Background

Lipids are critical for metabolism, signaling, transport across membranes, regulating body temperature, and inflammation. The dysregulation of lipid metabolism or lipid transport can thus disrupt the cell homeostasis and promote oxidative stress that leads to excessive inflammation and cell death. Interestingly, some viruses such as the enveloped viruses have evolved to hijack host cell lipids to augment viral fusion, formation of replication complexes, assembly, and egress. In addition, viruses can also modulate cellular lipid metabolic pathways and leverage intracellular lipid stores to promote viral replication. For instance, dengue virus (DENV) infection can upregulate and re-localize fatty acid synthase (FASN) to the replication complex to increase beta-oxidation for energy production (Heaton et al., 2010; Tang et al., 2014). DENV can also target lipid droplet stores to utilize triglycerides for the production of fatty acids, which are subsequently transported to the mitochondria where they undergo beta-oxidation to synthesize ATP required for viral replication (Zhang et al., 2017). However, much work remains to be done to better understand the virus and lipid interactions responsible for productive virus infection.

RNA interference and CRISPR technologies are useful tools to probe into the lipid metabolic processes required for viral infection. For example, we have recently identified that TMEM41B and VMP1 play a central role in lipid mobilization, mitochondrial beta-oxidation, and global metabolic regulations, to facilitate the replication of flaviviruses and coronaviruses (Yousefi et al., 2022). Deficiency of these proteins resulted in impaired beta-oxidation capacity and dysregulation of the cell lipidome that severely compromised viral infection. To gain insights into the lipid metabolism pathways that are differentially modulated before and after virus infection, we can leverage mass spectrometry to characterize the lipidome. The current protocol describes the detailed procedures required for mass spectrometry in target cells. We have used liquid chromatography–mass spectrometry (LC–MS), the preferred technique for use in metabolomics and lipidomics due to its ability to identify metabolites, even at low concentrations. The method also allows for untargeted lipidomics analysis, so users can simultaneously quantify a variety of lipids in host cells. Furthermore, to evaluate the effects of fatty acid oxidation on cellular bioenergetics, a modified Seahorse assay can be used to ascertain the lipid dependencies for energy production. In combination, these assays provide molecular insights into the role of lipid metabolism in cellular energetics and their potential contribution to virus infection outcome. Finally, we developed a web tool (https://kuanrongchan-lipid-metabolite-analysis-app-k4im47.streamlit.app/) that allows users to plot clustergrams and heatmaps based on the intensity measurements from the LC–MS. While we have used these methods to study virus–lipid interactions in human cells, as lipids are involved in multiple cell processes, we believe that these methods can be more broadly applied to study the role of lipids for other diseases, such as diabetes, obesity, atherosclerosis, and cancer.

In summary, we will describe the detailed protocols for:

1. Sample preparation and cell extraction for LC–MS

2. LC–MS analysis and interpretation

3. Bioinformatics analysis and web tool for data analysis

4. Seahorse bioenergetics analysis for fatty acid oxidation