key: cord-0988656-zmqvh4r7 authors: Weissenhorn, Winfried; Poudevigne, Emilie; Effantin, Gregory; Bassereau, Patricia title: How to get out: ssRNA enveloped viruses and membrane fission date: 2013-04-11 journal: Curr Opin Virol DOI: 10.1016/j.coviro.2013.03.011 sha: b2dd46982e2e794dc182e0daedefd75b6489d5ad doc_id: 988656 cord_uid: zmqvh4r7 Enveloped viruses acquire their membrane from the host cell and accordingly need to separate their envelope from cellular membranes via membrane fission. Although some of the enveloped viruses recruit the endosomal sorting complex required for transport (ESCRT) to catalyze the final fission reaction, many enveloped viruses seem to bud in an ESCRT-independent manner. Here we describe the principles that govern membrane fission reactions in general and review progress in the understanding of ESCRT-mediated membrane fission. We relate ESCRT function to budding of single stranded RNA viruses and discuss alternative ways to mediate membrane fission that may govern ESCRT-independent budding. Enveloped viruses assemble at cellular membranes, which provide the newly formed virus envelope. Viral proteins bend membranes into vesicular structures during assembly and use either their own or cellular host proteins to catalyze membrane fission, an important energetic barrier associated with virion release. The host cell may provide the blueprint for fission, since many membrane trafficking processes require a membrane fission step. Schematically, cellular vesicles have an extracellular, luminal or cytoplasmic content and come in two flavors. First, protein-coated vesicles, such as clathrincoated endocytotic vesicles [1] , COPI-coated or COPIIcoated vesicles that bud off Golgi and ER membranes, respectively [2, 3] or retromer-coated vesicles in the retrograde pathway [4] . Second, non-coated vesicles such as those retrieving for instance GPI-linked or glycolipid receptors from the plasma membrane [5] or vesicles that bud into the lumen of endosomes producing multivesicular bodies [6] . Clathrin-coated vesicles recruit helical dynamin polymers at the membrane neck connecting the vesicle and the donor membrane [7 ,8 ] . GTP hydrolysis then leads to polymer twisting and neck constriction [9] , which sets the stage for membrane fission occurring at the frontier between the narrow dynamin coated membrane and the coexisting bare membrane with a larger diameter. Additionally, fission kinetics is affected by the mechanical properties of the membrane (tension and bending rigidity) [10 ] . Dynamin has been also implicated in the budding of caveolae [11] and dynamin-like molecules catalyze mitochondrial fission [12] . An alternative model for vesicle fission stipulates that proteins containing amphipathic helices can induce fission by hydrophobic insertion; in the case of N-BAR domain proteins, this effect is modulated, by an antagonistic relationship between these helices and BAR-domain scaffolds [13 ,14] . In contrast, vesicle budding into the lumen of endosomes is organized by endosomal sorting complexes for transport (ESCRT) [6, 15] . Because endosomal vesicle budding is topologically similar to enveloped virus budding, some of the enveloped viruses hijack part of the ESCRT machinery to escape from host cells [16] [17] [18] . Here we review membrane fission release of enveloped ssRNA viruses catalyzed by the ESCRT machinery or by ESCRT-independent pathways. In order to place virus release and membrane fission into the context of membrane biophysics, we first summarize general physical principles of membranes favoring fission and relate these principles to ESCRT-dependent and ESCRT-independent budding processes. In vitro experiments and theoretical approaches have shown that lipid domains in membranes can induce membrane bending and fission from quasi-flat membranes due to the constrictive line tension at the edge of the domains [19 ,20] . Lateral tension in membranes limits this pinching-off effect, as it favors flat membranes [19 ] . In the case of tubular geometries reminiscent of the neck of a budding virion, fission also occurs at the edge of domains and can be accelerated by membrane tension [21] . Proteins are in principle not required for this process and the membrane domains can be made of lipids only [22] . However, proteins including actin may contribute to induce phase separation in membranes [23, 24] leading to membrane fission [25 ] and forces generated by actin polymerization pushing on the membrane can help fission [22] . Although this line-tension fission mechanism has been documented in vitro and in some cases in vivo, it is not clear to which extent it contributes to in vivo membrane fission reactions such as for example ESCRT-driven membrane fission. Mechanisms for the entry and exit of enveloped virus from cells have been proposed by physicists, in the framework of colloid-membrane interactions. The virus is modeled as a solid particle with a spherical or elongated shape that can be wrapped by the interacting membrane. Theoretical models have been calculated describing the conditions under which particles can spontaneously bud off independently of fission proteins as a function of adhesion energy, particle size and shape or membrane tension. Membrane destabilization leading to fission is expected to occur when wrapping is total. An optimal particle size for budding depending on particle concentration and on the relative densities of interacting molecules on the particle has been derived from these models [26] [27] [28] . However, to date numerical simulations are limited to the effect of a single particle and to membranes of small size [29,30]. The ESCRT machinery is composed of five different complexes, ESCRT-0, ESCRT-I, ESCRT-II, ESCRT-III and VPS4, which assemble sequentially on the endosomal membrane to concentrate cargo, sort it into vesicles, pinch off the vesicles and recycle ESCRTs [6, 15, 31, 32] . Because ESCRT-III constitutes the membrane fission machinery [33 ,34 ] , together with VPS4 [6, 35 ] they are also recruited to topologically similar processes such enveloped virus budding [36] and cytokinesis [37, 38] . Higher eukaryotes express 12 ESCRT-III proteins named CHMP (CHarged Multivesicular body Protein) and IST1 (Increased Sodium Tolerance 1 gene product) [39] [40] [41] [42] . CHMP6 (Vps20), CHMP4 (A, B or C) (snf7), CHMP3 (Vps24) and CHMP2 (A or B) (Vps2) are recruited in this order and may form a core complex in yeast [31], while the remaining ESCRT-III (CHMP1A, B, CHMP5, CHMP7 and IST1) exerts regulatory functions. All ESCRT-III proteins are required for MVB biogenesis [6] ESCRT-III proteins are small helical assemblies [48] [49] [50] and their autoinhibited conformation is controlled by the C-terminal region [49, [51] [52] [53] . Displacement or artificial removal of the C-terminus leads to polymerization in vitro and on cellular membranes. Direct ESCRT-III-driven membrane deformation in vivo has been only observed twice. Expression of truncated CHMP4 together with dominant negative VPS4 produced membrane tubes that contained helical arrays of presumably CHMP4 [54] and expression of CHMP2B wild type led to the formation of long membrane tubes emanating from the plasma membrane. CHMP2B tube formation required CHMP4 at the base, depended on VPS4 and produced a tight helical layer of CHMP2B filaments spaced by 30 Å [55] similar to the CHMP2A-CHMP3 helical tubes [46] ( Figure 1 ). In Figure 1 ). However, it should be noted that yeast Vps24 (CHMP3) assembles into two stranded filaments [57] and the spiral ESCRT-III structures imaged at the midbody are much thicker [60] than those shown in Figure 1 . A modification of the dome-model proposes the symmetric ring-like arrangement of ESCRT-I-ESCRT-II complexes in the bud neck of intraluminal endosomal vesicles that provide the platform for ESCRT-III filaments projecting into the pore in a whorl-like arrangement that themselves serve as docking site for a domelike polymer or evolve into a dome-like polymer that executes fission [68, 69] . ESCRT-III-induced lipid phase separation may play an additional role [70] thus contributing to constrictive line tension at the edge of membrane domains and permitting spontaneous fission [21] . It is thus possible that the ESCRT-III-dome may function as a scaffold that induces local lipid redistribution and phase separation. As a final touch VPS4 ATP hydrolysis may couple mechanical stress imposed on the membrane with physical properties of ssRNA enveloped viruses and membrane fission Weissenhorn et al. 161 Orthomyxoviruses (influenza virus), negative-sense segmented ssRNA viruses, bud independently of the ESCRT machinery from the plasma membrane (Table 1 ) [82] . Influenza virus budding is especially interesting because fission is induced by the viral transmembrane protein M2, which functions otherwise as an ion channel. M2 localizes to the neck of the bud and contains a highly conserved amphipathic helix within its cytoplasmic tail that affects membrane curvature in a cholesterol-dependent way. In fact, the short amphipathic helix sufficed to induce budding from GUVs [83 ] . The M2 peptide may destabilize the membrane thereby providing the force for changes in membrane curvature [84] . Thus the function of M2 resembles aspects of fission reactions catalyzed by hydrophobic insertion [13 ], although M2-driven fission is controlled by the local cholesterol concentration and no protein scaffolds seem to be required. Although Coronaviridae, positive-strand ssRNA viruses also bud independent of ESCRTs (Table 1 ), no definite viral or cellular fission factor has yet been described. Coronaviruses obtain their viral envelope containing the glycoprotein S, the major membrane glycoprotein M and a minor membrane protein E by budding through membranes of the ER-Golgi intermediate compartment (ERGIC) [85] . The E protein contains one or two transmembrane regions and was suggested to play a role in membrane bending during assembly and fission [86, 87] . However, E may not be essential for MHV (mouse hepatitis virus) and SARS-CoV virion production [88, 89] . On the other hand, E-depleted TGEV (transmissible gastroenteritis coronavirus) produces non-infectious immature virions that are stuck in the secretory pathway [90] , indicating that the final mechanism still needs to be determined. Furthermore a number of ssRNA viruses, togaviridae, bunyaviridae and flaviviridae distinguish themselves from other ssRNA viruses by having their glycoproteins organized into protein coats that cover the viral membrane [91, 92] . Such coats may drive budding and may contribute to the final membrane fission process by aiding bud neck constriction. Budding may follow physically based budding principles described above, which stipulate that membrane destabilization leading to scission 162 Virus structure and function Model for ESCRT-driven membrane fission. In case of HIV-1 CHMP4B (or CHMP4A, to a lesser extend CHMP4C) can be recruited to the budding site via a classical ESCRT-I-ESCRT-II-CHMP6 sequence or directly via Alix or via a yet unknown process. Membrane recruitment will induce CHMP4B filament formation that assembles inside the bud neck; this may induce a first constriction of the neck (a). This first constriction may set the stage for CHMP3-CHMP2A or CHMP2B recruitment, which assemble upon interaction with CHMP4B (b). CHMP2A-CHMP3 or CHMP2B polymers could grow into dome-like structures that attract the neck membrane and constrict it. Upon completion of assembly, VPS4 may start to disassemble the ESCRT-III coat, which may further destabilize the membrane and thus catalyze fission concomitantly with disassembly. may occur when the whole viral particle is wrapped by proteins. Togaviridae, genus alphaviruses (Semliki Forest virus (SFV), Chikungunya, Ross River, and Venezuelan Encephalitis virus) and rubivirus (Rubella virus) are plus sense ssRNA viruses. Rubella virus and alphaviruses anchor their RNA synthesis in membranes of a cell organelle known as the cytopathic vacuole (CPV) that derives from modified endosomes and lysosomes [93, 94] . Budding is then organized from the plasma membrane and driven by nucleocapsid-E2 glycoprotein interactions [95] , which together with E1 forms a protein shell covering the viral membrane [ the glycoprotein coat may drive budding and release [102] . Flaviviridae, with its genera flavivirus (Dengue virus, yellow fever, west Nile virus, Japanese encephalitis virus), Hepacivirus (Hepatitis C virus (HCV), Hepatitis G virus) and pestivirus (swine fever) contain a positive sense ssRNA genome. HCV buds form endosomal membranes and despite the fact that most non-structural proteins of Flaviviridae have been implicated in virus morphogenesis none of them has an assigned role in membrane fission [106, 107] . Immature virions are transported in vesicles from the ER to the Golgi where furin cleavage of prM induces the formation of mature virions, which are released from the plasma membrane via vesicular transport [107] . This latter process depends on ESCRTs [108, 109] and dominant-negative VPS4 or ESCRT-III components as well as siRNA knockdowns (Tsg101, Alix, CHMP4B, VPS4) inhibited HCV production (Table 1 ) [110, 111] albeit without affecting the accumulation of intracellular infectious particles [112] . Thus ESCRT-dependency may be required for HCV release via the exosomal secretion pathway [113] restricting the role of ESCRTs to post-budding transport and release steps rather than membrane fission at ER membranes. Much progress has been made over the last decade to understand how enveloped viruses pinch off from their host cells. In fact much insight into ESCRT function has come from studying enveloped virus budding. Today we know some of the principles that govern ESCRT-III function, but a detailed picture of how it catalyzes fission in conjunction with the ATPase VPS4 is still lacking. The effect of ESCRT-III polymers on lipids and the generation or maintenance of membrane domains is yet unexplored. It is tempting to speculate that some aspects of ESCRT-III-driven fission will resemble those of dynamin-catalyzed fission; these include the role of the helical protein scaffold, although one is assembled on the outside of a membrane neck (dynamin) and the other one on the inside of a membrane neck (ESCRT-III), thus requiring completely different modes of inducing neck narrowing. However, the final constriction may be achieved by using mechanical forces generated either by GTPase (dynamin) or ATPase (VPS4) activities. Furthermore membrane tension and rigidity, which affect the kinetics of dynamin-catalyzed fission, may also play a role during ESCRT-driven fission reactions. It is yet unclear why some of the enveloped viruses bud independently of ESCRTs. Obviously their genomes would be flexible enough to include short late domain sequences to recruit ESCRTs. In the absence of ESCRTs, one or several factors, viral or yet unknown cellular factors, may set the stage for membrane fission. In case of influenza virus, the mechanism of hydrophobic insertion by parts of M2 plays a crucial role to deform the membrane neck leading to fission. In a potential third class of viruses such as for example flaviviridae, the formation of a glycoprotein coat may be an important contributor to fission. The glycoprotein network could generate bending forces on the membrane that may lead to bud neck narrowing and eventually fission, which, however, might involve additional cellular or viral factors. Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest Molecular mechanism and physiological functions of clathrin-mediated endocytosis COPI budding within the Golgi stack COPII and the regulation of protein sorting in mammals Retrograde transport: two (or more) roads diverged in an endosomal tree? Traffic Driving membrane curvature in clathrin-dependent and clathrin-independent endocytosis The ESCRT pathway Retrovirus budding ESCRT machinery and cytokinesis: the road to daughter cell separation ESCRT-III polymers in membrane neck constriction Biochemical analyses of human IST1 and its function in cytokinesis Essential role of hIST1 in cytokinesis Ist1 regulates vps4 localization and assembly Structural basis for autoinhibition of ESCRT-III CHMP3 Plasma membrane deformation by circular arrays of ESCRT-III protein filaments Charged multivesicular body protein 2B (CHMP2B) of the endosomal sorting complex required for transport-III (ESCRT-III) polymerizes into helical structures deforming the plasma membrane A crescent-shaped ALIX dimer targets ESCRT-III CHMP4 filaments Structure and disassembly of filaments formed by the ESCRT-III subunit Vps24 Helical structures of ESCRT-III are disassembled by VPS4 This paper reveals the helical tubular architecture ESCRT-III and suggests The endosomal sorting complex ESCRT-II mediates the assembly and architecture of ESCRT-III helices Cortical constriction during abscission involves helices of ESCRT-III-dependent filaments Dynamics of ESCRT proteins Structure and function of ESCRT-III Membrane buckling induced by curved filaments Computational model of membrane fission catalyzed by ESCRT-III Regulation of Vps4 during MVB sorting and cytokinesis Dynamics of endosomal sorting complex required for transport (ESCRT) machinery during cytokinesis and its role in abscission Computational model of cytokinetic abscission driven by ESCRT-III polymerization and remodeling Solution structure of the ESCRT-I and -II supercomplex: implications for membrane budding and scission Membrane-elasticity model of coatless vesicle budding induced by ESCRT complexes Endosomal sorting complex required for transport (ESCRT) complexes induce phase-separated microdomains in supported lipid bilayers The cell biology of HIV-1 virion genesis The role of cellular factors in promoting HIV budding The cell biology of HIV-1 and other retroviruses Late budding domains and host proteins in enveloped virus release Rabies virus assembly and budding Filovirus assembly and budding Filoviruses: interactions with the host cell Viral and host proteins that modulate filovirus budding Paramyxovirus assembly and budding: building particles that transmit infections Efficient budding of the tacaribe virus matrix protein z requires the nucleoprotein Arenavirus reverse genetics: new approaches for the investigation of arenavirus biology and development of antiviral strategies Influenza virus budding does not require a functional AAA+ ATPase, VPS4 Influenza virus M2 protein mediates ESCRT-independent membrane scission This paper reveals that the amphipathic helix of M2 suffices to induce fission Influenza virus assembly and budding The coronavirus E protein: assembly and beyond. Viruses Characterization of the coronavirus mouse hepatitis virus strain A59 small membrane protein E Arkin IT: A highly unusual palindromic transmembrane helical hairpin formed by SARS coronavirus E protein The small envelope protein E is not essential for murine coronavirus replication A severe acute respiratory syndrome coronavirus that lacks the E gene is attenuated in vitro and in vivo Absence of E protein arrests transmissible gastroenteritis coronavirus maturation in the secretory pathway A structural perspective of the flavivirus life cycle Class II enveloped viruses Novel replication complex architecture in rubella replicon-transfected cells Biogenesis of the Semliki Forest virus RNA replication complex A structural and functional perspective of alphavirus replication and assembly The fusion glycoprotein shell of Semliki Forest virus: an icosahedral assembly primed for fusogenic activation at endosomal pH Glycoprotein organization of Chikungunya virus particles revealed by X-ray crystallography Ubiquitin depletion and dominant-negative VPS4 inhibit rhabdovirus budding without affecting alphavirus budding The unique architecture of Bunyamwera virus factories around the Golgi complex Cytoplasmic tails of hantavirus glycoproteins interact with the nucleocapsid protein The cytoplasmic tail of hantavirus Gn glycoprotein interacts with RNA Electron cryotomography of Tula hantavirus suggests a unique assembly paradigm for enveloped viruses Insights into bunyavirus architecture from electron cryotomography of Uukuniemi virus Three-dimensional organization of Rift Valley fever virus revealed by cryoelectron tomography Electron cryomicroscopy and single-particle averaging of Rift Valley fever virus: evidence for GN-GC glycoprotein heterodimers Assembly of infectious hepatitis C virus particles Architects of assembly: roles of Flaviviridae non-structural proteins in virion morphogenesis Interaction between the yellow fever virus nonstructural protein NS3 and the host protein Alix contributes to the release of infectious particles Association of Japanese encephalitis virus NS3 protein with microtubules and tumour susceptibility gene 101 (TSG101) protein The ESCRT system is required for hepatitis C virus production Generation of a recombinant reporter hepatitis C virus useful for the analyses of virus entry, intra-cellular replication and virion production Vps4 and the ESCRT-III complex are required for the release of infectious hepatitis C virus particles Regulation of hepatitis C virus secretion by the Hrs-dependent exosomal pathway Work in the laboratory of the authors is supported by the ANR (Agence Nationale de la Recherche) (W.W.), the FINOVI Foundation (W.W.), the LabEx GRAL (W.W.) and the Institut Curie (P.B.). G.E. is supported by a post doctoral fellowship from the ANRS (Agence Nationale de Recherche sur le Sida et les hé patites virales).