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.