Methods for Lipid Droplet Biophysical Characterization in Flaviviridae Infections

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.

Keywords: Flaviviridae; LDs-associated proteins; light scattering; lipid droplet; microscopy; viral proteins.

FIGURE 1

Major LDs morphological features and biogenesis. (A) LDs are composed by a neutral core of triacylglycerols (TAGs) and sterol esters (SEs), surrounded by a monolayer of phospholipids and unesterified sterol, with several proteins at the surface and/or partially integrated within their structure, mainly from the PAT family: perilipin 1 (PLIN1), perilipin 2 (PLIN2), and perilipin 3 (PLIN3). (B) LDs are derived from the endoplasmic reticulum (ER), as a result of TAG and SE molecules accumulation between the two leaflets of the ER membrane. The nascent droplets grow into mature LDs, with the help of curvature-inducing agents, and may remain attached to the ER (not shown) or detach from the ER into the cytosol.

FIGURE 2

Flavivirus life cycle. Flavivirus enter in the host cell by receptor-mediated endocytosis. Acidification of the endosomal vesicle leads to the fusion of the viral and cell membranes, enabling the release of the viral genome ss(+)RNA into the cytosol. The ss(+)RNA is translated into a polyprotein that is processed by viral and host proteases, originating seven non-structural and three structural proteins (not shown). Virus replication and assembly occur near the ER and LDs. After virion maturation by the host protease furin, the mature virion follows the secretion pathway and is subsequently released by exocytosis.

FIGURE 3

LDs as platform for DENV assembly. LDs (1) at the ER-Golgi intermediate compartment (ERGIC), ARF1 and its guanine nucleotide exchange factor (GEF) GBF1, together with COPI, deliver adipose triacylglycerol lipase (ATGL) and ADRP (perilipin 2) from ER export sites (ERES) to the surface of LDs. DENV subverts this process for the transportation of the C protein to LDs surface. (2) The accumulation of DENV C on LDs depends on perilipin 3 and intracellular K⁺ concentration. (3) Replicated viral genomes are released through the vesicle pore and then engaged into nucleocapsids that bud through the ER membrane in close proximity. (4) DENV C can be released from LDs to the cytosol or other cellular compartments for subsequent viral assembly (gray-dashed frame and enlarged panel). (5) Packed virions accumulate within the lumen of the vesicle packets-containing ER network before being transported to the Golgi (adapted from Zhang et al., 2017).

FIGURE 4

Schematic representation of LDs isolation and purification.

FIGURE 5

ζ-potential of LDs in different conditions. (A) LDs ζ-potential values determined in the absence and in the presence of distinct DENV C concentrations, at different KCl concentrations (adapted from Carvalho et al., 2012). (B) LDs ζ-potential values determined before (filled symbols) or after limited proteolysis (empty symbol), in the presence of DENV C (squares) or pep14-23 (circles) (adapted from Martins et al., 2012).

FIGURE 6

Representative fusion event of two lipid droplets. Fusion of two lipid droplets observed in mammary epithelial cells (MEC) treated with 100 μM free palmitic acid + 10 μM 3-deazaadenosine. LDs were stained with Nile red and MEC were imaged on a time-lapse system. Scale bar: 5 μm [reprinted by permission from Springer Nature: Springer United States (Cohen et al., 2017), Copyright (2017)].

FIGURE 7

DENV infected cells accumulate the C protein around LDs. (A) DENV C is targeted to LDs in BHK, HepG2, and C6/36 cells infected with DENV. (B) DENV infection increases the number of LDs. (C) DENV C expression increases the number of LDs (adapted from Samsa et al., 2009).

FIGURE 8

Transmission electron microscopy of cells infected with viruses, showing LDs morphological changes. (A) Tamarin liver tissue uninfected and (B–D) infected with GB virus B, an unclassified flavivirus similar to HCV. Lipid droplets (pointed by →, and with electron-dense surface deposits), mitochondria () and the nucleus (N) can be identified. The scale bar corresponds to 1 μm. Most hepatocytes of the infected animal contain multiple small (ca. 2 μm diameter) LDs. They may be indicative of ongoing viral replication [reprinted from Martin et al. (2003). Copyright (2003) National Academy of Sciences, United States].

FIGURE 9

Stimulated Raman scattering image processing. (A) The intensity and size of each LD can be measured. (B) For each cell, cultured with different concentrations of oleic acid, the total size, intensity, and number of LDs can be quantified. The relation of these LD morphology parameters among single cells can be inferred under various culture conditions. A dot-plot shows a possible relation among those parameters. The different colors of the dots represent different culture conditions [adapted with permission from Cao et al. (2016). Copyright (2016) American Chemical Society].

FIGURE 10

Atomic force microscopy image of LDs isolated from HepG2 cells. LDs were deposited onto freshly cleaved muscovite mica. The inset shows a cross-section profile of two LDs with a diameter of approximately 200 nm and height of 15–20 nm (adapted from Carvalho et al., 2012).

FIGURE 11

AFM-based force spectroscopy results for DENV C interaction with LDs. (A) Typical force-extension curves of a control experiment with an unmodified silicon nitride AFM tip interacting with the mica surface (gray line), where adhesion forces were undetectable, and of a DENV C-functionalized tip interacting with a LD isolated from HepG2 cells (black line). Force rupture histograms of DENV C–LDs binding in buffer with (B) 10 mM KCl, (C) 100 mM KCl, (D) 400 mM KCl (adapted from Carvalho et al., 2012) or (E) 100 mM KCl plus 100 μM pep14-23 (adapted from Martins et al., 2012).