containing DMSO or GCA (10 μm) (12 h samples). For the 12 h sample, cells were incubated 12 h in the presence of DMSO or GCA, and then intracellular RNA was harvested. When the 12 h sample was harvested, the cells in the 24 h sample were washed to remove extracellular virus, and medium with either DMSO or GCA was added as for the 12 h sample. This process was repeated at 12 h intervals until the final harvest. DENV RNA was then quantified as in A. The normalized level of NS2A in DMSO-treated cells at 12 h was set to 1 for each experiment, and levels of NS2A in all other conditions are expressed relative to this value. Data are expressed as means ± S.D. (n = 4). * = p < 0.05. n.s., not significant. RQ, relative quantification.

Fig. 6.

DENV infection of GBF1-KD cells is sensitized to GCA in a manner independent of Golgi disruption. A, DENV infection in GBF1-KD HepG2 cells. HepG2 cells were transduced with nonreplicating lentiviral vectors harboring either nonsilencing control shRNA, or shRNA targeting GBF1. Stable cell lines were obtained with puromycin selection (1.5 μg/ml) and were generated fresh for each experiment. After induction of shRNA expression with doxycycline (2 μg/ml for 48 h), the cells in at least two representative wells of each cell line were detached by trypsinization and counted at least in duplicate to ensure that infections were set up at the same moi. Cell viabilities were also determined by trypan blue exclusion and determined to be > 95% at the time of infection in all experiments (data not shown). Cells were then infected with DENV (moi = 1) and incubated for 16 h, after which time intracellular RNA was extracted and/or cell lysates were made. After synthesis of cDNA, the amounts of DENV NS2A and GBF1 were quantified by qPCR and normalized to the internal control RPL13A. The relative quantification amount of either NS2A or GBF1 in the nonsilencing control was then set to 1. Data are expressed as relative quantification means ± S.D. (n = 3 for each experiment). Although relative quantification results are shown, t-tests were performed on the 2^ΔC_T values, as described under “Experimental Procedures.” Left panel: DENV infection in GBF1-KD cells versus DENV infection in control cells. Horizontal bars represent means; individual experiments are shown with black triangles. Vertical error bars represent ± S.D. (n = 3). n.s. = not significant. Right lower panel: Extent of GBF1 KD in GBF1-KD cells. Means ± S.D. (n = 3) are shown for the cells used in the left panel. Upper right panel: Equal amounts of protein were resolved by SDS-PAGE and transferred to nitrocellulose membranes. Membranes were then probed with anti-GBF1 antibodies. n.s., not significant; RQ, relative quantification. B, GBF1-KD cells are sensitized to GCA-mediated inhibition of DENV infection. HepG2 cells with stable integration of tetracycline-inducible shRNA directed against GBF1 were generated as above. Cells were then incubated either in the absence or presence of doxycycline (2 μg/ml) for 48 h. Before DENV infection, the cells in at least two representative wells of each treatment were detached by trypsinization and counted at least in duplicate to ensure that infections were set up at the same moi. Cells were then infected with DENV (moi = 1–2), after which time viral inoculum was removed and replaced with medium containing DMSO or GCA (0.1 μm, 0.5 μm, 1 μm, 5 μm, or 10 μm). Cells were incubated for 16 h, after which time intracellular RNA was extracted. After synthesis of cDNA, DENV NS2A RNA and GBF1 mRNA were quantified by qPCR and normalized to the internal control RPL13A. DENV NS2A values were expressed as relative quantification values, with the amount of NS2A in DMSO-treated cells set to one for each cell line in each experiment. Half maximal inhibitory concentration (IC₅₀) values were obtained from log₁₀[GCA] and relative NS2A values using GraphPad Prism, as described under “Experimental Procedures.” Error bars represent ± the standard error of the mean. Values shown represent the IC₅₀ values obtained using all replicates of each experiment (n = 4, 0 μg/ml; n = 3, 2 μg/ml) ± the S.D. of the IC₅₀ values obtained by calculating the IC₅₀ value of each experiment individually. The IC₅₀ values were significantly different between the two cell lines (p = 0.031, unpaired two-tailed t test, unequal variance, performed using the IC₅₀ values). The R² values for each curve were: 0.64, control cells; 0.91, GBF1-KD cells. Doxycycline-mediated induction of shRNA expression efficiently reduced the level of GBF1 mRNA to ∼30% that of non-induced cells (right panel). C, GCA-mediated inhibition of DENV infection is not caused by general Golgi disruption. Cells from the experiment described in Fig. 6B, incubated in the absence of doxycycline, were seeded onto cover slips and treated with DMSO or GCA (1 μm). After 16 h, cells were fixed with formaldehyde, permeabilized, and immunolabeled with anti-GM130 primary antibodies followed by Alexa Fluor 488-conjugated anti-mouse secondary antibodies. Slides were mounted in SlowFade Gold reagent and visualized on a DeltaVision microscope (40x magnification; green, GM130; blue, nuclei). Because of the difference in visualizing punctate versus diffuse structures, the intensity settings were not the same for cells treated with DMSO versus 1 μm GCA. Using the same settings, GM130 could not be visualized in the 1 μm GCA-treated cells without saturating the GM130 in the DMSO-treated cells. Results shown are representative of two experiments in which cells were incubated in the presence of drugs for 16 h and one experiment in which cells were incubated in the presence of drugs for 1.5 h. Scale bars, 20 μm.