key: cord-0009859-ykw7x2gp authors: Aktepe, Turgut E.; Mackenzie, Jason M. title: Shaping the flavivirus replication complex: It is curvaceous! date: 2018-06-29 journal: Cell Microbiol DOI: 10.1111/cmi.12884 sha: 05427026b5365c77d390b06c4e7abe2e16a69bd2 doc_id: 9859 cord_uid: ykw7x2gp Flavivirus replication is intimately involved with remodelled membrane organelles that are compartmentalised for different functions during their life cycle. Recent advances in lipid analyses and gene depletion have identified a number of host components that enable efficient virus replication in infected cells. Here, we describe the current understanding on the role and contribution of host lipids and membrane bending proteins to flavivirus replication, with a particular focus on the components that bend and shape the membrane bilayer to induce the flavivirus‐induced organelles characteristic of infection. The Flavivirus genus is the largest of the Flaviviridae family, which are among some of the most significant emergent or re-emergent human pathogens worldwide. Examples include West Nile (WNV), dengue (DENV), Zika (ZIKV), yellow fever (YFV), and Japanese encephalitis (JEV) viruses. These viruses require an arthropod vector where they are transmitted to vertebrates through an infected mosquito during its blood-feeding cycle. Although flaviviruses are responsible for hundreds of millions of infections worldwide, global eradication of flaviviruses remain a challenging task mainly due to the mosquito intermediate vector. Interestingly, disturbances within the vector-vertebrate equilibrium have resulted in a significant interregional spread of these viruses (Chambers, Hahn, Galler, & Rice, 1990) . Most flavivirus infections are asymptomatic however, also provoke a range of clinical manifestations from mild flu-like symptoms to severe complications. More specifically, WNV causes meningitis and encephalitis (Sejvar & Marfin, 2006) , DENV promotes dengue haemorrhagic fever and dengue shock syndrome (Gubler, 1998) , and ZIKV induces microcephaly and Guillain-Barré syndrome (Mlakar et al., 2016) . Currently, the only effective vaccines available towards flaviviruses are the tick-borne encephalitis virus and JEV purified, inactivated virus vaccines and the YFV 17D live attenuated virus vaccine (Chambers et al., 1990; Leyssen, De Clercq, & Neyts, 2000; Monath, 1987; Stock, Boschetti, Herzog, Appelhans, & Niedrig, 2012) . The (+)RNA translocates to the cytoplasmic surface of the endoplasmic reticulum (ER) where it is then translated by the host ribosome into a polyprotein and processed into 10 proteins (Pijlman et al., 2008) . Due to the limited number of viral proteins, host factors such as lipids and proteins are sequestered and exploited to assist in viral replication. Viruses require these host factors for entry, transcription and translation, immune evasion, and finally egress, which are vital stages of the viral life cycle and frequent targets during the design of "novel" antiviral drugs. However, an aspect that is generally overlooked and is a significant hallmark of almost every (+)RNA virus is the formation of virus-induced membrane structures (Mackenzie, 2005 ; Martín-Acebes, Vázquez-Calvo, & Saiz, 2016; Nagy & Pogany, 2011; Neufeldt, Cortese, Acosta, & Bartenschlager, 2018) . In this review, we will focus on the role of these virus-induced membrane structures during flavivirus replication, examine the host factors (lipids and proteins) required for the biogenesis of these structures, and discuss the importance of targeting these structures during antiviral therapy. A common feature of arguably all (+)RNA viruses is the formation of virus-induced membrane "organelles" during replication (Table 1) . Barajas, Jiang, & Nagy, 2009 Vps4p AAA+ ATPase Aids in the viral RC formation by interacting with the viral RNA Barajas, de Castro Martín, Pogany, Risco, & Nagy, 2014 Erg25, SMO1, and 2 Sterol synthesis and RC formation Sharma, Sasvari, & Nagy, 2010 (Continues) Pestivirus and hepacivirus replication occurs within a perinuclear matrix defined as the membranous web (Egger et al., 2002) ; bromoviruses induce numerous intraluminal ER membrane invaginations (Lee & Ahlquist, 2003) ; and alphaviruses replicate in spherulelined cytoplasmic vacuoles derived from the lysosome and endosomes (Froshauer, Kartenbeck, & Helenius, 1988) . Flaviviruses are no exception to this process as they induce the formation of vesicle packets (VPs) and convoluted membranes/paracrystalline arrays (CM/PC) (Mackenzie, Jones, & Young, 1996) . WNV-induced ER rearrangements are first observed before the end of the latent period (9 to 10 h.p.i) and is apparent with an increase in cytoplasmic vacuoles (Ishak, Tovey, & Howard, 1988; Ng, 1987; Ng & Hong, 1989; Westaway, Mackenzie, Kenney, Jones, & Khromykh, 1997) . As the infection progresses, membrane structures protruding the rough ER membranes proliferate with whorls of fibres in various vacuoles (Ng, 1987 ; later confirmed as replicating RNA by ultrastructural studies) and are followed by the formation of microtubule paracrystals (Ng & Hong, 1989 ). These structures have been confirmed as three continuous membranous structures: VP, CM/PC (Mackenzie et al., 1996; Westaway et al., 1997) . The VPs are groups of 70-to 100-nm vesicles proliferating and enclosed by the rough ER. They contain electron dense material (viral dsRNA) and viral proteins (NS1, NS2A, NS3, NS4A, and NS5; Cortese et al., 2017; Junjhon et al., 2014; Mackenzie et al., 1996; Mackenzie, Khromykh, Jones, & Westaway, 1998; Miorin et al., 2013; Welsch et al., 2009; Westaway et al., 1997; Westaway, Khromykh, & Mackenzie, 1999) , which are proposed to form the rep- to the CM/PC where it is proposed to undergo translation and proteolytic processing by the viral protease NS2B-3 and host signalase (Westaway et al., 1997) . CM/PC structures are continuous with the rough ER with the WNV CM/PC containing markers from the ER-Golgi intermediate compartment (Mackenzie, Jones, & Westaway, 1999) . Interestingly, WNV-infected mammalian and insect cells contain both VP and CM structures (Ng, 1987) ; however, these structures were only observed in DENV-infected mammalian cells. structures (Junjhon et al., 2014) . is not entirely understood but believed to play similar roles between different viruses. The VPs will conceal the dsRNA and the RCs, preventing detection by pathogen recognition receptors (Overby, Popov, Niedrig, & Weber, 2010; Uchida et al., 2014) , as well as antiviral proteins such as PKR (Samuel et al., 2006) and MxA (Hoenen et al., 2014; Hoenen, Liu, Kochs, Khromykh, & Mackenzie, 2007) . Furthermore, the process of compartmentalisation increases the local concentration of replicative components, narrows RNA replication and translation to specific sites, acts as a scaffolding for RC anchoring to membranes, and tethers the viral RNA during unwinding (Mackenzie, 2005; Miller & Krijnse-Locker, 2008) . Combined, these functions act as a central hub for viral replication that promotes exponential replication; however, the identification and role of host lipid and protein factors required to form these structures are not entirely understood. In addition to modulating the innate immune response during infection, viral proteins manipulate multiple pathways to regulate cellular homeostasis. Genome-wide RNA interference and CRISPR screens in Lipids are a diverse group of naturally occurring organic compounds that are synthesised from fatty acids and their derivatives. Lipids are one of the most abundant type of cellular molecules that display innumerable amounts of biochemical and physiological cellular functions. They are the main constituent of cellular membranes (plasma membrane, ER, Golgi, endosome, and lysosomes); however, the lipid composition constructing these structures vary among tissue types (Klose, Surma, & Simons, 2013; Muro, Atilla-Gokcumen, & Eggert, 2014) . Historically, it was believed that the primary role of lipids was limited to membrane morphogenesis and energy production; however, advances in lipidomics has led to the discovery of lipids in various cellular functions. These include structural changes and stability (induce and stabilise membrane curvature), protein modification (glycosylation), signalling platforms (such as lipid rafts), and inflammation (Kusumi et al., 2012; Muro et al., 2014) . The integration of specific lipid classes into membrane leaflets allows them to adjust their fluidity, plasticity, and topology, which further aids in maintaining membrane curvature and regulates signalling. Viral replication is a complex process that requires and regulates many host factors including lipid metabolism and redistribution (Stapleford & Miller, 2010) . Viruses interact with host lipids to enhance replication; however, certain lipids that are advantageous for one virus could be detrimental for another. WNV (Medigeshi, Hirsch, Streblow, Nikolich-Zugich, & Nelson, 2008) Fatty acid synthase (FASN) is a cytoplasmic, multifunctional protein that catalyses fatty acid synthesis. In the presence of NADPH, FASN primarily synthesises palmitate-a long-chain fatty acid (C 16:0)-from acetyl-CoA and malonyl-CoA (Wakil, 1989) . Martín-Acebes et al. Mammalian cellular membranes are composed of lipid bilayers containing phospholipids and cholesterol. Modification of membrane fatty acids, phospholipids, and cholesterol content disrupts membrane fluidity and affects a variety of cellular functions (Smith, 1994) . Cholesterol biosynthesis is regulated within the ER by the sterol regulatory element-binding protein, a membrane-bound transcription factor. Sterol regulatory element-binding proteins play a key role in activating genes that upregulate cholesterol synthesis and uptake, such as 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) and FASN (Simons & Ikonen, 2000) . Additionally, cellular cholesterol is further regulated by the intake of extracellular cholesterol (Goldstein & Brown, 1984) and the efflux of intracellular cholesterol (Schmitt & Tampé, 2002 Sphingolipids are generally composed of a long-chain sphingoid base, amide-linked fatty acids, and a polar (carbohydrate) head group at the 1-position, except for ceramide and sphingomyelin, which contains a hydroxy at the 1-position and a phosphorylcholine head group, respectively (Hannun & Bell, 1989) . Sphingolipids consist of four main members: sphingomyelin, ceramide, sphingosine, and sphingosine-1phosphate. Due to the central position occupied by ceramide in the sphingolipid pathway, it is considered as the central metabolic hub for sphingolipid biosynthesis and catabolism (Hannun & Obeid, 2008) . Removal of phosphorylcholine from sphingomyelin by the hydrolytic activity of acid sphingomyelinase results in the production of ceramide through the sphingomyelinase pathway (Utermöhlen, Herz, Schramm, & Krönke, 2008) . Ceramide is also synthesised in the ER through the de novo pathway by the catalysis of serine and palmitoyl-CoA by serine-palmitoyl-coenzyme A transferase, which acts as the first committed step in ceramide biosynthesis. Finally, recycling of complex sphingolipids through the salvage pathway can convert sphingosine into ceramide by the enzymatic activity of ceramide synthase (Merrill & Wang, 1992) . Over the past few decades, sphingolipids have been shown to regulate cellular homeostasis almost at every level. Ceramide has been shown to regulate cell senescence (Venable, Lee, Smyth, Bielawska, & Obeid, 1995) , cell stress responses such as differentiation, cell-cycle arrest, and apoptosis and induces membrane curvatures (Obeid, Linardic, Karolak, & Hannun, 1993) . Sphingomyelin does not have the tendency to form membrane curvature due to the phosphorylcholine head group. However, the formation of ceramide by cleaving the phosphorylcholine head group causes a structural change resulting in a cone-shaped lipid structure, which has the tendency to induce spontaneous negative curvature. The presence of ceramide on one leaflet of a lipid bilayer enhances membrane bending (negative curvature) and the tendency to form hexagonal phases II structures (Goñi & Alonso, 2002; Krönke, 1999; Utermöhlen et al., 2008) . The unique structure of ceramide can also influence cellular signalling by effecting membrane microdomain function (such as lipid rafts) and vesicular transport. Reduction of intracellular ceramide levels attenuated WNV replication but enhanced DENV replication (Aktepe et al., 2015) . These results suggest that a regulated flux of sphingomyelin-to-ceramide conservation is essential for flavivirus infection, where a regulated balance of sphingolipids is required during specific stages of viral replication. Caution should be taken when designing therapeutic agents against flaviviruses as clearance of one virus may be detrimental for another. Two lipidomic analyses on WNV-infected mammalian cells and DENVinfected mosquito cells have revealed a distinct modulation of the phospholipid landscape upon infection (Liebscher et al., 2018; Perera et al., 2012) . Both these studies observed a significant decrease in phosphatidylcholine (PChol) abundance with an increase in lyso-PChol. This change is indicative of increased activity of the host enzyme Phospholipase A2, a role that has been recently shown for at least WNV (Liebscher et al., 2018) . In contrast to ceramide, lyso-PChol exhibits the capacity to induce positive membrane curvature due to its relatively large hydrophilic head group in relation to its hydrocarbon tail (Fuller & Rand, 2001) . Therefore, the combination of ceramide and lyso-PChol to induce both negative and positive curvature would align with the requirements of the invagination from the ER to form the VP. A more recent study has also revealed a highly dynamic regulation of lipid homeostasis in DENV-infected mosquitoes (Chotiwan et al., 2018) , which again showed major changes in phospholipids, glycerophospholipids, glycerolipids, and sterol lipids. This study additionally showed that the entire repertoire of lipid biochemistry contributes to DENV replication including lipid synthesis, lipolysis, lipid conversion, β-oxidation, and/or redistribution to provide the optimal lipid environment and platforms facilitating efficient virus replication. In addition to lipids, host proteins are crucial in almost every step of (+) The RTN family of proteins are membrane-associated proteins that are prominently found on the ER membrane, where they induce ER membrane shaping and stabilise highly curved ER membrane tubules (Voeltz, Prinz, Shibata, Rist, & Rapoport, 2006 and maintain an open channel to the cytoplasm (Diaz, Wang, & Ahlquist, 2010) . Furthermore, the E71 2C protein, which associates with host membrane vesicles to induce viral RCs, interacts with host RTN3 protein to aid in infectivity, viral protein, and dsRNA synthesis (Tang et al., 2007) . Translation of viral proteins on the surface of the ER acts as a platform for protein-protein and protein-lipid interactions. Viral proteins interact with DNAJC14, which acts as a chaperone to modulate VP formation (Yi et al., 2011 , Yi et al., 2012 , potentially on cholesterol-rich microdomains . We speculate that the viral protein NS4A (based on its predicted topology, structure, and membrane remodelling capacity [Roosendaal et al., 2006 , Miller et al., 2007 ) induces membrane curvature while interacting with the host RTN3.1A protein (Aktepe et al., 2017) . We suggest that the biogenesis and recruitment of the cone-shaped lipids ceramide (Aktepe et al., 2015) and lyso-PChol (Liebscher et al., 2018) (Yi et al., 2012) . This characterisation of DNAJC14 highlights the importance of host factors in regulating flavivirus replication and the protein-protein and protein-lipid interactions that drive and shape RC formation and stabilisation. Flaviviruses have evolved to utilise host lipids and proteins to generate virus-induced membrane compartments to assist in replication. WNV and DENV co-opt FASN, phospholipids, cholesterol, and RTN3.1A to aid in membrane biogenesis, however differentially require ceramide during this process. FASN is recruited to sites of viral replication to stimulate an increase in fatty acid biosynthesis, which are modified and/or incorporated into the ER. Free fatty acids and cholesterol within the ER may lead to the expansion of the viral membranes and regulate membrane fluidity and curvature. Furthermore, we speculate that sphingolipids may have differential roles during flavivirus membrane morphogenesis. Cone-shaped and inverted cone-shaped lipids possess a tendency to change the physical properties of membranes by inducing stress onto the inserted side of the bilayer to form either positive or negative membrane curvature (Dowhan & Bogdanov, 2002) . The insertion of cone shaped ceramide lipids on one leaflet of the lipid bilayer enforces spontaneous negative membrane curvature ( Figure 1 ; Goñi & Alonso, 2002; Krönke, 1999; Utermöhlen et al., 2008) . The attenuation observed during WNV replication in the absence of ceramide may be linked to CM/PC and VP abnormalities. Although Perera et al. 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We thank all the researchers who have contributed to this exciting area of virology. Due to space and size constraints, we apologise for not citing all the literature related to this field. Our research was supported by a Project Grant (1081756)