Graphical abstract

Alphaviruses, including Chikungunya (CHIKV) and Venezuelan equine encephalitis virus (VEEV), are among the leading causes of recurrent epidemics all over the world. Alphaviral nonstructural protein 1 (nsP1) orchestrates the capping of nascent viral RNA via its S‐adenosyl methionine‐dependent N‐7‐methyltransferase (MTase) and guanylyltransferase activities. Here, we developed and validated a novel capillary electrophoresis (CE)‐based assay for measuring the MTase activity of purified VEEV and CHIKV nsP1. We employed the assay to assess the MTase inhibition efficiency of a few adenosine analogs and identified 5‐iodotubercidin (5‐IT) as an inhibitor of nsP1. The antiviral potency of 5‐IT was evaluated in vitro using a combination of cell‐based assays, which suggest that 5‐IT is efficacious against CHIKV in cell culture (EC50: 0.409 µm).

Arthropod-borne viruses of the alphavirus and flavivirus genera are human pathogens of significant concern, and currently, no specific antiviral treatment is available for these viruses. In this study, the antiviral mechanisms of natural small molecules against Dengue virus (DENV) and Chikungunya virus (CHIKV) have been investigated. Herbacetin (HC) and Caffeic acid phenethyl ester (CAPE) showed depletion of polyamine levels in Vero cells as demonstrated by thin-layer chromatography (TLC). As polyamines are known to play a role in viral replication and transcription, HC and CAPE were expected to inhibit virus replication by reducing polyamine levels. To test this hypothesis, HC and CAPE were evaluated for antiviral activities using a cell-based virus yield assay by quantitative reverse transcription-polymerase chain reaction (qRT-PCR), plaque reduction assay, and immunofluorescence assay (IFA). HC and CAPE displayed potent inhibition with EC50 of 463 nM and 0.417 nM for CHIKV and 8.5 uM and 1.15 uM for DENV, respectively. Interestingly, however, the addition of exogenous polyamines did not completely rescue the virus titer in both CHIKV and DENV infected cells and this indicated additional antiviral mechanisms for HC and CAPE. Further, in silico analysis indicated that HC and CAPE directly target the viral methyltransferases (MTase) of CHIKV and DENV. A high throughput ELISA-based assay that quantifies m7GMP-nsP1 adduct was employed to validate inhibition of CHIKV nsP1 MTase and IC50 was calculated to be 0.009 uM and 0.08 uM for CAPE and HC respectively. Altogether, the identification of natural small molecules as antivirals opens the door for the development of antiviral therapies for the treatment of CHIKV and DENV infections.

No abstract

Molecular interactions of Chikungunya virus (CHIKV) non-structural protein 3 (nsP3) with GTPase Activating Protein SH3 Domain Binding Protein 1 (G3BP1) host protein is important for efficient replication of CHIKV. CHIKV nsP3 protein binds to the nuclear transport factor 2 (NTF2)-like domain of G3BP1 via two tandem FGDF motifs and disrupts the stress granules (SGs) formation. Interestingly, this makes the G3BP1 host protein an additional drug target for antiviral research. In this study, seven potential small molecules targeting FGDF motif binding pocket of G3BP1 have been identified using structure-based virtual screening approach. Binding energies and binding modes of the molecules were further analyzed in detail through molecular docking, dynamics and simulations. Surface Plasmon Resonance (SPR) and Isothermal Titration Calorimetry (ITC) experiments confirmed the binding of identified molecules to purified G3BP1 with binding affinities in micromolar (μM) range. The antiviral efficacy of molecules targeting G3BP1 was evaluated by in vitro cell culture-based antiviral studies. All seven molecules L-7, WIN, SB2, NAL, DHD, GSK and FLU effectively diminished the CHIKV replication with EC50 values of 1.996, 0.403, 5.387, 1.528, 7.394, 3.664, and 0.618 μM, respectively. Moreover, the CHIKV infected cells treated with these molecules seemed to have fewer virus-induced SGs than virus infected control cells. Interestingly, the inhibitors showed no adverse effect on SG formation in oxidative stress condition. It further substantiated that these inhibitors effectively bind to the NTF2-like domain of G3BP1, obstruct the nsP3-G3BP1 interactions, and halts virus replication. This is the first report of small molecules targeting G3BP1, the host protein playing a crucial role in CHIKV viral replication and in the formation of SGs for host antiviral response.