Protein-driven membrane remodeling: Molecular perspectives from Flaviviridae infections

Abstract

The mammalian cell membrane consists of thousands of different lipid species, and this variety is critical for biological function. Alterations to this balance can be dangerous as they can lead to permanent disruption of lipid metabolism, a hallmark in several viral diseases. The Flaviviridae family is made up of positive single-stranded RNA viruses that assemble at or near the location of lipid droplet formation in the endoplasmic reticulum. These viruses are known to interfere with lipid metabolism during the onset of liver disease, albeit to different extents. Pathogenesis of these infections involves specific protein-lipid interactions that alter lipid sorting and metabolism to sustain propagation of the viral infection. Recent experimental studies identify a correlation between viral proteins and lipid content or location in the cell, but these do not assess membrane-embedded interactions. Molecular modeling, specifically molecular dynamics simulations, can provide molecular-level spatial and temporal resolution for characterization of biomolecular interactions. This review focuses on recent advancements and current knowledge gaps in the molecular mechanisms of lipid-mediated liver disease preceded by viral infection. We discuss three viruses from the Flaviviridae family: dengue, zika, and hepatitis C, with a particular focus on lipid interactions with their respective ion channels, known as viroporins.

Graphical abstract

Significance

Lipids are important players in the regulation of cellular processes; their roles often involve modulating interactions with proteins. Disruption to lipid dynamics is common to several viral infections. Pathogenesis from the Flaviviridae family of viruses is particularly linked to alterations in lipid homeostasis in the cell. Left untreated, chronic infections can lead to lipid dysregulation and complications in liver function, from steatosis to fibrosis, cirrhosis, and hepatic carcinoma in some cases. Interactions between viral proteins and the local membrane environment substantially change its lipid content and distribution. Recent studies show a correlation between viral proteins and lipid metabolism but often cannot provide molecular-level details to explain local changes observed during viral infection. This review highlights molecular modeling contributions to the understanding of molecular mechanisms involving viroporins and lipid membranes.

Introduction

The dynamic structure of cell membranes consists of a matrix of lipids—amphiphilic molecules that self-assemble into a bilayer with varying levels of lateral order—and proteins—molecules responsible for a variety of important cellular functions (1,2). Membranes act as barriers for the cell, where the interactions between lipids and proteins play important roles in cellular processes (3,4,5). The chemical structure of lipids is divided into headgroup, backbone, and fatty acid tails (Fig. 1). The chemical diversity of groups across lipid species affects the structural, mechanical, and biochemical properties of membranes and determines their roles in various cellular processes (6,7). In recent years, lipids have received renewed attention as active players in illnesses, often implicated in cell signaling cascades, making them potential biomarkers of disease (8,9,10). Specifically, the impact of viral infection on lipid metabolism has become a well-known phenomenon that precedes chronic diseases (11,12,13,14).

Figure 1.

Classification of lipids according to their chemical groups. Common backbones are shown in blue: glycerol for glycerophospholipids and triglyceride groups, and sphingosine for sphingolipids and ceramides. Phosphate groups are shown in red circles. Common headgroups are shown in green boxes: phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylinositol-4-phosphate (PI4P), and phosphatidylserine (PS); the sugar headgroup for the ganglioside GM3 is also boxed in green. To see this figure in color, go online.

Alterations in lipid metabolism tend to affect liver cells to a larger extent; certain viruses are known to target primarily the liver and its metabolism (15,16,17,18). In virus-induced liver pathologies, protein-lipid interactions play a larger role in the viral life cycle (13,19). Membrane lipid diversity provides a platform for specific interactions with viral proteins, which in turn change the local distribution of lipids in the membrane and its physical properties. In the liver, such changes are the gateway to nonalcoholic fatty liver diseases (NAFLDs). NAFLDs have a median prevalence of 20% worldwide and are characterized by steatosis, fat mismanagement in the liver. Left untreated, these diseases can progress to nonalcoholic steatohepatitis, hepatocellular carcinoma, and liver failure (20,21). Though some risk factors have been identified, the connection that underlies viral infection and NAFLD progression to nonalcoholic steatohepatitis is complex and not well understood (20,22). Current experimental methods study correlations between viral proteins and lipid content in different cellular locations. However, the links are often shown indirectly due to limitations in transmembrane (TM) protein imaging, reconstruction of physiologically accurate lipid membranes, and the lack of dynamics, to name a few (23,24).

To overcome these challenges, an increasingly utilized in silico method is molecular dynamics (MD) simulations. It allows the study of biomolecular processes at the all-atom (AA) or coarse-grained (CG) resolution to model macroscopic properties based on microscale interactions (25). MD simulations can be a powerful tool to predict particle interactions, protein conformational changes, and explore molecular mechanisms of action (26,27). However, simulation parameters, time scales, and sampling still pose a limitation, especially when there are high energy barriers 4(27).

Here, we present advances and current gaps in the understanding of molecular mechanisms of viral-induced liver disease. We focus our discussion on changes in lipid-lipid interactions due to the presence of viral ion channels, known as viroporins, from members of the Flaviviridae viral family, namely dengue, zika, and hepatitis C. We present a summary of MD simulation studies that provide molecular insights into viroporin-lipid interactions. Better understanding of the molecular mechanisms during viral infection that exacerbate liver disease can aid in the design of targeted therapeutics.

Flaviviridae viral family and lipid metabolism

Viruses from the Flaviviridae family have an enveloped, positive, single-stranded RNA (28). The family is split into four genera: Flavivirus, Hepacivirus, Pegivirus, and Pestivirus (29). These viruses are known to cause infectious diseases in humans and other mammals (30). Among these, viruses belonging to the Hepacivirus and Flavivirus genera have been well studied; this review focuses on dengue virus (DENV) and zika virus (ZIKV) of the Flavivirus genus and hepatitis C virus (HCV) of the Hepacivirus genus. All of these hijack regulatory networks of lipid homeostasis in the host (31,32).

DENV is estimated to infect about 390 million people a year, with about half of cases being asymptomatic, while ZIKV varies in number of infections, ranging from thousands to millions yearly (33). HCV chronic infection affects an estimated 58 million of the world’s population, with about half of infections leading to NAFLD-related problems (34,35). The progression of HCV to NAFLDs can be silent and asymptomatic, and treatment is primarily related to lifestyle adjustments (36,37). Currently, approved vaccines are available for DENV (38) but not for HCV or ZIKV, though some are in clinical trials (39,40). Antiviral therapies that target key nonstructural viral proteins are under investigation (33,41). Detailed studies of the molecular mechanisms of these viruses are needed to aid in the design of diagnostic and therapeutic targets.

Effect on lipid regulation and sorting

Viruses are known to hijack cholesterol and fatty acids synthesis pathways. DENV and HCV both form replication complexes at the endoplasmic reticulum (ER) through membrane remodeling events induced by their nonstructural proteins. The viral infection propagates through autophagy and lipid biosynthesis upregulation in the sterol regulatory element-binding protein pathway (42,43,44,45,46). Treatments for DENV, ZIKV, and HCV infections target interferon pathways, lipid synthesis trafficking, and transport pathways to eliminate the infection (47,48).

Changes in lipid content are also prevalent in all these infections. Studies have reported changes to ceramide, sphingomyelin (SM), phosphatidylcholine (PC), phosphatidylserine (PS), plasmalogens (PL), and dicarboxylic acid levels in ZIKV-infected cells (32). HCV infection increases levels of PC, phosphatidylinositol (PI), phosphatidylethanolamine (PE), and cholesterol, as well as fatty acids with longer acyl tails, and decreases levels of polyunsaturated fatty acids (9). Inhibiting the production of ceramides, phosphatidylinositol-4-phosphate (PI4P), and sterols decreases viral proliferation in DENV and HCV infections (42,49). Given the specificity of lipid species involved, targeting only those critical for virus life cycles has the potential of greatly reducing treatment toxicity; more studies are needed to refine promising candidates (49).

Effect on lipid droplet homeostasis

Lipid droplets (LDs) are triacylglycerol- and sterol-ester-containing aggregates that play active roles in viral life cycles and associate with intracellular membranes at viral assembly sites (31). LD metabolism is critical for viral replication and assembly of HCV, DENV, and ZIKV (18). Viral proteins such as capsid and NS5A of HCV, and NS4A and NS4B of DENV, interact with LDs during virus production (31,50,51). Interestingly, there have been sightings of LDs in the inner nuclear membrane with Flaviviridae viral proteins attached to them (18). This raises questions about unknown alternative or additional functions of viral proteins, a version of the proposed protein-specific phenomenon of “moonlighting” (52). These ties between viruses and LDs have motivated focus on them as therapeutic targets. Modulating LDs without toxic effects is possible (53), though the therapeutic efficacy of this approach is yet to be verified in vivo (31).

It is evident that specific viral proteins play roles in altering lipid metabolism pathways during infection, many of which are still being evaluated to fully understand their function in promoting virus infectivity (54). The rest of this review focuses on viral TM proteins called viroporins, molecular assemblies that act as ion channels for new viral particles and intervene during viral assembly.

Emerging relevance of viroporins

Viroporins are proteins that consist of small hydrophobic helices of less than 100 amino acids and are usually found embedded into the ER (55). They alter ion concentration gradients and traffic in the cell (56), leading to favorable environments for newly formed viral particles during egress. To combat this, antivirals have been designed to block some viroporin channels such as M2 of influenza A virus (IAV), p7 of HCV, and Vpu of HIV-1 (54,55).

Apart from regulating ion transport, viroporins are known to enable proper viral assembly and release (57). They can also contribute to the formation of vesicle-like structures, called viroplasms, through regulation of Ca²⁺ stores and gradients, as seen in rotavirus NSP4 protein (58). MD simulations can provide an atomistic perspective to understand the role of viroporins during viral infection. Well-studied examples include the M2 channel from IAV (5,54,59) and the Vpu protein from HIV-1 (60,61). MD simulations are able to predict key interactions between these viroporins and lipid membranes, revealing information about their mechanism of action at the molecular level. The rest of this review focuses on interactions of DENV, ZIKV, and HCV viroporins with membrane lipids in the context of local lipid redistribution and membrane remodeling. Table 1 summarizes key computational works and relevant experimental studies.

Table 1.

Summary of molecular studies of DENV, ZIKV, and HCV viroporins

Virus	Study	Protein	PDB	Ref.
DENV	CG	E/M – whole envelope	3J27	(62)
	AA and CG	E/M tetramer – 1, 3, and 15 clusters	3J27	(63)
	CG	E/M – whole envelope	3J27	(64)
	mutagenesis	prM/E complex	4B03	(65)
	cryo-EM	E/M single complex	1P58	(66)
	CG	E/M trimer	3J27	(67)
ZIKV	AA – accel. MD	E/M dimer	5IZ7	(68)
	mutagenesis	M – TM domains	6CO8, 5IRE	(69)
	CG	E/M trimer	5IRE	(67)
	AA	M	5IRE	(70)
	cryo-EM, mutagenesis	E/M dimer	7JYI	(71)
HCV	mutagenesis	p7 monomer	strain 2a sequence	(72)
	AA	p7 channel	2M6X	(73)
	AA	p7 monomers and channels	3ZD0, 2M6X, strain 1a sequence	(74)
	AA	p7 monomer TM domains	strain 1a sequence	(75)

AA, all-atom; CG, coarse-grained; cryo-EM, cryoelectron microscopy; accel. MD, accelerated MD simulations; TM, transmembrane.

Studies on viroporins from the Flaviviridae family

DENV M protein

DENV affects a broad range of human functions, including capillary leakage and liver damage (76). The virus has three structural proteins, envelope (E), precursor membrane (prM), and capsid, and seven nonstructural proteins, NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 (77,78). This is a typical setup for flaviviruses and is also seen in ZIKV. prM, the precursor form of the M protein, is found in immature virions; it matures into the M protein after passing through the trans-Golgi network through cleavage of the prM protein by furin cellular protease (78). The precursor portion of M (pr) is necessary for preventing fusion of the E protein before the virions are ready for release (79).

Studies of the structure and behavior of the M protein in lipid membranes have been largely done experimentally. Cryo-electron microscopy identified three alpha helices: one of them amphipathic (AP) and protruding toward the cytosolic environment at a 20° angle, and two TM domains in antiparallel configuration in the outer membrane leaflet (see Fig. 2 d) (66). A mutagenesis study showed that Gln114, Gln124, Trp126, Lys128, and Arg129 are highly conserved residues of the AP domain that are tightly linked to virus infectivity (65).

Figure 2.

DENV E/M protein dimer complex (PDB: 3J27). E protein ectodomain in blue, stem in green, and TM sections in red. M protein stem in purple, and TM sections in yellow. Amino acids with a positive charge are shown in blue, negatively charged in red, polar in green, and nonpolar in white. Outer and inner membrane leaflets are represented by top and bottom red lines, respectively. (A) E protein ectodomain amino acids interact with anionic PS lipids. (B) E protein amino acids lining membrane interact with adjacent lipids to increase virus stability and robustness. (C) Arg60 of M protein and Arg471 of E protein, both in blue, anchor protein by interacting with lower leaflet lipid headgroups. (D) Trp26, in white, and His28 and His57, both in blue, of M protein preferentially interact with PE lipids. To see this figure in color, go online.

MD simulation studies of this protein with lipid bilayers are limited but reveal important details about membrane structural changes that stabilize protein structure and virus formation. Fig. 2 shows the dimeric E/M protein complex of DENV and its relative position in the membrane. The spatial configuration was used because an ion channel conformation for the M protein or E/M complex is not yet available. A CG study of an entire DENV envelope with PC, PE, and PS lipids showed that the TM portions of both M and E proteins induce positive curvature in the inner leaflet through interactions with lipid headgroups, with at least 45% of normalized protein-lipid contacts occurring with PS lipids (64). A viral envelope model containing only POPC lipids leads to a threefold greater root-mean-square deviation of the TM region, revealing the importance of membrane charge and specific lipid species in determining TM configuration. Enrichment of PS near the protein can be achieved with just the ectodomain (Fig. 2 a). Another study on a membrane model based on lipidomics of DENV-infected cells shows that 70% of lipids in outer leaflet interact with the protein, leading to a 7 Å decrease in bilayer thickness near the protein, ∼25% denser lipid packing in the inner leaflet, and low lipid diffusion seen in robust and stable viruses such as IAV (Fig. 2 b) (62). This is interesting, given that IAV robustness comes from the presence of 40%–50% cholesterol composition (80), while that of the DENV models did not, highlighting the role of the protein complexes in stabilizing the DENV envelope.

Simulations of a PC, PE, PS, and cholesterol-containing membrane shows that only three units of the E/M complex are needed to generate a stable curvature that resembles that of the virus (R = 25 nm). Curvature generation is enabled through protein-lipid interactions at the locations of Arg60 and Arg471 in the M and E proteins, respectively (Fig. 2 c) (63). Mishra et al. further emphasize the role of arginine residues and their interactions with anionic lipids on mechanisms of pore formation in viruses (81). In addition, Trp26, His28, and His57 were shown to induce lipid re-sorting and recruit PE lipids to protein-binding sites in membranes containing PC, PE, PS, SM, and PL lipids, leading to a ∼5 Å decrease in membrane thickness and ∼3 Å positive curvature near the protein (Fig. 2 d) (67). Finally, oligomers of 15 E/M units are needed for complete vesiculation of a rectangular membrane patch through a gradual increase in curvature and compensation of tension by migration of 18% of inner leaflet lipids to the outer leaflet (63). These studies highlight the specificity of protein residues and lipid species that modulate the large-scale process of membrane vesiculation and must operate in concerted dynamics.

ZIKV M protein

The Zika fever epidemic of 2015 incited a pronounced interest to understand ZIKV behavior due to serious side effects on neurological function, the reproductive system, and fetal development (82,83). As part of the Flavivirus genus, ZIKV contains the same set of structural and nonstructural proteins as DENV, including the prM protein attached to the E protein (see Fig. 3) (83).

Figure 3.

ZIKV E/M protein dimer complex (PDB: 5IRE). The conformation and partitioning of the peripheral and TM regions of E and M are effectively the same as in Fig. 2; whole E and M protein structures are colored in ice-blue and orange, respectively. Amino acids with a positive charge are shown in blue, polar in green, and nonpolar in white. Outer and inner membrane leaflets are represented by top and bottom red lines, respectively. (A) M protein: Arg10, Lys11, Arg15, and Arg23, all in blue and found in the N-terminus, interact with anionic PS lipids. (B) Thr57 and Ser58 from the M protein, both in green, and Trp474 from the E protein, shown in white, form one of two lipid-binding pockets needed for virus replication. (C) M protein residues Gly54 and Gln59, both in green, stabilize the channel conformations. To see this figure in color, go online.

Cryo-electron microscopy and mutagenesis studies revealed two lipid-binding pockets inside the E/M protein complex of ZIKV (71). One of them is shown in the inner membrane leaflet, formed by conserved residues Thr57, Ser58, and Trp474 (Fig. 3 b). This pocket halts viral replication when the polarity of the residues is removed or changed, likely due to loss of interactions with inner leaflet lipids. A mutagenesis study showed that Gly54, Gln59, and the entire second helix of the M protein, all in the membrane hydrophobic core, are necessary for the ion channel conductivity of the protein, suggesting that the oligomeric protein structure is greatly stabilized by the interactions of TM residues with the surrounding lipid environment (Fig. 3 c) (69).

Few simulation works have studied the role of ZIKV M protein interactions with lipids as they pertain to viral infectivity. A CG study of E/M protein trimers in a membrane containing PC, PE, PS, SM, and PL lipids showed a lipid-sorting effect that recruits PS lipids to the protein site through hydrophilic interactions with basic charged residues concentrated in the AP protruding helix of the M protein (Fig. 3 a) (67). This leads to a ∼10 Å decrease in bilayer thickness and a ∼3 Å positive curvature near and below the protein. Accelerated MD simulations with a POPC membrane model showed that low pH, a condition that occurs during E-protein-mediated fusion and prM dissociation, leads to protonation of His288, His323, and His446 residues, causing loss of interactions between E/M dimers. This further increases by ∼20% the energy required for protein-membrane binding and reduces protein compactness, as evaluated from center-of-mass distances between monomers and tilt angles between the protein and the membrane surface (68). In contrast, membrane response is consistent at both low and high pH, characterized by a 5 Å decrease in membrane thickness next to the protein due to lipid-protein interactions in the bilayer core. A study using both experiments and modeling examined the formation of M protein ion channels in virions (70). The AA simulations showed that the M protein favors a hexameric channel conformation in a POPC membrane model, with its AP helix resting at the membrane-water interface to promote pore stability, lest the channel pore closes within 50 ns of simulation (70).

In general, MD simulations of DENV and ZIKV viroporins have revealed how key residues interact with specific lipid species to change the local membrane structure and composition. However, most works focus on their behavior and function in the viral envelope, with an emphasis on the E protein still attached to it, rather than on the viroporin itself. The E/M complex is assumed to separate before forming functional M channels (65,70), and too few details are known about this. More studies are needed to determine if the channels form while in the prM conformation or only when M is free from the pr domain. These studies should aim to elucidate the relevance of protein-lipid interactions in stabilizing the M channel structure and to characterize the subsequent ER membrane remodeling that contributes to virus assembly.

HCV p7 protein

HCV, belonging to a different genus of Flaviviridae than DENV and ZIKV, has a different set of viral proteins; these include structural proteins core, E1, and E2 and nonstructural proteins p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B (84). p7 oligomerizes to form a hexameric channel inside the ER, with its N and C termini both facing the ER lumen, and its mid-region pointing toward the cytosol (84,85,86,87). A hexameric structure of the channel from the 5a viral strain of HCV was resolved using nuclear magnetic resonance (NMR) in 2013 (see Fig. 4) and is the only available channel structure of the viroporin (88). In silico predictions have suggested alternate monomeric conformations that remain stable in a channel conformation during simulation but have not been observed experimentally (89). Different ion-gating mechanisms have been proposed for p7, potentially due to the use of different virus strains, suggesting a unique and strain-dependent behavior for this viroporin (55).

Figure 4.

HCV strain 5a p7 hexameric channel (PDB: 2M6X). All monomers are colored in cyan. Amino acids with a positive charge are shown in blue, polar in green, and nonpolar in white. Outer and inner membrane leaflets are represented by top and bottom red lines, respectively. (A) Lys33 (Arg33 in strain 2a) and Arg35, shown in blue, interact with anionic lipids to stabilize TM orientation. (B) Tyr42 and Tyr45 in TM domain 2 (Tyr7 and Tyr10 when visualized with PDB: 2K8J), shown in green, interact with hydrophilic lipid headgroups to form a kink in the helix. (C) Amino acids in helix 3, the C-terminus of a monomer, interact with cholesterol, enabling the shift of His17 in helix 1 toward the inside of channel pore. To see this figure in color, go online.

Most of what is known about the role of p7 in HCV infection has been revealed through experiments. p7 is involved in viral assembly, budding, and possibly LD formation in collaboration with NS5B (90). It is also involved with NS2 and some structural E proteins in the recruitment of LDs during virus assembly and maturation (91,92). PS lipids enhance the ion channel function of p7 by improving channel permeabilization activity (93). The detailed interactions that underlie lipid resorting and recruitment remain understudied. Despite this, a mutagenesis study showed Arg33 and Arg35 interacting with anionic lipid headgroups to stabilize the TM orientation of p7 sourced from virus strain 2a (Fig. 4 a; note that Arg33 corresponds to Lys33 in the 5a strain structure shown in the center of this figure) (72). The study also showed that different viral strains of p7 promote virus assembly and release with varying potency, confirming how unique amino acid sequences modulate virus infectivity. One last experimental work showed that PE-rich lipid environments promote long-lasting ion release, compared with PC-rich environments that disrupt channel shape and promote short-burst patterns, illustrating the effect of lipid composition on channel conformation and function (94).

MD simulations have mostly examined the role of specific amino acids on the spatial conformation of p7 TM helices and resolved discrepancies in experimental studies. The NMR study by Ouyang et al. showed that His17, a critical amino acid for channel cationic selectivity, embeds into the protein matrix away from the channel pore (88), while biochemical studies concluded that it points inside the channel (95). AA simulations comparing POPC bilayers with and without cholesterol show that interactions of helix 3 of the protein monomers with cholesterol leads to spontaneous repositioning of a pair of His17 residues inside the ion channel (Fig. 4 c) (73). Upon cholesterol binding, the helices experience a 10° shift of their tilt angle with respect to the bilayer normal, which eventually causes the adjacent helix 1 His17 residue and the one opposite it in the channel pore to face one another. This offered a plausible explanation for the contrasting reports from experiment and showed that protein configuration is dependent on lipid environment.

A separate AA study of a p7 monomer from HCV strain 1a was done with a POPC membrane to examine the flexibility in the second helix of the p7 monomer sequence (75). Specifically, the role of Tyr residues in kink formation as the protein interacts and attracts charged lipids was evaluated (Fig. 4 b; note: these residues are unseen in strain 5a but are identifiable as Tyr7 and Tyr10 in strain 1b-based model 2K8J). Replacing Tyr42 and Tyr45 with hydrophobic residues enhances the kink by ∼7%, altering the secondary structure of the monomer within the membrane (75), which could affect oligomerization of the viroporin or its ion-gating function in the long run. To further discriminate among the monomer structures proposed for p7 from different virus strains, one study compares dynamics for a computationally generated p7 structure (96), and two others derived from NMR (74,88,89). At low pH conditions, the channel configuration from NMR (seen in Fig. 4) closes upon interactions between lipid headgroups and protein residues in the N terminus, accompanied by slight membrane thinning near the viroporin. Other structures show less interactions that result in an increased pore size due to a ∼10° increase in kink angles of their second TM helices (74).

Together, these works clearly show the dependency of p7 conformation on local lipid environment and viral strain origin and, by extension, its function and role in virus production. Notoriously, most of these studies are conducted with at most a binary bilayer composition, emphasizing the urgent need for studies that evaluate the effect of specific lipid species on p7 protein-lipid interactions and resulting membrane response. Such studies will help to characterize lipid recruitment patterns around p7 viroporins and determine the mechanical and structural factors that underlie membrane remodeling for viral assembly and long-term disruptions to lipid content in the ER observed during NAFLDs.

Outlook

This review presents studies of protein-lipid interactions during infections of the Flaviviridae viral family. We specifically discuss DENV, ZIKV, and HCV, including insights into molecular mechanisms that alter lipid-lipid integrations and membrane remodeling as viral assembly takes place. We examined these particular viruses as they are closely related to lipid dysregulation and liver metabolism that can lead to NAFLDs, especially when the infection is chronic. Despite the challenges in modeling large-scale dynamics of viral membrane fusion, budding, and vesiculation and their effect on membrane lipid redistribution, MD simulations allow us to examine molecular interactions and dynamics at high resolution.

Special attention is given to viroporins from the Flaviviridae family of viruses, which are known to assemble very close to the nucleation site of LDs and disrupt lipid synthesis pathways. There are only a handful of well-studied viroporins, like M2 of IAV and Vpu of HIV-1. Here, we present current knowledge on the viroporins from DENV, ZIKV, and HCV and the associated membrane response. Protein-lipid interactions consistently stabilize the viroporin conformation inside the membrane and drive changes in local membrane composition, structure, and dynamics. There is still a need for more studies of isolated protein-membrane systems to elucidate the mechanisms of membrane remodeling that promote viral infection. Leveraging molecular modeling techniques to this end will help in the design of effective therapeutics that target fundamental aspects of the viral life cycle and NALFD onset and progression.

Author contributions

O.C. and V.M.-G. discussed and decided on the content and organization of the review. O.C. reviewed literature and drafted the initial content. V.M.-G. supervised the writing process, guided discussions, and edited the final draft of the manuscript.

Editor: Meyer Jackson.