The role of galactosyleceramides in flavivirus infection

Abstract

Mosquito-borne flaviviruses are an intensifying threat to global health without specific antiviral therapies or a widely available vaccine. Between 2000 and 2019, there was a 10-fold surge in dengue virus cases reported globally [1]. 2024 was a record year with more than 14 million dengue cases reported worldwide [2]. Flaviviruses are reliant upon host cell membranes to complete their life cycle. Preliminary data for this work included an siRNA loss of function screen of each enzyme in the sphingolipid metabolic pathway performed in liver cells (Huh7) infected with dengue virus 2. This screen identified numerous enzymes of interest in the metabolic pathway. This dissertation investigates the roles of two key enzymes in the sphingolipid metabolic pathway that affect synthesis and degradation of galactosylceramides, UDP-galactosyltransferase 8 (UGT8) and galactocerebrosidase (GALC), and how they affect flavivirus infections in vitro. We studied how modulating the expression of UGT8 and GALC during infection with dengue virus, serotype 2 (DENV2) and Zika virus (ZIKV) impact the virus life cycle in human hepatoma cells (Huh7). We additionally studied how UGT8 knockdown affected ZIKV titer in neuroblastoma cells (SH-SY5Y). Chapter 2 focuses on the effect of UGT8 during DENV2 infection of Huh7 cells. We showed that with UGT8 knockdown, there was an increase in titer, while overexpression caused a decrease in titer. Mechanistic studies demonstrated that modulating UGT8 expression affected entry and release of DENV2 particles. We did not observe any effect on DENV2 genome replication or the specific infectivity of the virus particles. We hypothesized that modulation of UGT8 expression alters the cell and viral membranes to influence virus release and virus entry. Chapter 3 focuses on GALC, which performs the reciprocal reaction as UGT8 and degrades galactosylceramides in the lysosome. We discovered that GALC knockdown increased both extracellular and intracellular DENV2 titers, but did not affect genome replication or specific infectivity. This suggested that GALC knockdown caused an increase in the efficiency of viral assembly. We proposed that the oxidative stress resulting from loss of function of the GALC enzyme upregulated sphingomyelinases to increase cellular pools of ceramide, resulting in an increase in efficiency of viral assembly. Chapter 4 explored the effects of UGT8 in ZIKV infection of both Huh7 cells and SH-SY5Y cells. Modulation of UGT8 expression in these cells showed a similar phenotype to DENV2. Together, the findings in this dissertation highlight the importance of sphingolipid metabolism in flavivirus infections and provides new insights into the roles of these specialized lipids during infection. Hypothesis of the study: The flavivirus life cycle is critically influenced by the balance of galactosylceramide synthesis and degradation, processes regulated by UDP-galactosyltransferase 8 (UGT8) and galactocerebrosidase (GALC) respectively.