Abstract
Lipid droplets (LDs) are intracellular organelles for neutral lipid storage, originated from the endoplasmic reticulum. They play an essential role in lipid metabolism and cellular homeostasis. In fact, LDs are complex organelles, involved in many more cellular processes than those initially proposed. They have been extensively studied in the context of LD-associated pathologies. In particular, LDs have emerged as critical for virus replication and assembly. Viruses from the Flaviviridae family, namely dengue virus (DENV), hepatitis C virus (HCV), West Nile virus (WNV), and Zika virus (ZIKV), interact with LDs to usurp the host lipid metabolism for their own viral replication and pathogenesis. In general, during Flaviviridae infections it is observed an increasing number of host intracellular LDs. Several viral proteins interact with LDs during different steps of the viral life cycle. The HCV core protein and DENV capsid protein, extensively interact with LDs to regulate their replication and assembly. Detailed studies of LDs in viral infections may contribute for the development of possible inhibitors of key steps of viral replication. Here, we reviewed different techniques that can be used to characterize LDs isolated from infected or non-infected cells. Microscopy studies have been commonly used to observe LDs accumulation and localization in infected cell cultures. Fluorescent dyes, which may affect LDs directly, are widely used to probe LDs but there are also approaches that do not require the use of fluorescence, namely stimulated Raman scattering, electron and atomic force microscopy-based approaches. These three are powerful techniques to characterize LDs morphology. Raman scattering microscopy allows studying LDs in a single cell. Electron and atomic force microscopies enable a better characterization of LDs in terms of structure and interaction with other organelles. Other biophysical techniques, such as dynamic light scattering and zeta potential are also excellent to characterize LDs in terms of size in a simple and fast way and test possible LDs interaction with viral proteins. These methodologies are reviewed in detail, in the context of viral studies.
Highlights
Lipid droplets (LDs) are intracellular organelles for neutral lipid storage (Walther and Farese, 2012; Hashemi and Goodman, 2015), originated from the endoplasmic reticulum (ER) where some enzymes involved on the generation of neutral lipids are located (Buhman et al, 2001; Hashemi and Goodman, 2015)
Several viruses of the Flaviviridae family have been associated to the deregulation of the lipid metabolism
To characterize LDs size, techniques based on light scattering such as AF4 and nanoparticle tracking analysis (NTA), may provide fast and accurate results
Summary
Lipid droplets (LDs) are intracellular organelles for neutral lipid storage (Walther and Farese, 2012; Hashemi and Goodman, 2015), originated from the endoplasmic reticulum (ER) where some enzymes involved on the generation of neutral lipids are located (Buhman et al, 2001; Hashemi and Goodman, 2015). Flaviviridae and other viruses, such as rotaviruses, use LDs as platforms for viral assembly (Figure 3) (Roingeard and Melo, 2017; Zhang et al, 2017) This is achieved through the interaction of LDs with viral proteins, namely the equivalent core and capsid (C) proteins from HCV and DENV, respectively, which play multiple roles during the viral life cycle (Roingeard and Melo, 2017; Zhang et al, 2017). This interaction originates loci, where viral RNA and non-structural proteins involved in genome replication were detected (McLauchlan, 2009) These evidences suggest that these loci may be where the assembly and production of nascent virions occurs. By inhibiting the Na+/K+-ATPase in DENV-infected cells, without affecting RNA replication, the potassium ions intracellular concentration can be lowered, which in turn prevents the C protein from interacting with LDs and, decreases the number of viral particles formed (Carvalho et al, 2012). Immunofluorescence studies may confirm if the protein presents cytoplasmic localization or if it co-localizes with LDs
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