The SKI complex is a broad-spectrum, host-directed antiviral drug target for coronaviruses, influenza, and filoviruses

Abstract

The SARS-CoV-2 pandemic has made it clear that we have a desperate need for antivirals. We present work that the mammalian SKI complex is a broad-spectrum, host-directed, antiviral drug target. Yeast suppressor screening was utilized to find a functional genetic interaction between proteins from influenza A virus (IAV) and Middle East respiratory syndrome coronavirus (MERS-CoV) with eukaryotic proteins that may be potential host factors involved in replication. This screening identified the SKI complex as a potential host factor for both viruses. In mammalian systems siRNA-mediated knockdown of SKI genes inhibited replication of IAV and MERS-CoV. In silico modeling and database screening identified a binding pocket on the SKI complex and compounds predicted to bind. Experimental assays of those compounds identified three chemical structures that were antiviral against IAV and MERS-CoV along with the filoviruses Ebola and Marburg and two further coronaviruses, SARS-CoV and SARS-CoV-2. The mechanism of antiviral activity is through inhibition of viral RNA production. This work defines the mammalian SKI complex as a broad-spectrum antiviral drug target and identifies lead compounds for further development.

Keywords: antiviral host factor; broad-spectrum antiviral; coronavirus; filovirus; influenza.

Fig. 1.

Yeast suppressor screening identifies the SKI complex as a suppressor of NS1 and ORF4a-mediated slow growth. (A) Yeast knockouts for each component of the SKI complex were transformed with the NS1 galactose-inducible expression plasmid or empty vector control (EV). Growth rate of these yeast was measured over a 48 h culture period. Mean OD600 between three independent colonies in two independent experiments is plotted with error bars being the SD. (B) As in A, but with an ORF4a expression plasmid. (C) HEK293T cells were cotransfected with plasmids to express HA-tagged human SKI genes (SKIV2L-HA, TTC37-HA, or WDR61-HA) with either IAV NS1-GFP or MERS-CoV ORF4a-GFP. After transfection, cells were lysed with RIPA lysis buffer and used to analyze whole cell lysate (WCL) or protein was used for HA immunoprecipitation, analyzing nonspecific bead binding (NS), flow through for unbound protein (FT) and the immunopreciptate (IP). In all cases, samples were separated by SDS/PAGE and Western blotted (WB) for GFP, HA, or tubulin (as loading and immunoprecipitation clearance control).

Fig. 2.

Knockdown of the SKI complex by siRNA inhibits replication of IAV and MERS-CoV. (A) A549 cells were transfected with siRNAs targeting the different components of the SKI complex using two unique sequences for each of the three target genes along with scrambled and mock controls. After 3 d of transfection, cells were infected with IAV at MOI 0.01. After 24 h, supernatant was collected, and viral titer assessed by plaque assay. Plotted is the mean plaque forming units (PFU)/mL from three independent experiments with error bars being SD. (B) As in A but with Huh7 cells and MERS-CoV infection at MOI 0.1. Virus titer was determined by TCID50 assay. Plotted is the mean TCID50/mL from three independent experiments with error bars being SD. (C) A third siRNA sequence for each of the three SKI genes was transfected into A549 cells for 3 d, at which point the cells were infected and assessed as in A. Plotted is a representative experiment of two showing the mean PFU/mL from triplicate wells of infection. (D) As in C but MERS-CoV infection of Huh7 cells. (E) A549 cells were transfected for 3 d as described and collected in TRIzol for qRT-PCR analysis of each of the SKI genes being targeted by siRNA (all three unique sequences). Data are a representative experiment of three performed in triplicate wells. PCR reads were normalized with GAPDH and fold change was set relative to scrambled siRNA transfected cells. (F) A549 and Huh7 cells were transfected with SKIV2L targeting siRNA (sequences 1 and 2) for 3 d prior to collection in RIPA lysis buffer. Samples were Western blotted for SKIV2L or tubulin as a loading control. Data are representative of two independent repeats. (G) A549 and (H) Huh7 cells were transfected with siRNAs targeting the SKI complex and cell viability was assessed over the 3-d period by CellTiter-Glo assay. Data are the mean relative luminescence set relative to scrambled control from a representative experiment performed in quadruplicate of three (A549) or two (Huh7) independent experiments. In all cases, t tests were performed for Scr control vs. siRNA transfected cells; ns, non-significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 3.

Modeling compounds to bind to the SKI complex and screening for antiviral activity. The 3D structure of SKI8 was subjected to SILCS simulations from which the (A) FragMaps (mesh representations for apolar [green, −0.9 kcal/mol], hydrogen-bond donor [blue, −0.6 kcal/mol], hydrogen bond acceptor [red, −0.6 kcal/mol], positive [cyan, −1.2 kcal/mol], and negative [orange −1.2 kcal/mol] functional groups) were calculated and the binding hotspots determined (all hotspots as blue spheres with those defining the identified binding site as larger spheres colored by ranking (low to high as blue to red). (B) Expanded view of putative binding site. (C) Putative binding site with the FragMaps, the pharmacophore features (spheres, aromatic [cyan] and hydrogen-bond donor [blue]), and the SILCS Monte Carlo conformational sampling docked orientation. Compounds predicted to bind were purchased and screened for antiviral activity. A549 cells were infected with IAV and treated with compounds as indicated. Virus was collected, and PFU/mL determined. (D) Compounds 1 to 20 were tested with single wells of infection. The 50 µM data are from one experiment, 10 µM data are from two independent experiments with error bars being SD. Dotted line to denote the DMSO control PFU/mL for ease of visualization. N.B. Compound 6 did not arrive with the order, it has not been deliberately omitted. (E) Compounds 21 to 40, data from two independent experiments with error bars being SD. UMB18 was included as a positive control. (F) Structural variants of UMB18 were investigated, data are from two independent experiments with error bars being SD. UMB18-2 was also blindly listed as UMB40. (G–J) Chemical structures of the four lead compounds.

Fig. 4.

SKI targeting compounds have antiviral activity against IAV and MERS-CoV. (A) A549 cells were infected with IAV at MOI 0.01 and treated with UMB18 at the indicated concentrations for 24 h. Based on the stock of compound, 0.5% DMSO acted as the vehicle control for 50 µM and 25 µM while 0.1% acted as the control for all other concentrations. Virus was collected after 24 h, and PFU/mL determined by plaque assay. Data are from three independent experiments performed in triplicate with mean PFU/mL displayed and error bars representing SD. (B) Huh7 cells were infected with MERS-CoV at MOI 0.1 and treated with UMB18 as in A. Virus was collected and titer determined by TCID50 assay. Data are from three independent experiments as in A. (C and D) Cells infected as in A and B but treated UMB18-2. Data are from a representative experiment of two, each performed in triplicate. (E) A549 cells were treated with UMB28 or UMB36 and compared with UMB18 at either 50 µM or 10 µM (with 0.5% or 0.1% DMSO being the appropriate negative controls) and infected with IAV at MOI 0.01. Virus was collected after 24 h, and PFU/mL determined by plaque assay. Data are from three independent experiments (one of a single well and two of triplicate wells) with the mean PFU/mL displayed and error bars being the SD. (F) As in E, but Huh7 cells were treated and infected with MERS-CoV at MOI 0.1 for 24 h. Virus titer was determined by TCID50 assay. Data are from three independent experiments all of triplicate wells with the mean TCID50/mL displayed and error bars being the SD. (G) A549 cells were siRNA transfected with sequence 3 of each of the SKI targeting siRNAs as used in Fig. 2C. Following the 3-d transfection, cells were infected with IAV (MOI 0.01) in the presence of 10 µM or 0.1% DMSO for 24 h. Virus was collected and PFU/mL determined by plaque assay. Data are from two independent experiments performed in triplicate wells. In all cases, except G, t tests were performed for vehicle control vs. drug-treated samples; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. For G, a two-way ANOVA Bonferroni multiple comparisons test was performed; NS, nonsignificant, *P < 0.05.

Fig. 5.

UMB18 inhibits filovirus infection. Huh7 cells were treated with UMB18 for test, with toremifene citrate (TOMF) as a positive control and DMSO as a negative control. Treatments were over an 8-point dose curve with threefold dilutions, each in triplicate. Cells were infected with (A) Ebola virus Makona strain (EBOV) or (B) Marburg virus Angola strain (MARV) for 48 h. Cells were fixed and labeled with antibodies to VP40 for each virus. Infected cells were detected by peroxidase secondary labeling to determine the percentage inhibition of infection by each treatment. Cytotoxicity is also displayed which was determined by CellTiter-Glo assay on uninfected samples. Data are from one representative experiment of two performed in triplicate. Dotted line is at 50% inhibition for determining IC₅₀ values.

Fig. 6.

SKI targeting lead compounds inhibit viral RNA and protein production. Time-of-addition experiments were performed with IAV infection and UMB18 (A) and UMB18-2 (B). A549 cells were plated and treated with drug 2 h prior to infection (−2 h), at the time of infection (0 h), or 2 h after virus was added to cells (+2 h). After 24 h infection supernatant was collected and titer determined by plaque assay. Mean PFU/mL and SD are displayed from two independent experiments performed in triplicate with error bars being SD. (C) Experimental setup as described in B, but with UMB18-2 or DMSO control added at 0 h or +6 h. Data are from a representative experiment of two performed in triplicate. A one-way ANOVA was performed; ns, nonsignificant, *P < 0.05, **P < 0.01. (D) A549 cells were infected with IAV at MOI 3 for 8 h with UMB18 or (E) UMB18-2 treatment. Supernatant was collected and used to titer by plaque assay. Data are from three independent experiments performed on triplicate wells with mean PFU/mL displayed. (F and G) The same infected cells from D and E were collected in TRIzol and NS1 mRNA transcript analyzed by qRT-PCR. Input levels were normalized to GAPDH and fold change of transcript was determined relative to DMSO control. (H) Using the same extracted RNA as in G, an M-RTPCR protocol was used to amplify all IAV segments which were analysed on an agarose gel. Displayed are the amplifications from two independent wells for UMB18-2 at 50 μM and 10 μM and three wells for DMSO controls. (I) Infected and treated cells were also collected in RIPA lysis buffer and used for Western blotting of NS1. Displayed is a representative blot of the three independent repeats for each compound.

Fig. 7.

UMB18-2 inhibits all highly pathogenic human coronaviruses in multiple cell lines. (A) A549-ACE2 cells were infected with SARS-CoV at MOI 0.1 and treated with UMB18-2 at 50 µM or 10 µM (with 0.5% or 0.1% DMSO being the appropriate negative controls) for 48 h. Supernatant was collected and used for TCID50 assay to determine viral titer. Mean TCID50/mL and SD are displayed from three independent experiments performed in triplicate. (B) As in A but infection at MOI 0.01. (C) Huh7-ACE2 cells were infected with SARS-CoV at MOI 0.1 and treated with UMB18-2. Viral production was analyzed as in A but after 24 h of infection. (D) Vero E6 cells were infected with SARS-CoV-2 at MOI 0.1 and treated with UMB18-2 as in A. Viral production was analyzed as in A, but after 24 h of infection. (E and F) As described in A and B, but with SARS-CoV-2 infection. (G and H) Cells that were infected in E and F were collected in TRIzol and used for qRT-PCR analysis. Primers targeting RdRp were used. Input levels were normalized to GAPDH RNA and fold change of transcript levels was determined relative to DMSO control for each concentration of compound. Data are from a representative experiment. (I and J) A549-DPP4 cells were infected with MERS-CoV in the same way as described for A and B. (K and L) MERS-CoV RNA analysis as described for G and H. In all cases t tests were performed for vehicle control vs. drug treated samples; *P < 0.05, **P < 0.01, ****P < 0.0001.