Viral protein engagement of GBF1 induces host cell vulnerability through synthetic lethality

Abstract

Viruses co-opt host proteins to carry out their lifecycle. Repurposed host proteins may thus become functionally compromised; a situation analogous to a loss-of-function mutation. We term such host proteins viral-induced hypomorphs. Cells bearing cancer driver loss-of-function mutations have successfully been targeted with drugs perturbing proteins encoded by the synthetic lethal partners of cancer-specific mutations. Synthetic lethal interactions of viral-induced hypomorphs have the potential to be similarly targeted for the development of host-based antiviral therapeutics. Here, we use GBF1, which supports the infection of many RNA viruses, as a proof-of-concept. GBF1 becomes a hypomorph upon interaction with the poliovirus protein 3A. Screening for synthetic lethal partners of GBF1 revealed ARF1 as the top hit, disruption of which, selectively killed cells that synthesize poliovirus 3A. Thus, viral protein interactions can induce hypomorphs that render host cells vulnerable to perturbations that leave uninfected cells intact. Exploiting viral-induced vulnerabilities could lead to broad-spectrum antivirals for many viruses, including SARS-CoV-2.

Summary: Using a viral-induced hypomorph of GBF1, Navare et al., demonstrate that the principle of synthetic lethality is a mechanism to selectively kill virus-infected cells.

Figure 1.. Extending the principle of synthetic lethal interactions to a virus-induced hypomorph.

(A) Synthetic lethality is an extreme negative genetic interaction occurring between two genes. Here, genes ‘A’ and ‘B’ are not essential, and the cell remains viable upon the loss of either gene, depicted by red dotted outline of ‘A’ or ‘B’, individually. However, when these deletions are combined in a single cell, as visualized in the third panel, this double loss of function critically impairs the cell, resulting in its death. Such gene-gene combinations are termed synthetic lethal (SL) partners. (B) The principle of synthetic lethality has been successfully exploited in the development of certain cancer therapies by targeting the synthetic lethal partner of the cancer-causing oncogene, depicted by a red ‘A’. In the cancerous cell, gene ‘A’ has been mutated, depicted as ‘A*’, leading to an enhanced dependency by the cancer cell for its synthetic lethal partner ‘B’. Drugs that target the otherwise nonessential gene B induce cell death when combined with its SL partner, A*. Therefore, inhibiting the function of B can selectively kill cancerous cells while sparing noncancerous bystander cells. (C) Like the example in cancer (B), a viral infection provides opportunities for specifically targeting infected cells by synthetic lethality. When a cell is infected, host factors, depicted as the red letter ‘A’, are recruited by viral proteins to support viral reproduction. The normal function of the host factor is thus attenuated by the presence of the virus, inducing a hypomorph, red letter ‘A’, which sensitizes the infected cell to inhibition of its synthetic lethal partner by an inhibitory drug.

Figure 2.. A chemogenomic screen identifies synthetic lethal partners of GBF1.

(A) A schematic of the experimental design for chemogenomic screening with the GBF1 inhibitor golgicide A (GCA). A CRISPR extended knockout (EKO) library of NALM6-Cas9 cells was treated with 2 μg/ml doxycycline to induce individual gene knockouts via Cas9 expression. The pooled library was split into individual flasks and grown over 8 days period in the presence or absence of 4 μM golgicide A (GCA). Following incubation, guide RNA frequencies were measured using Illumina sequencing, and log₂ fold changes between GCA and control samples were compared. (B) A plot of relative sgRNA frequencies of all genes showing genes passing a 0.05 FDR cutoff in white circles. The 53 genes with negative sgRNA fold change from GCA treatment represent putative synthetic lethal interactors of GBF1. The 17 genes with overrepresented sgRNAs and positive sgRNA fold change represent GBF1 suppressors that may confer protection against GCA. (C) Gene ontology functional enrichment analysis of synthetic lethal partners of GBF1. The 53 putative synthetic lethal partners of GBF1 were analyzed in clusterProfiler against the entire KO gene collection from the CRISPR library to functionally classify the SL genes. Significantly enriched gene ontologies are plotted and ranked by their −log₁₀, FDR-adjusted enrichment p-value. The number of putative synthetic lethal genes in each gene ontology is coded by the heatmap and ranges from 3 (yellow) to 9 (pink). (D) A combined PPI network of the 53 synthetic lethal interactors of GBF1 (green circles) and the 17 GBF1 suppressors (orange circles) was obtained from the STRING database and visualized using Cytoscape. Edges between two circles denote evidence-based interaction between the connecting proteins. Circles with red outlines highlight known targets of viral proteins, as per the VirHostNet (v2.0) virus-host PPIs database. Gene names of the proteins and their gene ontology functions are color matched.

Figure 3.. Validation of putative synthetic lethal interactions in HeLa cells.

(A) ARF1 displays a robust synthetic lethal interaction with GBF1. ARF1, HSP90, CSK, PRKAA1, the control gene MSMO1, and the top GBF1 suppressor gene ARF4 were silenced in HeLa cells with shRNA-mediated lentivirus transductions and incubated with 1.5 or 4 μM golgicide A (GCA) or DMSO for 48 h. CellTiterBlue reagent was added and fluorescence measurements were collected. Live cells metabolize the reagent into fluorescent products and an increase in the fluorescence signal is directly proportional to the number of living cells. The percent viability at each GCA concentration was calculated by dividing the fluorescence from a GCA-treated sample by its matched DMSO-treated control. Changes in cell viabilities for each knockdown (KD) cell line were determined by comparing the respective percent viabilities to the MSMO1 KD control using a Brown Forsythe and Welch ANOVA multiple comparison test (Brown and Forsythe, 1974; Welch, 1951), with statistically significant differences are indicated as: * if p-value < 0.01; ** if p-value < 0.001; *** if p-value <0.0001. Error bars represent the SEM from three biological replicates. (B) ARF1 KD cells show enhanced sensitivity in a GCA dose-response curve. A GCA or DMSO working solution (200 μM) was serially diluted and co-plated with 20,000 cells per well of ARF1 KD and MSMO1 KD cells in a 96-well plate, with final GCA or DMSO concentrations ranging from 0 – 100 μM. After 48 h, cell viability was measured with CellTiterBlue and the normalized fluorescence, relative to the DMSO-treated samples, was calculated using the smallest and largest mean values to define 0% and 100%, respectively. A dose reponse curve of the normalized fluorescence was plotted against the log₁₀ GCA concentration and IC₅₀ values were calculated using the a four parameter logistic regression model in Graphpad. Error bars represent the SEM from three biological replicates.

Figure 4.. Poliovirus nonstructural protein 3A induces a vi-hypomorph of GBF1.

(A) Poliovirus 3A physically interacts with GBF1. HeLa cells were transfected with FLAG* tagged poliovirus 3A or an empty control plasmid for 24 h. Equal amounts of lysates were prepared, and immunoaffinity enriched for bound protein complexes to 3A-FLAG* protein. Affinity captured proteins were eluted and resolved on SDS-PAGE along with 1% of the total input lysate and the final wash. Resolved proteins were transferred to a nitrocellulose membrane and immunoblotted using anti-GBF1 (top panel) and anti-FLAG (bottom panel) antibodies. (B) Poliovirus 3A redistributes GBF1 away from its perinuclear localization. HeLa cells transfected with FLAG* tagged poliovirus 3A or an empty control plasmid were fixed, stained with fluorescently labeled antibodies against FLAG and GBF1, and imaged by wide-field fluorescence microscopy. Bar 5 μM. (C-D) Images of GBF1 were analyzed and the distances of each GBF1 puncta to the nearest nucleus was determined and plotted across the entire distance range (C) and between 20 μM to 40 μM (D) for 42 control cells and 13 cells transfected with 3A-FLAG*. The corresponding box plots show statistically significant differences in GBF1 distribution between the two samples with *** representing a p-value <0.0001.

Figure 5.. Synthetic lethal killing of a vi-hypomorph of GBF1.

(A) Expression of poliovirus 3A-FLAG* in ARF1 and MSMO1. Each gene was stably silenced in HeLa cells and transfected with FLAG*-tagged poliovirus 3A. The expression levels of 3A-FLAG 48 h post transfection were measured by flow cytometry. (B) Poliovirus 3A induces cell death in ARF1 KD cells. Cell viabilities of ARF1 KD and MSMO1 KD cells transfected with 3A-FLAG* or an empty plasmid control were measured using the fluorescence readout of CellTiterBlue. A change in fluorescence is directly proportional to the number of living cells, and a decrease in the absolute fluorescence indicates reduced cell viability. Percent viabilities of ARF1 KD and MSMO1 KD cells were calculated by dividing the absolute fluorescence values of 3A-transfected samples by the matched empty-transfected samples. Multiple t-test was used to compare percent viability between the 3A-treated ARF1 KD and MSMO1 KD cells with ****** representing P-value < 0.000001. Error bars represent the SEM from six biological replicates.