Nodosome Inhibition as a Novel Broad-Spectrum Antiviral Strategy against Arboviruses, Enteroviruses, and SARS-CoV-2

Abstract

In the present report, we describe two small molecules with broad-spectrum antiviral activity. These drugs block the formation of the nodosome. The studies were prompted by the observation that infection of human fetal brain cells with Zika virus (ZIKV) induces the expression of nucleotide-binding oligomerization domain-containing protein 2 (NOD2), a host factor that was found to promote ZIKV replication and spread. A drug that targets NOD2 was shown to have potent broad-spectrum antiviral activity against other flaviviruses, alphaviruses, enteroviruses, and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19). Another drug that inhibits receptor-interacting serine/threonine protein kinase 2 (RIPK2), which functions downstream of NOD2, also decreased the replication of these pathogenic RNA viruses. The antiviral effect of this drug was particularly potent against enteroviruses. The broad-spectrum action of nodosome-targeting drugs is mediated in part by the enhancement of the interferon response. Together, these results suggest that further preclinical investigation of nodosome inhibitors as potential broad-spectrum antivirals is warranted.

Keywords: COVID-19; NOD2; RIPK2; SARS-CoV-2; antiviral; arbovirus; broad spectrum; coxsackievirus; interferon; nodosome.

FIG 1

Inflammasome gene expression in HFAs is induced by ZIKV, IFN-α, and poly(I·C). (A) Relative inflammasome gene expression in HFAs infected with ZIKV PRVABC-59 (MOI = 0.3) was determined by qRT-PCR at 48 h postinfection. (B) HFAs were treated with human recombinant IFN-α (rIFN-α) for 4, 8, and 12 h, after which relative NOD2 expression was determined. (C) HFAs were transfected with poly(I·C) for 12 h, after which relative inflammasome gene expression was determined. Error bars represent standard errors of the means. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (by Student’s t test).

FIG 2

NOD2 silencing suppresses ZIKV multiplication and enhances the expression of interferon-stimulated and inflammasome genes. HFAs were transfected with NOD2-specific or nonsilencing siRNAs for 24 h and then infected with ZIKV (MOI = 0.05). (A) Cell culture medium or total cellular RNA was harvested after 24 and 48 h for plaque assays or qRT-PCR at 48 h postinfection. (B to D) Relative levels of viral genome (B); the interferon-stimulated genes Viperin, 2′-5′-oligoadenylate synthetase 1 (OAS1), and myxovirus resistance protein 2 (MX2) (C); as well as the inflammasome genes gasdermin D (GSDMD) and caspase 1 (Casp1) (D). Values are expressed as the means from three independent experiments. Error bars represent standard errors of the means. *, P < 0.05; ***, P < 0.001 (by Student’s t test).

FIG 3

The anti-NOD2 drug GSK717 inhibits ZIKV replication. ZIKV-infected HFAs (MOI = 0.05 to 5) were treated with DMSO or the NOD2-blocking agent GSK717, after which viral titers were determined daily up to 72 h postinfection. (A and B) ZIKV titers as relative fold changes (MOI = 0.05) at 72 h (A) and as PFU per milliliter at 48 h postinfection (MOI = 5) (B). (C) Cellular ATP levels were determined in uninfected HFAs treated with GSK717 or DMSO for 72 h. Values are expressed as the means from three independent experiments. Error bars represent standard errors of the means. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (by Student’s t test).

FIG 4

The anti-NOD2 drug GSK717 blocks the spread of ZIKV infection. (A) Representative confocal imaging (magnification, ×20) showing the antiviral effect of GSK717 at 20 μM and 40 μM. A549 cells were infected with ZIKV (MOI = 1) followed by treatment with DMSO or GSK717 at 20 or 40 μM for 48 h before processing for indirect immunofluorescence. ZIKV-infected cells were identified using a mouse monoclonal antibody (4G2) to the envelope protein and Alexa Fluor 488 donkey anti-mouse to detect the primary antibody. Nuclei were stained with DAPI. Images were acquired using a spinning-disk confocal microscope equipped with Volocity 6.2.1 software. (B) Cellular ATP levels in A549 cells after 48 h of GSK717 or DMSO treatment. Values are expressed as the means from three independent experiments. Error bars represent standard errors of the means.

FIG 5

The anti-NOD2 drug GSK717 inhibits DENV replication. A549 cells were infected with DENV-2 (MOI = 0.05 to 5) and treated with GSK717 or DMSO for 48 h, after which cell culture media were harvested for plaque assays. (A and B) Viral titers as relative fold changes at an MOI of 0.05 or 5 (A) and as PFU per milliliter at an MOI of 0.05 (B). (C) Representative confocal imaging (magnification, ×20) of DENV-2-infected cells treated with GSK717. A549 cells were infected with DENV-2 (MOI = 1) followed by treatment with DMSO or GSK717 at 20 or 40 μM for 48 h before fixation and immunostaining. DENV-infected cells were detected using mouse monoclonal antibody (4G2) to the envelope protein and Alexa Fluor 488 donkey anti-mouse. Nuclei were stained with DAPI. Images were acquired using a spinning-disk confocal microscope equipped with Volocity 6.2.1 software. Values are expressed as the means from three independent experiments. Error bars represent standard errors of the means. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (by Student’s t test).

FIG 6

The anti-NOD2 drug GSK717 inhibits the replication of MAYV, CVB5, and SARS-CoV-2. A549 cells infected with a low MOI (0.05) and a high MOI (5) of MAYV were treated with GSK717 or DMSO for 48 h, followed by collection of supernatants for plaque assays. (A and B) Relative (A) and absolute (B) viral titers at both MOIs and the MOI of 0.05, respectively. (C) A549 cells were infected with CVB5 (MOI = 0.1) followed by treatment with GSK717 or DMSO as a control for 24 h before total cellular RNA was collected for viral genome quantification by qRT-PCR. Viral RNA levels relative to mock are shown. ACE2-SK-N-SH cells were infected with SARS-CoV-2 (MOI = 0.05 to 5) and treated with GSK717 or DMSO for 48 h, after which culture supernatants were harvested for plaque assays. (D and E) Viral titers as relative fold changes (D) and as PFU per milliliter (E) at both MOIs and the MOI of 0.05, respectively. (F) Cellular ATP levels in ACE2-SK-N-SH cells after 48 h of GSK717 or DMSO treatment. Values are expressed as the means from three independent experiments. Error bars represent standard errors of the means. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (by Student’s t test).

FIG 7

The anti-RIPK2 drug GSK583 has broad-spectrum antiviral activity. (A) A549 cells infected separately with three different arboviruses (MOI = 0.1) were treated with the RIPK2 inhibitor GSK583 or DMSO alone for 24 h, followed by supernatant collection for plaque assays. Arbovirus titers as PFU per milliliter are presented. (B) A549 cells infected with CVB5 (MOI = 0.1) were treated with GSK583 (15 μM) or DMSO alone, and total cellular RNA was then collected at 4, 8, 12, and 24 h postinfection, followed by qRT-PCR to quantify relative levels of viral genomic RNA. (C) A549 cells infected with CVB5 (MOI = 0.1) were treated with increasing concentrations of GSK583 or DMSO. At 24 h postinfection, total cellular RNA was collected and then subjected to qRT-PCR to quantify the relative levels of viral genome. ACE2-SK-N-SH cells infected with SARS-CoV-2 at a low MOI (0.05) and a high MOI (5) were treated with GSK583 or DMSO for 24 h, followed by supernatant and total cellular RNA collection. (D and E) Viral titers at both MOIs as relative fold changes (D) and at the MOI of 0.05 as PFU per milliliter (E). (F) Viral RNA levels relative to the mock level at the MOI of 0.05. (G) Cellular ATP levels were measured in uninfected A549 and ACE2-SK-N-SH cells after 48 h of treatment with GSK583 or DMSO alone. Values are expressed as the means from three independent experiments. Error bars represent standard errors of the means. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (by Student’s t test).

FIG 8

Antinodosome drugs enhance the IFN response. A549 cells were treated with GSK583, GSK717, or DMSO alone for 16 h, followed by the addition of human recombinant IFN-α (100 U/ml). Total cellular RNA was harvested for qRT-PCR at 4 and 8 h posttreatment. Relative expression levels of transcripts for eight ISGs (Casp1, GBP5, NLRC5, NLRP3, NLRC4, MX2, IFIT1, and OAS1) in drug- and DMSO-treated cells are shown. Values are expressed as the means from three independent experiments. Error bars represent standard errors of the means. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (by Student’s t test).