Viruses are a major threat to human health and economic well-being. In recent years Ebola, Zika, influenza, and chikungunya virus epidemics have raised awareness that infections can spread rapidly before vaccines or specific antagonists can be made available. Broad-spectrum antivirals are drugs with the potential to inhibit infection by viruses from different groups or families, which may be deployed during outbreaks when specific diagnostics, vaccines or directly acting antivirals are not available. While pathogen-directed approaches are generally effective against a few closely related viruses, targeting cellular pathways used by multiple viral agents can have broad-spectrum efficacy. Virus entry, particularly clathrin-mediated endocytosis, constitutes an attractive target as it is used by many viruses. Using a phenotypic screening strategy where the inhibitory activity of small molecules was sequentially tested against different viruses, we identified 12 compounds with broad-spectrum activity, and found a subset blocking viral internalisation and/or fusion. Importantly, we show that compounds identified with this approach can reduce viral replication in a mouse model of Zika infection. This work provides proof of concept that it is possible to identify broad-spectrum inhibitors by iterative phenotypic screenings, and that inhibition of host-pathways critical for viral life cycles can be an effective antiviral strategy.
Transcription of integrated DNA from viruses or transposable elements is tightly regulated to prevent pathogenesis. The Human Silencing Hub (HUSH), composed of Periphilin, TASOR and MPP8, silences transcriptionally active viral and endogenous transgenes. HUSH recruits effectors that alter the epigenetic landscape and chromatin structure, but how HUSH recognizes target loci and represses their expression remains unclear. We identify the physicochemical properties of Periphilin necessary for HUSH assembly and silencing. A disordered N-terminal domain (NTD) and structured C-terminal domain are essential for silencing. A crystal structure of the Periphilin-TASOR core complex shows Periphilin forms a-helical homodimers, which each bind a single TASOR molecule. The NTD binds RNA non-specifically and forms insoluble aggregates through an arginine/tyrosine-rich sequence reminiscent of low-complexity regions from self-associating RNA-binding proteins. Residues required for TASOR binding and aggregation were required for HUSH-dependent silencing and genome-wide deposition of repressive mark H3K9me3. The NTD was functionally complemented by low-complexity regions from certain RNA-binding proteins and proteins that form condensates or fibrils. Our work suggests the associative properties of Periphilin promote HUSH aggregation on nascent transcripts. Running title: Function of Periphilin in HUSH silencingKeywords: Transcriptional repression; epigenetic silencing; histone H3 lysine 9 methylation (H3K9me3); transposable element (TE); long interspersed nuclear element-1 (LINE-1); lowcomplexity sequence; disordered region; arginine/tyrosine-rich region; biomolecular condensate; liquid phase separation; membraneless compartment; RNA-binding protein syncytiotrophoblast fusion in placental development (Dupressoir et al, 2012;Friedli & Trono, 2015).A subset of TEs can autonomously replicate through an RNA intermediate and reintegrate into the genome like retroviruses. Some of these TEs are endogenous retrovirus (ERV) genomes inherited from ancestral infections of germline. The other type of autonomously replicating TE in humans are the non-viral LINE-1 (long interspersed nuclear element-1) retroelements. Active ERVs and LINE-1s are transcribed and encode reverse transcriptase and integrase enzymes,
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