Role of lipids in virus replication

Viruses intricately interact with and modulate cellular membranes at several stages of their replication, but much less is known about the role of viral lipids compared to proteins and nucleic acids. All animal viruses have to cross membranes for cell entry and exit, which occurs by membrane fusion (in enveloped viruses), by transient local disruption of membrane integrity, or by cell lysis. Furthermore, many viruses interact with cellular membrane compartments during their replication and often induce cytoplasmic membrane structures, in which genome replication and assembly occurs. Recent studies revealed details of membrane interaction, membrane bending, fission, and fusion for a number of viruses and unraveled the lipid composition of raft-dependent and -independent viruses. Alterations of membrane lipid composition can block viral release and entry, and certain lipids act as fusion inhibitors, suggesting a potential as antiviral drugs. Here, we review viral interactions with cellular membranes important for virus entry, cytoplasmic genome replication, and virus egress.     

Virus entry, assembly, budding, and membrane rafts.

As intracellular parasites, viruses rely heavily on the use of numerous cellular machineries for completion of their replication cycle. The recent discovery of the heterogeneous distribution of the various lipids within cell membranes has led to the proposal that sphingolipids and cholesterol tend to segregate in microdomains called membrane rafts. The involvement of membrane rafts in biosynthetic traffic, signal transduction, and endocytosis has suggested that viruses may also take advantage of rafts for completion of some steps of their replication cycle, such as entry into their cell host, assembly, and budding. In this review, we have attempted to delineate all the reliable data sustaining this hypothesis and to build some models of how rafts are used as platforms for assembly of some viruses. Indeed, if in many cases a formal proof of raft involvement in a virus replication cycle is still lacking, one can reasonably suggest that, owing to their ability to specifically attract some proteins, lipid microdomains provide a particular milieu suitable for increasing the efficiency of many protein-protein interactions which are crucial for virus infection and growth.     

Virus Entry, Assembly, Budding, and Membrane Rafts

As intracellular parasites, viruses rely heavily on the use of numerous cellular machineries for completion of their replication cycle. The recent discovery of the heterogeneous distribution of the various lipids within cell membranes has led to the proposal that sphingolipids and cholesterol tend to segregate in microdomains called membrane rafts. The involvement of membrane rafts in biosynthetic traffic, signal transduction, and endocytosis has suggested that viruses may also take advantage of rafts for completion of some steps of their replication cycle, such as entry into their cell host, assembly, and budding. In this review, we have attempted to delineate all the reliable data sustaining this hypothesis and to build some models of how rafts are used as platforms for assembly of some viruses. Indeed, if in many cases a formal proof of raft involvement in a virus replication cycle is still lacking, one can reasonably suggest that, owing to their ability to specifically attract some proteins, lipid microdomains provide a particular milieu suitable for increasing the efficiency of many protein-protein interactions which are crucial for virus infection and growth.     

脂筏在病毒感染中的作用

脂筏是细胞膜上富含鞘脂和胆固醇的微区结构,广泛分布于细胞的膜系统.脂筏中含有诸多信号分子和免疫受体,在细胞的生命活动中扮演非常重要的角色.更为重要的是,脂筏为细胞表面发生的蛋白质-蛋白质和蛋白质-脂类分子间的相互作用提供了平台.研究表明,很多病毒可以利用细胞膜表面的脂筏结构介导其侵入宿主细胞,一些病毒可以借助脂筏结构完成病毒颗粒的组装和出芽.本文将综述不同类型的病毒如SV40、HIV等借助脂筏完成入侵以及流感病毒等利用脂筏完成组装和出芽的证据及机理,并概述目前研究病毒与脂筏相互作用的方法及存在的问题.深入研究脂筏在病毒感染中的作用,将有助于对病毒与宿主细胞的相互作用的理解,从而可能发现新的、有效的对抗病毒的方法。     

Lipids ‒ A key for hepatitis C virus entry and a potential target for antiviral strategies

Viruses have evolved to complex relationship with their host cells. Many viruses modulate the lipid composition, lipid synthesis and signaling of their host cell. Lipids are also an essential part of the life cycle of the hepatitis C virus (HCV). HCV is a major human pathogen, persistently infecting 170 million people worldwide, with no currently effective treatment available for all patients. HCV appears to make use of the host lipid metabolism and one common feature of chronic hepatitis C is the steatosis, characterized by excessive accumulation of triglycerides and lipid content in the liver. Thus, HCV lifecycle appears to be closely connected to host cell lipid metabolism, from cell entry, through viral RNA replication to viral particle production and formation/assembly.     

Lipids and flaviviruses,present and future perspectives for the control of dengue,Zika, and West Nile viruses

Flaviviruses are emerging arthropod-borne pathogens that cause life-threatening diseases such as yellow fever, dengue, West Nile encephalitis, tick-borne encephalitis, Kyasanur Forest disease, tick-borne encephalitis, or Zika disease. This viral genus groups > 50 viral species of small enveloped plus strand RNA virus that are phylogenetically closely related to hepatitis C virus. Importantly, the flavivirus life cycle is intimately associated to host cell lipids. Along this line, flaviviruses rearrange intracellular membranes from the endoplasmic-reticulum of the infected cells to develop adequate platforms for viral replication and particle biogenesis. Moreover, flaviviruses dramatically orchestrate a profound reorganization of the host cell lipid metabolism to create a favorable environment for viral multiplication. Consistently, recent work has shown the importance of specific lipid classes in flavivirus infections. For instances, fatty acid synthesis is linked to viral replication, phosphatidylserine and phosphatidylethanolamine are involved on the entry of flaviviruses, sphingolipids (ceramide and sphingomyelin) play a key role on virus assembly and pathogenesis, and cholesterol is essential for innate immunity evasion in flavivirus-infected cells. Here, we revise the current knowledge on the interactions of the flaviviruses with the cellular lipid metabolism to identify potential targets for future antiviral development aimed to combat these relevant health-threatening pathogens.     

Membrane-containing viruses with icosahedrally symmetric capsids

Viruses with an icosahedrally symmetric protein capsid and a membrane infect hosts from all three domains of life. Similar architectural principles are shared by different viral families, as exemplified by double-stranded DNA viruses such as PRD1 and STIV. During virus assembly, the membrane lipids are selectively acquired from the host cell. The X-ray structure of bacteriophage PRD1 revealed that the lipids are asymmetrically distributed between the two leaflets and facet length is controlled by a tape-measure protein. In most membrane-containing viruses, viral and host membranes fuse during viral entry. In the best-understood systems of the alphaviruses, flaviviruses and herpes viruses, fusion is mediated by viral glycoproteins. Recent structural advances reveal how very different protein architectures can be used to form trimeric extensions that extend into the target cell membrane and then fold back to mediate fusion of the target and viral membranes.     

Canine distemper virus infection requires cholesterol in the viral envelope

Cholesterol is known to play an important role in stabilizing particular cellular membrane structures, so-called lipid or membrane rafts. For several viruses, a dependence on cholesterol for virus entry and/or morphogenesis has been shown. Using flow cytometry and fluorescence microscopy, we demonstrate that infection of cells by canine distemper virus (CDV) was not impaired after cellular cholesterol had been depleted by the drug methyl-beta-cyclodextrin. This effect was independent of the multiplicity of infection and the cellular receptor used for infection. However, cholesterol depletion of the viral envelope significantly reduced CDV infectivity. Replenishment by addition of exogenous cholesterol restored infectivity up to 80%. Thus, we conclude that CDV entry is dependent on cholesterol in the viral envelope. Furthermore, reduced syncytium formation was observed when the cells were cholesterol depleted during the course of the infection. This may be related to the observation that CDV envelope proteins H and F partitioned into cellular detergent-resistant membranes. Therefore, a role for lipid rafts during virus assembly and release as well is suggested.     

Organelle biogenesis and intracellular lipid transport in eukaryotes.

The inter- and intramembrane transport of phospholipids, sphingolipids, and sterols involves the most fundamental processes of membrane biogenesis. Identification of the mechanisms involved in these lipid transport reactions has lagged significantly behind that for intermembrane protein traffic until recently. Application of methods that include fluorescently labeled and spin-labeled lipid analogs, new cellular fractionation techniques, topographically specific chemical modification techniques, the identification of organelle-specific metabolism, permeabilized cell methodology, and yeast molecular genetics has contributed to revealing a diverse biochemical array of transport processes for lipids. Compelling evidence now exists for ATP-dependent, ATP-independent, vesicle-dependent, and vesicle-independent transport processes that are lipid and membrane specific. ATP-dependent transport processes include the transbilayer movement of phosphatidylserine and phosphatidylethanolamine at the plasma membrane and the transport of phosphatidylserine from its site of synthesis to the mitochondria. ATP-independent processes include the transbilayer movement of virtually all lipids at the endoplasmic reticulum, the movement of phosphatidylserine between the inner and outer mitochondrial membranes, and the transfer of nascent phosphatidylcholine and phosphatidylethanolamine to the plasma membrane. The ATP-independent movement of lipids between organelles is believed to be due to the action of lipid transfer proteins, but this still remains to be proved. Vesicle-based transport mechanisms (which are also inherently ATP dependent) include the transport of nascent cholesterol, sphingomyelin, and glycosphingolipids from the Golgi apparatus to the plasma membrane and the recycling of sphingolipids and selected pools of phosphatidylcholine from the plasma membrane to the cell interior. The vesicles involved in cholesterol transport to the plasma membrane are different from those involved in bulk protein transport to the cell surface. The vesicles involved in recycling sphingomyelin to and from the cell surface are different from those involved in the assembly of newly synthesized sphingolipids into the plasma membrane. The preliminary characterization of these lipid translocation processes suggests divergent rather than unifying mechanisms for lipid transport in organelle assembly.     

Integrity of membrane lipid rafts is necessary for the ordered assembly and release of infectious Newcastle disease virus particles

Membrane lipid raft domains are thought to be sites of assembly for many enveloped viruses. The roles of both classical lipid rafts and lipid rafts associated with the membrane cytoskeleton in the assembly of Newcastle disease virus (NDV) were investigated. The lipid raft-associated proteins caveolin-1, flotillin-2, and actin were incorporated into virions, while the non-lipid raft-associated transferrin receptor was excluded. Kinetic analyses of the distribution of viral proteins in lipid rafts, as defined by detergent-resistant membranes (DRMs), in non-lipid raft membranes, and in virions showed an accumulation of HN, F, and NP viral proteins in lipid rafts early after synthesis. Subsequently, these proteins exited the DRMs and were recovered quantitatively in purified virions, while levels of these proteins in detergent-soluble cell fractions remained relatively constant. Cholesterol depletion of infected cells drastically altered the association of viral proteins with DRMs and resulted in an enhanced release of virus particles with reduced infectivity. Decreased infectivity was not due to effects on subsequent virus entry, since the extraction of cholesterol from intact virus did not significantly reduce infectivity. Particles released from cholesterol-depleted cells had very heterogeneous densities and altered ratios of NP and glycoproteins, demonstrating structural abnormalities which potentially contributed to their lowered infectivity. Taken together, these results indicate that lipid rafts, including cytoskeleton-associated lipid rafts, are sites of NDV assembly and that these domains are important for ordered assembly and release of infectious Newcastle disease virus particles.     

Comprehensive proteomic analysis of host cell lipid rafts modified by HBV infection

Lipid rafts are cholesterol- and sphingolipid-rich membrane microdomains that have been shown to participate in the entry, assembly and budding of various viruses. However, their involvement in HBV replication remains poorly characterized. In a preliminary study, we observed that HBV release could be markedly impaired by methyl-β-cyclodextrin mediated depletion of cholesterol in lipid rafts, and that this effect could be reversed by replenishment of exogenous cholesterol, suggesting that lipid rafts play an important role in the HBV life cycle. To further understanding how HBV exploited host cell lipid rafts to benefit replication, comprehensive proteomic approaches were used to profile the proteome changes of host cell lipid rafts in response to HBV infection using 2DE-MS/MS, in combination with SILAC-based quantitative proteomics. Using these approaches, a total of 97 differentially expressed proteins were identified. Bioinformatics analysis suggested that multiple host cell pathways were involved in the HBV infection processes including signal transduction, metabolism, immune response, transport, vesicle trafficking, cell adhesion and cellular ion homeostasis. These data will provide valuable clues for further investigation of HBV pathogenesis.     

The ins and outs of endoplasmic reticulum‐controlled lipid biosynthesis

Endoplasmic reticulum (ER)‐localized enzymes synthesize the vast majority of cellular lipids. The ER therefore has a major influence on cellular lipid biomass and balances the production of different lipid categories, classes, and species. Signals from outside and inside the cell are directed to ER‐localized enzymes, and lipid enzyme activities are defined by the integration of internal, homeostatic, and external information. This allows ER‐localized lipid synthesis to provide the cell with membrane lipids for growth, proliferation, and differentiation‐based changes in morphology and structure, and to maintain membrane homeostasis across the cell. ER enzymes also respond to physiological signals to drive carbohydrates and nutritionally derived lipids into energy‐storing triglycerides. In this review, we highlight some key regulatory mechanisms that control ER‐localized enzyme activities in animal cells. We also discuss how they act in concert to maintain cellular lipid homeostasis, as well as how their dysregulation contributes to human disease.     

How Cells Tune Viral Mechanics—Insights from Biophysical Measurements of Influenza Virus

During replication, the physical state of a virus is controlled by assembly and disassembly processes, when particles are put together and dismantled by cellular cues, respectively. A fundamental question has been how a cell can assemble an infectious virus, and dismantle a virus entering an uninfected cell and thereby trigger a new round of infection. This apparent paradox might be explained by considering that infected and uninfected cells are functionally different, or that assembly and disassembly take place along different cellular pathways. A third possibility is that the physical properties of newly assembled viruses are different from the infection-ready viruses. Recent biophysical experiments measured the stiffness of single Influenza viruses and combined this with biochemical measurements and cell biological assays. Besides inducing the fusogenic state of hemagglutinin, low pH cues softened the virus and precluded aggregation of viral ribonucleoprotein particles with the matrix protein M1. The recent experiments suggest a two-step model for Influenza virus entry and uncoating involving low pH in early and late endosomes, respectively. I conclude with a short outlook into how combined biophysical and cell biological approaches might lead to the identification of new cellular cues controlling viral uncoating and infection.     

Effects of lipid rafts on dynamics of retroviral entry and trafficking: Quantitative analysis

The association of cell surface receptors with sterol-sphingolipid-enriched microdomains of the plasma membrane, so-called lipid rafts, may affect the receptor-mediated entry and trafficking dynamics of viruses. A model retrovirus, subgroup A avian sarcoma and leukosis virus (ASLV-A), can initiate infection by binding to either of two forms of the tumor virus subgroup A (TVA) receptor, a lipid-raft-associated glycosylphosphatidylinositol (GPI)-anchored receptor (TVA800) or a transmembrane receptor (TVA950). Narayan et al. previously found that virus particles bound to TVA950 were more rapidly internalized than virions bound to TVA800, and the internalization via TVA950 exhibited biphasic kinetics. To explore potential molecular mechanisms for these results we developed a mathematical model that accounts for internalization of viruses through cellular pits, trafficking to an endosomal compartment where fusion occurs, and viral DNA synthesis. By fitting the model to experimental data we found that viruses bound to TVA950 were internalized up to 2.6-fold more rapidly than viruses bound to TVA800. Two- to threefold greater lateral diffusivities of transmembrane proteins, relative to GPI-anchored proteins, observed in other systems, suggest that the internalization rate of ASLV-A is diffusion-limited. Furthermore, by allowing for recycling of internalized TVA950-bound viruses back to the cell surface, we can account for the observed biphasic internalization kinetics. This mechanism is also consistent with the observed slower rate of DNA synthesis for viruses that enter via TVA950. Overall, the model provides a means to generate new experimentally testable hypotheses and sets a foundation for building a quantitative and integrated understanding of viral entry, trafficking, and intracellular dynamics.     

How Cells Tune Viral Mechanics—Insights from Biophysical Measurements of Influenza Virus

During replication, the physical state of a virus is controlled by assembly and disassembly processes, when particles are put together and dismantled by cellular cues, respectively. A fundamental question has been how a cell can assemble an infectious virus, and dismantle a virus entering an uninfected cell and thereby trigger a new round of infection. This apparent paradox might be explained by considering that infected and uninfected cells are functionally different, or that assembly and disassembly take place along different cellular pathways. A third possibility is that the physical properties of newly assembled viruses are different from the infection-ready viruses. Recent biophysical experiments measured the stiffness of single Influenza viruses and combined this with biochemical measurements and cell biological assays. Besides inducing the fusogenic state of hemagglutinin, low pH cues softened the virus and precluded aggregation of viral ribonucleoprotein particles with the matrix protein M1. The recent experiments suggest a two-step model for Influenza virus entry and uncoating involving low pH in early and late endosomes, respectively. I conclude with a short outlook into how combined biophysical and cell biological approaches might lead to the identification of new cellular cues controlling viral uncoating and infection.     

Multifaceted roles for lipids in viral infection

Viruses have evolved complex and dynamic interactions with their host cell. In recent years we have gained insight into the expanding roles for host lipids in the virus life cycle. In particular, viruses target lipid signaling, synthesis, and metabolism to remodel their host cells into an optimal environment for their replication. This review highlights examples from different viruses that illustrate the importance of these diverse virus-lipid interactions.     

Thylakoid membrane biogenesis in Chlamydomonas reinhardtii 137+: cell cycle variations in the synthesis and assembly of polar glycerolipid

The synthesis and assembly of thylakoid membrane polar glycerolipid (glycolipid, phospholipid, and ether lipid) have been monitored in synchronous cultures of the green alga Chlamydomonas reinhardtii 137+. A "pulse" protocol using radioactive acetate as the lipogenic precursor was devised to allow assessment of both processes during the 24-h (12-h light/12-h dark) vegetative cell cycle. Under these conditions, acetate incorporation into each chromatographically resolved lipid at the cellular level reliably reflects lipid synthesis, and the appearance of radiolabeled lipid in purified photosynthetic membrane is indicative of the lipid assembly attendant to thylakoid biogenesis. Our results demonstrate that polar glycerolipid is synthesized by the alga and is assembled into its thylakoid membrane continuously, but differentially, with respect to cell cycle time. Synthesis and assembly are most rapid during the photoperiod (mid-to-late G1), reach maximum rates at mid- photoperiod, and are comparatively negligible in the dark (S, M, and early-to-mid G1). The extent to which synthesis and assembly vary within this general kinetic pattern, though, is characteristic of each thylakoid lipid, suggesting that the processes take place in a multistep manner with some temporal coordination among the different lipid types. Parallelism between the cyclic patterns of polar lipid synthesis at the cellular level and of polar lipid assembly into photosynthetic membrane at the subcellular level indicates that lipid production is not only essential to continuing thylakoid biogenesis but is also the critical determinant of the kinetics of thylakoid lipid assembly.     

Cholesterol dependence of Newcastle Disease Virus entry

Lipid rafts are membrane microdomains enriched in cholesterol, sphingolipids, and glycolipids that have been implicated in many biological processes. Since cholesterol is known to play a key role in the entry of some other viruses, we investigated the role of cholesterol and lipid rafts in the host cell plasma membrane in Newcastle Disease Virus (NDV) entry. We used methyl-β-cyclodextrin (MβCD) to deplete cellular cholesterol and disrupt lipid rafts. Our results show that the removal of cellular cholesterol partially reduces viral binding, fusion and infectivity. MβCD had no effect on the expression of sialic acid containing molecule expression, the NDV receptors in the target cell. All the above-described effects were reversed by restoring cholesterol levels in the target cell membrane. The HN viral attachment protein partially localized to detergent-resistant membrane microdomains (DRMs) at 4°C and then shifted to detergent-soluble fractions at 37°C. These results indicate that cellular cholesterol may be required for optimal cell entry in NDV infection cycle.     

Membrane lipids as signaling molecules

PURPOSE OF REVIEW: Membrane lipids play important roles in signaling reactions. They are involved in most if not all cellular signaling cascades and in a wide variety of tissue and cell types. The purpose of this review is to highlight major pathways of signaling originating in membrane lipids. Details of lipid metabolism, and its relation to protein function, will thus advance understanding of the role of lipids in health and disease. RECENT FINDINGS: Major classes of lipids including glycerophospholipids, their metabolites (eicosanoids, endocannabinoids), and sphingolipids have recently generated interest in the field of signal transduction. These lipids are tightly regulated and have an impact on various physiological functions. Importantly, aberrant lipid metabolism often leads to onset of pathology, and thus the precise balance of signaling lipids and their effectors can serve as biomarkers. SUMMARY: Membrane lipids form precursors for second messengers and functional assembly matrices on membrane domains during cellular stimulation. Many of these modifications are rapid reactions at lipid headgroups. Metabolism of the fatty acyl portion of membrane lipids leads to the generation of a bewildering complexity of lipid mediators with extended effects in space and time.     

Lipid metabolism in cancer

Lipids form a diverse group of water-insoluble molecules that include triacylglycerides, phosphoglycerides, sterols and sphingolipids. They play several important roles at cellular and organismal levels. Fatty acids are the major building blocks for the synthesis of triacylglycerides, which are mainly used for energy storage. Phosphoglycerides, together with sterols and sphingolipids, represent the major structural components of biological membranes. Lipids can also have important roles in signalling, functioning as second messengers and as hormones. There is increasing evidence that cancer cells show specific alterations in different aspects of lipid metabolism. These alterations can affect the availability of structural lipids for the synthesis of membranes, the synthesis and degradation of lipids that contribute to energy homeostasis and the abundance of lipids with signalling functions. Changes in lipid metabolism can affect numerous cellular processes, including cell growth, proliferation, differentiation and motility. This review will examine some of the alterations in lipid metabolism that have been reported in cancer, at both cellular and organismal levels, and discuss how they contribute to different aspects of tumourigenesis.