Recent RNA virus outbreaks such as Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and Ebola virus (EBOV) have caused worldwide health emergencies highlighting the urgent need for new antiviral strategies. Targeting host cell pathways supporting viral replication is an attractive approach for development of antiviral compounds, especially with new, unexplored viruses where knowledge of virus biology is limited. Here, we present a strategy to identify host-targeted small molecule inhibitors using an image-based phenotypic antiviral screening assay followed by extensive target identification efforts revealing altered cellular pathways upon antiviral compound treatment. The newly discovered antiviral compounds showed broad-range antiviral activity against pathogenic RNA viruses such as SARS-CoV-2, EBOV and Crimean-Congo hemorrhagic fever virus (CCHFV). Target identification of the antiviral compounds by thermal protein profiling revealed major effects on proteostasis pathways and disturbance in interactions between cellular HSP70 complex and viral proteins, illustrating the supportive role of HSP70 on many RNA viruses across virus families. Collectively, this strategy identifies new small molecule inhibitors with broad antiviral activity against pathogenic RNA viruses, but also uncovers novel virus biology urgently needed for design of new antiviral therapies.
Background The emergence and continued global spread of the current COVID-19 pandemic has highlighted the need for methods to identify novel or repurposed therapeutic drugs in a fast and effective way. Despite the availability of methods for the discovery of antiviral drugs, the majority tend to focus on the effects of such drugs on a given virus, its constituent proteins, or enzymatic activity, often neglecting the consequences on host cells. This may lead to partial assessment of the efficacy of the tested anti-viral compounds, as potential toxicity impacting the overall physiology of host cells may mask the effects of both viral infection and drug candidates. Here we present a method able to assess the general health of host cells based on morphological profiling, for untargeted phenotypic drug screening against viral infections. Results We combine Cell Painting with antibody-based detection of viral infection in a single assay. We designed an image analysis pipeline for segmentation and classification of virus-infected and non-infected cells, followed by extraction of morphological properties. We show that this methodology can successfully capture virus-induced phenotypic signatures of MRC-5 human lung fibroblasts infected with human coronavirus 229E (CoV-229E). Moreover, we demonstrate that our method can be used in phenotypic drug screening using a panel of nine host- and virus-targeting antivirals. Treatment with effective antiviral compounds reversed the morphological profile of the host cells towards a non-infected state. Conclusions The phenomics approach presented here, which makes use of a modified Cell Painting protocol by incorporating an anti-virus antibody stain, can be used for the unbiased morphological profiling of virus infection on host cells. The method can identify antiviral reference compounds, as well as novel antivirals, demonstrating its suitability to be implemented as a strategy for antiviral drug repurposing and drug discovery.
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