Browsing by Author "Perera, Rushika, advisor"

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Open Access
Characterization of changes in metabolic pathways during dengue virus serotype 2 infection of the Aedes aegypti mosquito vector to identify control points for interrupting virus transmission
(Colorado State University. Libraries, 2018) Chotiwan, Nunya, author; Perera, Rushika, advisor; Blair, Carol, committee member; Foy, Brian, committee member; Huang, Claire, committee member; Di Pietro, Santiago, committee member
Dengue viruses (DENV) are mosquito-borne viruses that cause a wide range of acute symptoms from mild fever to lethal dengue shock syndrome in humans. DENV are transmitted primarily by Aedes aegypti (Ae. aegypti). These mosquitoes are widely distributed throughout tropical and subtropical areas around the world. Increasing globalization, urbanization and global warming are factors that enhance the spread of these vectors placing over 2.5 billion people at risk of contracting these viruses. Transmission of these viruses depends on their ability to infect, replicate and disseminate into several tissues in the mosquito vector. During DENV infection of its human and mosquito hosts, a visible rearrangement of lipid membrane architecture and alterations of the metabolic repertoire is induced. These events occur to facilitate efficient viral replication and virus assembly within the cell and to circumvent antiviral responses from the host. Interference with these virus-induced processes can be detrimental to virus replication and can prevent viral transmission. In this dissertation, we present the first insight into the metabolic environment induced during DENV serotype 2 (DENV2) replication in Ae. aegypti. Using untargeted high-resolution liquid chromatography-mass spectrometry, we explored the temporal metabolic perturbations that occur following dengue virus infection of the midgut, the primary site of the virus infection in the mosquito vector. Temporal changes of metabolites across early-, mid- and late-infection time points were identified. A marked increase in the /content of glycerophospholipids, sphingolipids and fatty acyls was coincident with the kinetics of viral replication. Elevation of glycerolipid levels and the accumulation of medium-chain acyl-carnitines suggested a diversion of resources during infection from energy storage to synthetic pathways and energy production. From the observations above, two active pathways, sphingolipid and de novo fatty acid synthesis pathways, were further validated to identify metabolic control hubs. Using inhibitor screening of the sphingolipid pathway, we determined that sphingolipid Δ-4 desaturase (DEGS), the enzyme that converts dihydroceramide to ceramide was important for DENV2 infection in cultured Ae. aegypti cells (Aag2). Long, double-stranded RNA-mediated knockdown of DEGS expression led to the imbalance of ceramide to dihydroceramide ratios and affected DENV2 infection in cell culture. However, the inhibitory effect to DENV2 replication was not observed during DEGS-knockdown in mosquito vectors. De novo fatty acid biosynthesis is the pathway that synthesizes the first lipid molecules, fatty acids, required in synthesizing complex lipid molecules, such as glycerophospholipids, glycerolipids and sphingolipids. As a result, this pathway serves as a bottle neck for the control of lipid metabolism. In this study, we annotated and characterized the expression of seven Ae. aegypti fatty acid synthase (AaFAS) genes in the different stages of mosquito development and upon exposure to different diets. We found that AaFAS1 shares the highest amino acid similarity to human fatty acid synthase (FAS) and is the dominant AaFAS that expressed in female mosquitoes. Knockdown expression of AaFAS1 expression showed a reduction in DENV2 replication in the Aag2 cells and in the midgut of Ae. aegypti mosquitoes during early infection. However, the correlation between viral infection and levels of AaFAS1 expression was difficult to elucidate. The work in this dissertation has highlighted metabolic pathways that are induced by DENV2 infection and the metabolic control points within these pathways that are critical for DENV2 infection in Ae. aegypti. Successful perturbation of metabolic homeostasis can potentially limit virus replication in the vector, presenting a novel avenue to block the transmission of DENV2 from the mosquitoes to humans.
Open Access
Flavivirus control of lipid metabolism: implications for virion formation, function and pathogenesis
(Colorado State University. Libraries, 2018) Gullberg, Rebekah, author; Perera, Rushika, advisor; Crick, Dean, committee member; Di Pietro, Santiago, committee member; Geiss, Brian, committee member; Wilusz, Jeff, committee member
Dengue viruses (DENV) are the most aggressive arthropod-born viruses worldwide with no currently available antivirals. There is a clear need to understand host viral interactions that can be exploited for therapeutic options. DENV are members of the Flaviviridae family with a positive sense single-stranded RNA genome surrounded by a virally encoded capsid protein, a host cell derived lipid envelope and an icosahedral shell of virally encoded glycoproteins. Its genome is replicated in virally–induced invaginations in the endoplasmic reticulum of the host cell that consistently develop in a time-dependent manner. These invaginations display a highly curved architecture and seem to increase the membrane contact sites within the ER and its vicinity. Functionally, these membranes condense the replication machinery, provide a scaffold to coordinate replication, and hide the viral double stranded RNA intermediate from the host cellular defenses. It has been shown that fatty acid synthesis is increased during infection to provide substrates for this membrane expansion. To identify further changes to cellular metabolism, we have profiled the metabolome of DENV serotype 2 (DENV2) infected Human Hepatoma cells (Huh7) cells at key time-points in virus replication. We have found time-dependent changes in cellular essential fatty acid metabolism. Furthermore, we have interrogated a library of siRNAs directed at the unsaturated fatty acid biosynthesis pathway to determine key enzymes involved in viral replication. We have identified that stearoyl Co-A desaturase 1 (SCD1), the rate-limiting enzyme responsible for converting stearic to oleic acid, is critical for viral replication, maturation and infectious particle formation. Finally, we have profiled the serum metabolome of acute-phase patients with dengue diseases, chikungunya virus infection, or an unknown febrile illness to identify metabolic changes with potential use as prognostic biomarkers. Hypothesis: Since dengue viruses are enveloped viruses, lipid metabolites in the human host are a critical resource hijacked by these viruses for their replicative advantage. Important metabolites will be altered during infection in a time dependent manner and can be quantified and correlated directly to their role in viral genome replication and infectious particle assembly and release. These metabolic changes could also be identified in human bio-fluids and could function as early biomarkers of disease manifestation.
Open Access
Reframing viral infections as acute metabolic disorders: dengue viruses and their dependency on host metabolic pathways
(Colorado State University. Libraries, 2022) St. Clair, Laura A., author; Perera, Rushika, advisor; Belisle, John, committee member; Nachappa, Punya, committee member; Wilusz, Jeff, committee member; Zabel, Mark, committee member
Dengue viruses (DENVs) are the etiological agent of the world's most aggressive arthropod-borne disease. At present, there are no available antivirals against DENVs. This fact underscores a dire need to examine host-virus interactions to identify and develop novel therapeutic approaches. As obligate intracellular parasites, DENVs are reliant upon and hijack several host metabolic pathways both to fulfill their replicative needs, and to evade the host immune response. We and others have previously established that infection with DENVs causes significant perturbation to host lipid metabolism, including elevations in sphingolipids in both the human and mosquito host. In addition, we and others previously discovered that the DENV NS1 protein increases sialidase activity in both in vitro and in vivo models leading to increased endothelial hyperpermeability and vascular leakage which are hallmarks of severe dengue. To further clarify and characterize these previous works, we have performed siRNA-mediated loss of function studies using human hepatoma cells (Huh7 cells) on several metabolic pathways altered during DENV2 infection. First, we examined the role of acyl-CoA thioesterases, enzymes responsible for controlling the intracellular balance of activated fatty acids and free fatty acids, on the DENV2 lifecycle. In these analyses, we determined that the cytosolic ACOT1 enzyme had an inhibitory effect on DENV2 replication and release, while mitochondrial ACOT (ACOTs 2 and 7) functionality was critical for viral replication and release. Moreover, we identified several enzymes within the ACOT family whose expression was dependent on ACOT2 and ACOT7 expression. These results highlighted complex relationships between ACOTs and DENVs, as well as identified yet unknown functional interdependence between ACOT enzymes. Next, we expanded our previous understanding of the relationship between DENVs and the human sialidase enzymes (NEU1-4). While previously studies linked upregulation of these enzymes with DENV2 pathology, we provide the first evidence showing that NEU1-4 functionality is vital for DENV2 genome replication and viral egress. Moreover, our analyses also revealed previously unknown functionality of NEU4 or its downstream products as transcriptional regulators for NEU1-3. Finally, we provide the first profile of the effect of loss of function of enzymes within the entire sphingolipid metabolic pathway (as identified through KEGG pathway database) on the DENV2 life cycle. In this study, we identified that enzymes involved the sphingomyelinase and salvage pathways of ceramide synthesis as opposed to de novo ceramide synthesis were critical to DENV2 release from Huh7 cells. In addition, we determined that enzymes involved in the synthesis and degradation of glycosphingolipids were vital for DENV2 release. An especially intriguing result within this arm of sphingolipid metabolism was that the two enzymes which hydrolyze GluCer had differential effects on DENV2 replication and release. GBA1 (lysosomal) had an antiviral effect on DENV2, while GBA2 (non-lysosomal) was required for DENV2 replication and release. This prompted us to profile the changes that occur to glycosphingolipids (GSLs) during infection, and we uncovered several species of GSLs that are elevated during infection. Moreover, we identified that Ambroxol HCl, a pharmaceutical GBA1 chaperone/GBA2 inhibitor, was able to abrogate these elevations in GSLs. Combined, our results allowed us to propose a novel function for GBA2 as a GluCer recycling enzyme during DENV2 infection. In conclusion, together, the work in this dissertation highlights critical metabolic nodes that impact virus replication and provides new directions for investigating viral infections as acute metabolic diseases.