Metabolism is a crucial frontier of host-virus interaction as viruses rely on their host cells to provide nutrients and energy for propagation. Vaccinia virus (VACV) is the prototype poxvirus. It makes intensive demands for energy and macromolecules in order to build hundreds and thousands of viral particles in a single cell within hours of infection. Our comprehensive metabolic profiling reveals profound reprogramming of cellular metabolism by VACV infection, including increased levels of the intermediates of the tri-carboxylic acid (TCA) cycle independent of glutaminolysis. By investigating the level of citrate, the first metabolite of the TCA cycle, we demonstrate that the elevation of citrate depends on VACV-encoded viral growth factor (VGF), a viral homolog of cellular epidermal growth factor. Further, the upregulation of citrate is dependent on STAT3 signaling, which is activated non-canonically at the serine727 upon VACV infection. The STAT3 activation is dependent on VGF, and VGF-dependent EGFR and MAPK signaling. Together our study reveals a novel mechanism by which VACV manipulates cellular metabolism through a specific viral factor and by selectively activating a series of cellular signaling pathways.

Cellular activities are finely regulated by numerous signaling pathways to support specific functions of complex life processes. Viruses are obligate intracellular parasites.

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Many poxviruses are significant human and animal pathogens, including viruses that cause smallpox and mpox. Identification of inhibitors of poxvirus replication is critical for drug development to manage poxvirus threats. Here we tested two compounds, nucleoside trifluridine and nucleotide adefovir dipivoxil, for antiviral activities against vaccinia virus (VACV) and mpox virus (MPXV) in physiologically relevant primary human fibroblasts. Both trifluridine and adefovir dipivoxil potently inhibited replication of VACV and MPXV (MA001 2022 isolate) in a plaque assay. Upon further characterization, they both conferred high potency in inhibiting VACV replication with half maximal effective concentrations (EC50) at low nanomolar levels in our recently developed assay based on a recombinant VACV secreted Gaussia luciferase. Our results further validated that the recombinant VACV with Gaussia luciferase secretion is a highly reliable, rapid, non-disruptive, and simple reporter tool for identification and chracterization of poxvirus inhibitors. Both compounds inhibited VACV DNA replication and downstream viral gene expression. Given that both compounds are FDA-approved drugs, and trifluridine is used to treat ocular vaccinia in medical practice due to its antiviral activity, our results suggest that it holds great promise to further test trifluridine and adefovir dipivoxil for countering poxvirus infection, including mpox.