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Scrambled or flipped: 5 facts about how cellular phosphatidylserine localization can mediate viral replication

['Marissa Danielle Acciani', 'Department Of Infectious Diseases', 'College Of Veterinary Medicine', 'University Of Georgia', 'Athens', 'Georgia', 'United States Of America', 'Melinda Ann Brindley', 'Department Of Population Health']

Date: 2022-04

Funding: Research reported in this publication was supported by the National Institute of Allergy And Infectious Diseases of the National Institutes of Health under Award Number R01AI139238 (MAB). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2022 Acciani, Brindley. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Introduction

Host cell lipids are intimately involved in virus replication. Enveloped viruses are enshrouded in lipids from host cell membranes, shielding particles from the immune system and extracellular environment. Viruses can up-regulate cell lipid synthases to stimulate lipid production or recruit synthases to sites of viral replication. Furthermore, some viruses can induce autophagy to free stored lipids and optimize replication. Viruses can also remodel the composition of cell membranes, altering the concentration or localization of components such as sterols, sphingolipids, and phospholipids [1]. Here, we discuss virus-directed localization of phosphatidylserine (PtdSer), through host flippases and scramblases.

PtdSer is an essential anionic phospholipid nonuniformly distributed throughout the cell. PtdSer synthases located in the mitochondria-associated membranes of the endoplasmic reticulum (ER) produce PtdSer, which is then transported primarily to the plasma membrane via vesicular trafficking. PtdSer distribution in the plasma membrane is asymmetric, as it is strictly found in the inner/cytoplasmic leaflet. While PtdSer makes up about 15 mol% of the total lipid in the plasma membrane, it accounts for about 30 mol% of the inner leaflet. Due to its negative charge, the concentration of inner leaflet PtdSer can impact the localization of plasma membrane-associating proteins, including those required for endocytosis, and is important for promoting membrane curvature [2]. Cellular enzymes termed flippases maintain the asymmetric distribution of PtdSer (Fig 1A). Plasma membrane flippase complexes include a Type 4 P-Type ATPase (P4-ATPase) family member, which binds and flips the phospholipids, and a cell division cycle protein 50 (CDC50) family member that facilitates folding and localization of the complex [2,3].

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TIFF original image Download: Fig 1. PtdSer flipping and scrambling in the plasma membrane. PtdSer is shown in orange, and all other phospholipid species are shown in gray. (A) In normal state cells, constitutively active flippase complexes transport outer leaflet PtdSer to the inner leaflet, resulting in highly asymmetric PtdSer distribution. (B, C) Flippase inactivation paired with scramblase activation results in high levels of exposed PtdSer on cell surfaces. (B) When cells undergo apoptosis, executioner caspases inactivate flippases and activate scramblase XKR8, which nonspecifically shuffles phospholipids between leaflets. (C) During calcium signaling, flippases are inactivated or down-regulated on the cell surface (mechanism is poorly characterized), while scramblase TMEM16F is activated by direct calcium binding. Like XKR8, TMEM16F nonspecifically shuffles phospholipids between leaflets. Created in BioRender.com. PtdSer, phosphatidylserine; TMEM16F, transmembrane protein 16F; XKR, XK related. https://doi.org/10.1371/journal.ppat.1010352.g001

Cells expose PtdSer during specific cell signaling events; platelet PtdSer exposure regulates blood coagulation, and apoptotic cell PtdSer exposure marks the cell for phagocytic uptake and clearance. Trans-bilayer movement of PtdSer, however, requires overcoming a high-energy barrier, with spontaneous translocation occurring at a very low rate [4]. To induce rapid PtdSer exposure, cells must inactivate flippases and activate scramblases to overcome the unfavorable action of translocating the polar head-groups across the hydrophobic bilayer (Fig 1B and 1C). Scramblases facilitate the trans-bilayer movement of phospholipids, enabling PtdSer exposure to occur rapidly [5]. Several plasma membrane-localized scramblase families have been identified. The transmembrane protein 16 (TMEM16) family includes calcium-dependent scramblases that facilitate temporary PtdSer exposure in response to an increase in intracellular calcium (Fig 1C) [6]. The XK-related (XKR) protein family includes scramblases activated by caspase cleavage. XKR8, the most well-characterized member, facilitates the nonreversible exposure of PtdSer after apoptosis induction (Fig 1B) [7]. Inside the cell, transmembrane protein 41B (TMEM41B) and vacuole membrane protein 1 (VMP1) were recently identified as an ER-localized scramblase that help distribute PtdSer in the ER membrane [8–10]. When incorporated into liposomes, these scramblases can shuffle phospholipids in an ATP- and calcium- independent manner.

In this review, we will briefly summarize how viruses utilize PtdSer-regulating enzymes during their replication cycle. While the majority of these studies investigated virus-induced PtdSer scrambling, we will also discuss several instances where flippases were linked to virus replication.

Entry through apoptotic mimicry Numerous enveloped viruses including dengue virus [11,12], Ebola virus (EBOV) [11,13–18], chikungunya virus [11,13,19], vaccinia virus [20–22], and others [11,13,19,23–33] utilize PtdSer receptors for initial host cell attachment, a mechanism termed apoptotic mimicry (Fig 2A). PtdSer within the outer leaflet of the viral envelope mediates binding to PtdSer receptors on phagocytic cells that typically recognize PtdSer-coated apoptotic bodies. Even the infectivities of nonenveloped enteroviruses and Hepatitis A virus are enhanced through PtdSer receptors because naked particles and RNA can be delivered into cells as cargo in PtdSer-rich exosomes [27,28]. Host cells produce many receptors that bind to PtdSer including T-cell immunoglobulin and mucin domain (TIM) proteins, Tyro3, Axl, and Mer (TAM), CD300 receptors, scavenger receptors, and integrins, which can be used by viruses with some redundancy [18,34]. PtdSer receptor utilization and virus entry have recently been reviewed [35]. PPT PowerPoint slide

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TIFF original image Download: Fig 2. PtdSer scrambling and virus replication. PtdSer is shown in orange and all other phospholipid species are shown in gray. (A) Apoptotic mimicry. Enveloped viruses containing exposed PtdSer can enter cells by attaching to cell surface apoptotic clearance receptors like TIMs, TAMs, CD300 receptors, scavenger receptors, and integrins, which typically bind and internalize PtdSer-coated apoptotic debris. (B) Enveloped viruses that fuse at the plasma membrane, including HIV, EHV-1, and SARS-CoV-2, trigger calcium-induced PtdSer scrambling upon cell surface binding, which enhances envelope-membrane fusion. TMEM16F was specifically shown to mediate this process for HIV and SARS-CoV-2. (C) (+) RNA viruses such as flaviviruses and coronaviruses rely on PtdSer scramblase TMEM41B for the formation of membranous ER RC, which are the primary sites of genome replication. (D) PtdSer scrambling mediated by caspase-cleaved XKR8 was shown to enhance VSV and EBOV budding. It was also shown that XKR8 is incorporated into EBOV-like particle envelopes. Created in BioRender.com. EBOV, Ebola virus; EHV-1, equine herpesvirus 1; ER, endoplasmic reticulum; HIV, human immunodeficiency virus; PtdSer, phosphatidylserine; RC, replication complex; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus 2; TAM, Tyro3, Axl, and Mer; TIM, T-cell immunoglobulin and mucin domain; TMEM16F, transmembrane protein 16F; TMEM41B, transmembrane protein 41B; VMP1, vacuole membrane protein 1; VSV, vesicular stomatitis virus; XKR, XK related. https://doi.org/10.1371/journal.ppat.1010352.g002 Viral envelope PtdSer is acquired during budding from either PtdSer-rich inner membranes or the plasma membrane. For viruses that bud from the plasma membrane, they must first alter the asymmetric PtdSer distribution to later engage PtdSer receptors. Many viruses induce apoptosis, thereby inactivating flippases and activating scramblases, increasing PtdSer on the plasma membrane and viral envelope. Some viruses also modulate cellular calcium signaling pathways to increase cytosolic calcium levels, which would trigger calcium-activated scramblases [36]. Studies examining EBOV PtdSer levels have implicated both apoptotic scramblase XKR8 [37,38] and calcium-activated scramblase TMEM16F [39] in EBOV envelope PtdSer and infectivity.

Scramblase TMEM16F and fusion Enveloped viruses must mediate fusion of viral and cellular membranes to initiate replication, and exposed PtdSer plays a role in this process (Fig 2B). Ptdser scrambling can alter the fusogenic properties of lipid bilayers either directly, as scrambled PtdSer can promote the formation of fusion intermediates, or indirectly by recruiting fusion machinery or restructuring fusion proteins [40]. Several studies suggest that PtdSer scrambling enhances viral fusion at the plasma membrane. For alphaherpesviruses equine herpesvirus 1 (EHV-1) and herpes simplex viruses 1 and 2 (HSV-1 and HSV-2), virus cell surface binding induced calcium signaling and PtdSer exposure, which enhanced viral fusion and entry [41,42]. Human immunodeficiency virus (HIV) and Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), which can also fuse at the plasma membrane, activate TMEM16F scrambling during the fusion process [43–45]. TMEM16F is localized in the plasma membrane and is activated during periods of high intracellular calcium [6]. Loss of TMEM16F at the plasma membrane, or functional inhibition of TMEM16F activity with small molecules, results in failure to produce fusion pores and therefore inhibits infection and/or syncytia formation [43,44].

Scramblases TMEM41B and VMP1 in (+)RNA viral replication During replication, positive-sense (+)RNA viruses remodel internal cellular membranes to form replication complexes (RCs), which is thought to improve replication efficiency and shield these sites from host antiviral defenses [46]. Recent evidence suggests that PtdSer scramblases are important for (+)RNA virus replication, likely due to their impact on RC formation (Fig 2C). Independent genome-wide screens for viral host factors identified ER scramblase TMEM41B as an essential component in flavivirus and coronavirus replication [47–51]. Cells deficient in TMEM41B failed to form flavivirus RC, while other viruses that do not replicate in ER RCs were unaffected [50]. Eliminating TMEM41B in mice also significantly delayed murine coronavirus disease progression in vivo [51]. ER scramblase VMP1, which shares a number of functional and structural similarities with TMEM41B [52], also appeared in 2 of these viral host factor screens [47,50]. Cells lacking VMP1 were significantly resistant to flavivirus infection [50] and somewhat resistant to coronavirus infection [47,51]; however, in-depth mechanistic studies on how VMP1 interferes with virus replication were not performed. Likely, the loss of trans-bilayer phospholipid movement in the ER, combined with altered lipid mobilization and cholesterol distribution observed in TMEM41B and VMP1 knockout cells [9,49,50], prevents ER membrane rearrangements that enable robust genome replication of (+)RNA viruses that replicate in this compartment.

Scramblase XKR8 and viral budding While investigating the role of scramblase XKR8 in EBOV entry through apoptotic mimicry, our group observed that XKR8-knockout human haploid HAP1 cells consistently released fewer recombinant vesicular stomatitis virus particles and EBOV virus-like particles (VLPs) [38]. VLP release was restored by transfecting XKR8-knockout HAP1 cells with exogenous XKR8. Our data further indicated that during VLP production, caspase-activated XKR8 scrambles PtdSer in the plasma membrane, resulting in enhanced particle release (Fig 2D). Because PtdSer can induce membrane curvature, it is likely that PtdSer scrambling in the plasma membrane promotes the egress of enveloped viruses. Curiously, EBOV matrix protein VP40 must first bind to inner leaflet PtdSer during particle assembly, followed by PtdSer exposure [53]. Thus, it will be interesting to examine the exact stimuli leading to scramblase activation and how this timing is achieved to enhance particle budding. Previous work by Nanbo and colleagues did not describe altered VLP production in XKR8 knockdown HEK293T cells, suggesting that either (1) minimal levels of XKR8 can support VLP budding and/or (2) XKR8 enhances VLP budding in a cell type–dependent manner [37]. These possibilities must also be explored to fully characterize the role of Ptdser scrambling in enveloped virus budding.

[END]

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