Due to its exceptional scale and spread, the 2022-2023 outbreak of monkeypox was declared a global emergency by the World Health Organization. The global spread of the monkeypox virus has demonstrated the need for in-depth characterization of the virus infection process and virus-host interactions. Multi-omics studies have helped to understand the fundamental principles of virus pathophysiology and thus guide the selection of virus- and host-directed therapies. In this study, we present a time-resolved multi-omics comparison of monkeypox viral infection in primary human cells.

Primary human foreskin fibroblast cells were infected with monkeypox virus for 6, 12, and 24 hours and subsequently processed for transcriptome and proteome analysis. Phosphopeptides were enriched using TiO2 beads. Liquid chromatography coupled mass spectrometry-based proteomics measurements were performed in data-dependent acquisition mode and searched using the MaxQuant/Andromeda peptide search engine.

We could show that crucial cellular processes are affected at different regulatory layers with distinct temporal profiles during monkeypox infection. To better understand the virus biology and to identify potentially druggable hot spots that the virus relies on, we performed an in-depth systems analysis by projecting the multi-omics data onto the known host biology. Using existing knowledge about host signaling cascades and interactome maps, we extracted infection-elicited molecular fingerprints and applied a graph-based approach to match these with potential anti-monkeypox virus drug targets. A selection of 52 drugs with diverse molecular modes of action was subsequently used in a proof-of-concept drug target validation screen.

With this approach, we showcase the clear benefit of integrating multi-omics dataset information to elucidate the molecular patterns of viral infection. These patterns, in turn, can be used to predict potential targets of host-directed antivirals.