Glucose and mannose analogs inhibit KSHV replication by blocking N-glycosylation and inducing the unfolded protein response.

Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiological agent for Kaposi's sarcoma (KS), an HIV/AIDS-associated malignancy. Effective treatments against KS remain to be developed. The sugar analog 2-deoxy- d-glucose (2-DG) is an anticancer agent that is well-tolerated and safe in patients and was recently demonstrated to be a potent antiviral, including KSHV and severe acute respiratory syndrome coronavirus 2. Because 2-DG inhibits glycolysis and N-glycosylation, identifying its molecular targets is challenging. Here we compare the antiviral effect of 2-DG with 2-fluoro-deoxy- d-glucose, a glycolysis inhibitor, and 2-deoxy-fluoro- d-mannose (2-DFM), a specific N-glycosylation inhibitor. At doses similar to those clinically achievable with 2-DG, the three drugs impair KSHV replication and virion production in iSLK.219 cells via downregulation of viral structural glycoprotein expression (K8.1 and gB), being 2-DFM the most potent KSHV inhibitor. Consistently with the higher potency of 2-DFM, we found that d-mannose rescues KSHV glycoprotein synthesis and virus production, indicating that inhibition of N-glycosylation is the main antiviral target using d-mannose competition experiments. Suppression of N-glycosylation by the sugar drugs triggers ER stress. It activates the host unfolded protein response (UPR), counteracting KSHV-induced inhibition of the protein kinase R-like endoplasmic reticulum kinase branch, particularly activating transcription factor 4 and C/EBP homologous protein expression. Finally, we demonstrate that sugar analogs induce autophagy (a prosurvival mechanism) and, thus, inhibit viral replication playing a protective role against KSHV-induced cell death, further supporting their direct antiviral effect and potential therapeutic use. Our work identifies inhibition of N-glycosylation leading to ER stress and UPR as an antienveloped virus target and sugar analogs such as 2-DG and the newly identified 2-DFM as antiviral drugs.

Tissue engineering of corneal stroma via melt electrowriting.

The cornea serves as the main refractive component of the eye with the corneal stroma constituting the thickest component in a stratified layered system of epithelia, stroma, and endothelium. Current treatment options for patients suffering from corneal diseases are limited to transplantation of a human donor cornea (keratoplasty) or to implantation of an artificial cornea (keratoprosthesis). Nevertheless, donor shortage and failure of artificial corneas to integrate with local tissue constitute important problems that have not been yet circumvented. Recent advances in biofabrication have made great progress toward the manufacture of tailored biomaterial templates with the potential of guiding partially or totally the regeneration process of the native cornea. However, the role of the corneal stroma on current tissue engineering strategies is often neglected. Here, we achieved a tissue-engineered corneal stroma substitute culturing primary keratocytes on scaffolds prepared via melt electrowriting (MEW). Scaffolds were designed to contain highly organized micrometric fibers to ensure transparency and encourage primary human keratocytes to self-orchestrate their own extracellular matrix deposition and remodeling. Results demonstrated reliable cell attachment and growth over a period of 5 weeks and confirmed the formation of a dense and highly organized de novo tissue containing collagen I, V, and VI as well as Keratocan, which resembled very closely the native corneal stoma. In summary, MEW brings us closer to the biofabrication of a viable corneal stroma substitute.