Identification of novel antivirals inhibiting recognition of Venezuelan equine encephalitis virus capsid protein by the Importin α/β1 heterodimer through high-throughput screening

Abstract

Although the alphavirus Venezuelan equine encephalitis virus (VEEV) has been the cause of multiple outbreaks resulting in extensive human and equine mortality and morbidity, there are currently no anti-VEEV therapeutics available. VEEV pathogenicity is largely dependent on targeting of the viral capsid protein (CP) to the host cell nucleus through the nuclear transporting importin (Imp) α/β1 heterodimer. Here we perform a high-throughput screen, combined with nested counterscreens to identify small molecules able to inhibit the Impα/β1:CP interaction for the first time. Several compounds were able to significantly reduce viral replication in infected cells. Compound G281-1564 in particular could inhibit VEEV replication at low μM concentration, while showing minimal toxicity, with steady state and dynamic quantitative microscopic measurements confirming its ability to inhibit CP nuclear import. This study establishes the principle that inhibitors of CP nucleocytoplasmic trafficking can have potent antiviral activity against VEEV, and represents a platform for future development of safe anti-VEEV compounds with high efficacy and specificity.

Ivermectin as a Broad-Spectrum Host-Directed Antiviral: The Real Deal?

“…Consistent with the fact that many viruses are known to rely on IMPα/β1-dependent nuclear import of specific viral proteins for robust infection [14,27,28], ivermectin has been confirmed in a body of in vitro studies to be active in limiting infection by a range of different RNA viruses [10,14], including HIV-1 [7], DENV (all four serotypes) and related flaviviruses [8,11,12], influenza, and alphaviruses such as Venezuelan equine encephalitis virus (VEEV) and chikungunya [9,15,16] (see Table 1); it is also active against DNA viruses [18][19][20]. Recent studies indicate it is a potent inhibitor of SARS-CoV-2 [17].…”

“…As has since been shown using a range of different small molecules, the critical importance of this interaction to dengue infection is the basis for the fact that multiple distinct small molecules that disrupt IMPα recognition of dengue NS5 are able to limit dengue infection [ 7 , 8 , 11 , 29 , 31 ]. In the case of ivermectin, this activity extends to a large number of different viruses (see below) [ 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 ], including SARS-CoV-2 [ 18 ]. Which SARS-CoV-2 proteins may access the nucleus in infected cells has not been examined in detail but, in terms of related coronaviruses, ORF6 (Open Reading Frame 6) protein from SARS-CoV-1 has been shown to bind IMPα [ 32 ], and ORF4b from MERS-CoV (Middle Eastern Respiratory Syndrome Coronavirus) is known to access the nucleus in NLS-dependent fashion [ 33 ].…”

“…Inhibits nuclear accumulation in a cellular context of IMPα/β1- but not β1-recognised viral proteins such as T-ag [ 7 , 16 ], DENV2 NS5 [ 7 ], VEEV Capsid [ 16 ], adenovirus E1A [ 20 ], PSV UL42 [ 18 ] as well as host cargoes (see [ 15 , 30 ])…”

“…From 2012 onwards, there have multiple reports that ivermectin has antiviral properties [ 4 , 5 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 ] towards a growing number of RNA viruses, including human immunodeficiency virus (HIV)-1, influenza, flaviruses such as dengue virus (DENV) and Zika virus (ZIKV) and, most notably, SARS-CoV-2 (COVID-19) [ 17 ]. Evidence for activity against DNA viruses is more limited, but encompasses Pseudorabies, polyoma and adenoviruses [ 18 , 19 , 20 ].…”

Venezuelan Equine Encephalitis Virus Capsid Implicated in Infection-Induced Cell Cycle Delay in vitro

“…This complex halts host nuclear import and downregulates transcription (Garmashova et al, 2007; Atasheva et al, 2008). Mutating the NLS alters capsid localization, retaining it primarily in the cytoplasm (Atasheva et al, 2010a; Lundberg et al, 2016); additionally, chemically inhibiting the interaction between capsid and importin α/β1 reduces viral titer and prolongs cell survival (Lundberg et al, 2013; Shechter et al, 2017; Thomas et al, 2018). Mutation of the NLS also prevents capsid from host cell transcriptional suppression (Atasheva et al, 2010b).…”