Role of vaccinia virus growth factor in stimulating the mTORC1-CAD axis of the de novo pyrimidine pathway under different nutritional cues.

Vaccinia virus (VACV), the prototype poxvirus, actively reprograms host cell metabolism upon infection. However, the nature and molecular mechanisms remain largely elusive. Given the diverse nutritional exposures of cells in different physiological contexts, it is essential to understand how VACV may alter various metabolic pathways in different nutritional conditions. In this study, we established the importance of de novo pyrimidine biosynthesis in VACV infection. We elucidated the significance of vaccinia growth factor (VGF), a viral early protein and a homolog of cellular epidermal growth factor, in enabling VACV to phosphorylate the key enzyme CAD of the de novo pyrimidine pathway at serine 1859, a site known to positively regulate CAD activity. While nutrient-poor conditions typically inhibit mTORC1 activation, VACV activates CAD via mTORC1-S6K1 signaling axis, in conditions where glutamine and asparagine are absent. However, unlike its cellular homolog, epidermal growth factor (EGF), VGF peptide alone in the absence of VACV infection has minimal ability to activate CAD, suggestive of the involvement of other viral factor(s) and differential functions to EGF acquired during poxvirus evolution. Our research provides a foundation for understanding the regulation of a significant metabolic pathway, namely, de novo pyrimidine synthesis during VACV infection, shedding new light on viral regulation under distinct nutritional environments. This study not only has the potential to contribute to the advancement of antiviral treatments but also improve the development of VACV as an oncolytic agent and vaccine vector.

Antiviral activities of two nucleos(t)ide analogs against vaccinia, mpox, and cowpox viruses in primary human fibroblasts.

Many poxviruses are significant human and animal pathogens, including viruses that cause smallpox and mpox (formerly monkeypox). Identifying novel and potent antiviral compounds is critical to successful drug development targeting poxviruses. Here we tested two compounds, nucleoside trifluridine, and nucleotide adefovir dipivoxil, for antiviral activities against vaccinia virus (VACV), mpox virus (MPXV), and cowpox virus (CPXV) in physiologically relevant primary human fibroblasts. Both compounds potently inhibited the replication of VACV, CPXV, and MPXV (MA001 2022 isolate) in plaque assays. In our recently developed assay based on a recombinant VACV expressing secreted Gaussia luciferase, they both exhibited high potency in inhibiting VACV replication with EC50s in the low nanomolar range. In addition, both trifluridine and adefovir dipivoxil inhibited VACV DNA replication and downstream viral gene expression. Our results characterized trifluridine and adefovir dipivoxil as strong poxvirus antiviral compounds and further validate the VACV Gaussia luciferase assay as a highly efficient and reliable reporter tool for identifying poxvirus inhibitors. Given that both compounds are FDA-approved drugs, and trifluridine is already used to treat ocular vaccinia, further development of trifluridine and adefovir dipivoxil holds great promise in treating poxvirus infections, including mpox.

Antiviral activities of two nucleos(t)ide analogs against vaccinia and mpox viruses in primary human fibroblasts.

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 (EC 50 ) 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.

Alteration in Cellular Signaling and Metabolic Reprogramming during Viral Infection.

Cellular activities are finely regulated by numerous signaling pathways to support specific functions of complex life processes. Viruses are obligate intracellular parasites. Each step of viral replication is ultimately governed by the interaction of a virus with its host cells. Because of the demands of viral replication, the nutritional needs of virus-infected cells differ from those of uninfected cells. To improve their chances of survival and replication, viruses have evolved to commandeer cellular processes, including cell metabolism, augmenting these processes to support their needs. This article summarizes recent findings regarding virus-induced alterations to major cellular metabolic pathways focusing on how viruses modulate various signaling cascades to induce these changes. We begin with a general introduction describing the role played by signaling pathways in cellular metabolism. We then discuss how different viruses target these signaling pathways to reprogram host metabolism to favor the viral needs. We highlight the gaps in understanding metabolism-related virus-host interactions and discuss how studying these changes will enhance our understanding of fundamental processes involved in metabolic regulation. Finally, we discuss the potential to harness these processes to combat viral diseases, as well as other diseases, including metabolic disorders and cancers.

Viral growth factor- and STAT3 signaling-dependent elevation of the TCA cycle intermediate levels during vaccinia virus infection.

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.