Ursolic Acid Inhibits Rotavirus Replication Through Modulation Of Lipid Droplet Homeostasis

Rotavirus (RV) replication occurs within viroplasms (VP), which are globular, membrane-less cytosolic inclusions primarily assembled by the viral NSP5 and NSP2 proteins. Among host factors, lipid droplets (LD) are strictly required for VP biogenesis. LD are ubiquitous organelles consisting of a neutral lipid core surrounded by a phospholipid monolayer and associated proteins. Ursolic acid (UA), a pentacyclic triterpenoid widely present in plants and fruits, displays multiple biological activities, including modulation of lipid metabolism, and exhibits antiviral activity against RV, as we have previously demonstrated. Here, we investigated the molecular mechanism underlying the antiviral effect of UA. Using biophysical approaches, we first examined the impact of UA on LD formation, finding that it impairs LD biogenesis, consistent with reduced LD budding from the endoplasmic reticulum. We then employed cell-based assays to assess LD turnover and observed that UA acts as a lipolytic stimulus, leading to a marked reduction in LD abundance. Notably, we found that autophagic pathways contribute to LD degradation in the presence of UA. Finally, molecular dynamics simulations proposed that UA, owing to its intrinsic lipid-partitioning capacity, inserts into the LD phospholipid monolayer, establishing interactions with interdigitated neutral lipids. Together, our results indicate that UA both hampers LD biogenesis and accelerates LD degradation, likely through its association with and destabilization of the LD membrane. This dual effect leads to LD depletion, thereby impairing VP formation and ultimately inhibiting RV replication. IMPORTANCEDiarrheal diseases caused by a wide range of pathogens, including bacteria and viruses, remain a major global public health concern, accounting for more than one million deaths annually. These diseases mainly affect children under five years of age, with RV being the primary cause of diarrheal mortality in this age group. The burden is particularly severe in low- and middle-income countries. To address RV-associated disease, coordinated public health interventions have been implemented worldwide, including improvements in water, sanitation, hygiene, the introduction of vaccines, and the widespread use of oral rehydration therapy. Although these measures have substantially reduced the global burden of diarrheal diseases, the lack of a specific antiviral treatment against RV highlights the need for continued research. The development of antiviral strategies could provide a valuable complementary approach to existing interventions and help further reduce morbidity and mortality in infected children.