Virus entry into host cells involves a cascade of sequen-tial events, including receptor binding, endocytosis and membrane fusion. A growing body of evidence is filling the gaps in our current understanding of these complex events. Recent studies of dynamin seem to connect these processes to present a more comprehensive picture of viral entry mechanisms.
Dynamin, a protein with a molecular weight of ~100 KD, was originally identified as a guanosine triphospha-tase (GTPase) associate with microtubules in calf brain (Shpetner et al., 1989), and then as a mammalian homo-logue of the shibire gene product that played a role in clath-rin-mediated endocytosis (CME) in Drosophila (Chen et al., 1991; Schnitzer, 1996). Dynamin was hypothesized to be involved in endocytosis because temperature-sensitive Shibire mutants were found to have an accumulation of invaginated pits at the plasma membrane (Kosaka et al., 1983). Electron microscopic observations revealed that
dynamin polymerized to form rings and spirals around the necks of the clathrin-coated pits (CCPs) suggesting that it had a role in the scission of newly formed clathrin-coated vesicles (CCVs) from the plasma membrane (Takei et al., 1995). This membrane scission process was depen-dent on dynamin’s efficient GTP hydrolysis of guanosine triphosphatase (GTP) and its subsequent conformational change (Marks et al., 2001). Although it was proposed that dynamin itself may be insufficient to provide the force needed for membrane scission during CCV genesis, it is now well established that this mechanochemical enzyme drives membrane fission (Schmid et al., 2011).
In recent decades there has been a surge of interest in exploring the mechanisms of virus entry into host cells. Experiments using short interfering RNA (SiRNA), the dynamin inhibitor dynasore (Macia et al., 2006) and the dominant negative mutant K44A-dynamin (Cao et al., 2000) suggest that an increasing number of viruses from numerous families use a dynamin-dependent cell
REVIEW ARTICLE
From endocytosis to membrane fusion: emerging roles of dynamin in virus entry
Yeping Sun and Po Tien
Center for Molecular Virology, CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China
AbstractDynamin, a large guanosine triphosphatase (GTPase), has been implicated in virus entry, but its mechanisms of action are controversial. The entry procedure of most enveloped viruses involves endocytosis and membrane fusion. Dynamin has been suggested to act both as a regulatory GTPase by controlling the early stages of clathrin-mediated endocytosis (CME), which is an important endocytic pathway utilized by many viruses, and as a mechanchemical enzyme that induces membrane fission and pinches endocytic vesicles off from the cellular plasma membrane in later stages in several endocytic pathways, including CME. In addition to its involvement in virus endocytosis, dynamin has also been proposed to participate in membrane fusion between the virus and endosomes following endocytosis. Crystal structures and cryo-electron micrography (cryo-EM) have elucidated the structure of dynamin, which led to development of a mechanochemical model of how dynamin-mediated membrane fission occurs. Based on this, we propose a hypothetical model that explains how dynamin facilitates virus membrane fusion and discuss its roles in virus entry.Keywords: Virus entry, dynamin, membrane fission, membrane fusion
Address for Correspondence: Po Tien, Center for Molecular Virology, CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China. E-mail: [email protected]
(Received 20 February 2012; revised 09 May 2012; accepted 14 May 2012)
entry process (Table 1). The initial role of dynamin in virus entry was considered as scission factor that pinches off membrane invaginations. Most of the studies listed in Table 1 were performed under this assumption. It is notable that the role of dynamin as a scission factor is not exclusive to the CME pathway. Several other viral endocytic pathways such as caveolin pathway and phagocytosis also require dynamin as a scission factor (Nichols, 2003; Marsh et al., 2006; Schelhaas, 2010). Macropinocytosis, another endocytic pathway frequently employed by viruses, has traditionally been considered to be dynamin-independent. However, certain macropinocytosis-like endocytic pathway, (e.g. fluid phase uptake), requires dynamin (Huang et al., 2008; Mercer et al., 2008, 2009). Dynamin was also shown to participate in other unidentified, non-clathrin, non-caveolin-dependent endocytic pathway, although its exact role remains unknown (Acosta et al., 2009). Therefore, the effect of dynamin inhibition is an important criterion for classifying endocytic pathways.
In addition to being a mechanochemical enzyme, dynamin has been proposed to be a regulatory GTPase that controls multiple early steps of CCV generation in CME, including cargo sorting, cargo capture and CCV maturation (Pucadyil et al., 2009). Besides membrane fission, mounting evidence suggests that dynamin and dynamin-like proteins (DLPs) are involved in membrane fusion (Orso et al., 2009; Burmann et al., 2011), but the exact mechanisms remain elusive. In this review, we discuss the current understanding of the roles played by dynamin in varios virus entry routes and propose that it is involved in multiple stages of virus entry, with multiple functions as both a mechanochemical enzyme and a regulatory GTPase.
Overview of virus entry pathways
Both enveloped and non-enveloped viruses share the main steps of entry, which begin with attachment to cell surface receptors and end with delivery of the viral
Table 1. Viruses that require dynamin for host cell entry.Family Virion type Virus Endocytic pathway ReferenceAdenoviridae Non-enveloped Adenovirus type 2 and 5 CME Gastaldelli et al., 2008Arenaviridae Enveloped Pichinde virus CME Vela et al., 2008Asfarviridae Enveloped African swine fever virus CME Hernaez et al., 2010Bunyaviridae Enveloped Hantaan virus CME Jin et al., 2002Caliciviridae Non-enveloped Murine norovirus-1 Lipid raft Gerondopoulos et al., 2010;
Perry et al., 2010Coronaviridae Enveloped Feline infectious peritonitis virus IL-2 pathway Van Hamme et al., 2008
SARS virus CME Yang et al., 2004; Inoue et al., 2007
Flaviviridae Enveloped Hepatitis C virus CME Meertens et al., 2006Dengue virus-1 CME Acosta et al., 2009Dengue virus-2 Undefined pathway; CME Acosta et al., 2009
Hepadnaviridae Enveloped Hepatitis B virus Caveolin pathway Macovei et al., 2010Herpesviridae Enveloped Herpes simplex virus 1 Lipid raft Gianni et al., 2010Iridoviridae Enveloped Tiger Frog Virus Caveolin pathway Guo et al., 2011Mimiviridae Enveloped Mimivirus phagocytosis Ghigo et al., 2008;
La Scola et al., 2008Orthomyxoviridae Enveloped Influenza A virus CME Rust et al., 2004Papillomavirida Non-enveloped Human papillomavirus type 31 Caveolin pathway Smith et al., 2007Parvoviridae Non-enveloped Canine parvovirus CME Cureton et al., 2012Picornaviridae Non-enveloped Coxsackievirus A9 Lipid raft Heikkila et al., 2010
Poliovirus Caveolin pathway Coyne et al., 2007Echovirus 1 Caveolin pathway Pietiainen et al., 2004
Laliberte et al., 2011Reoviridae Non-enveloped Avian reovirus Caveolin pathway Huang et al., 2011Retroviridae Enveloped HIV-1 CME Daecke et al., 2005;
Miyauchi et al., 2009; de la Vega et al., 2011
Enveloped Equine infectious anemia virus CME Brindley et al., 2008Rhabdoviridae Enveloped Vesicular stomatitis virus CME Johannsdottir et al., 2009Reoviridae Non-enveloped Rotavirus CME Lopez et al., 2004; 2006;
Gutierrez et al., 2010Togaviridae enveloped Semliki Forest virus CME Vonderheit et al., 2005
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genomes into the cytoplasm. Most viruses must make use of certain endocytic pathways to enter primary endocytic vesicles (PEVs) and then traffic to proper endosomal com-partments and release their genetic materials (Figure 1).
I. Dynamin-dependent endocytosisExperiments that deplete dynamin with SiRNA, per-turb dynamin function with Dynamin-K44A dominant
negative mutant or inhibit dynamin GTPase activity using dynasore have demonstrated that CME, the caveolin-mediated pathway, phagocytosis and IL-2 pathway are dynamin-dependent (Mercer et al., 2010b). Most viruses that have been assessed to-date utilize these dynamin-dependent endocytic pathways (Table 1).
More viruses access host cells by CME than any other endocytic pathway (see Table 1 and Figure 1). Although
Figure 1. Overview of virus entry pathways. Viruses use a variety of dynamin-dependent endocytic pathways or dynamin-independent endocytic pathways to enter the primary endocytic vesicles (PEVs). Then, the virus cargoes traffic through and penetrate in endosomal system, including early endosomes (EE), maturing endosome (ME), late endosomes (LE). Enveloped viruses penetrate by membrane fusion while nonenveloped viruses penetrate by lysing the endosomal membrane. Abbreviations: Adeno 2/5, adenovirus 2/5; ASFV, African swine fever virus; EIAV, Equine infectious anemia virus; VSV, Vesicular stomatitis virus; SFV, Semliki Forest virus; HPV 31, Human papillomaviruse type 31; HSV-1, Herpes simplex virus 1; MNV-1, Murine norovirus-1; CAV9, Coxsackievirus A9; FIPV, Feline infectious peritonitis virus; AAV2, Adeno-Associated Virus 2; Ad3/35, Adenovirus type 3/35.
the primary endocytic vesicles (CCVs) have average internal diameters of ~35–42 nm (Takamori et al., 2006; Cheng et al., 2007), which is significantly smaller than dimension of many viruses that use the CME pathway, CCVs can adjust their size and shape to accommodate them. The largest CCV diameter is about 200 nm, which is large enough to contain vesicular stomatitis virus (VSV) (Cureton et al., 2009; McMahon et al., 2011).
The caveolin pathway is also dynamin-dependent. This endocytic pathway is best studied in the case of SV40 in the polyomavirus family (Pelkmans et al., 2001). The viruses associate with glycolipid-, cholesterol- and caveo-lin1 (CAV1)-enriched caveolaes in the plasma membrane of cells and induce membrane curvature, thus promot-ing the formation of CAV1-coated caveosomes (Nichols, 2003; Parton et al., 2007). The role of dynamin in pinching off the endocytic vesicles from the plasma membrane has been established by some early studies (Schnitzer, 1996; Oh et al., 1998; Pelkmans et al., 2002). SV40 may also use raft-dependent, caveolar-, and CAV1-independent endo-cytic pathways as an alternative to the CAV1 pathway. CAV1 and dynamin have regulatory function, and CAV1 is most likely inhibitory; its expression tends to suppress endocytosis (Kirkham et al., 2005; Lajoie et al., 2007).
Phagocytosis is a dynamin-dependent endocytic pathway used by some large viruses. For instance, mini-virus (400 nm in diameter) can enter macrophages and amoebae by phagocytosis (Ghigo et al., 2008; La Scola et al., 2008). Experiments demonstrate that perturbation of dynamin function inhibits phagocytosis at the stage of membrane extension around the forming phagosomes, supporting the role of dynamin as a membrane fission factor (Gold et al., 1999).
Some viruses, including feline infectious peritonitis virus, may enter cells via the IL-2 pathway, which is clath-rin- and caveolin-independent but requires dynamin as the scission factor (Van Hamme et al., 2008). Herpes simplex virus 1 (HSV-1), however, utilizes a non-cave-olin-dependent, cholesterol-rich lipid raft-dependent endocytic pathway which requires dynamin to enter αVβ3-integrin–expressing CHO Cells (Gianni et al., 2010). Murine norovirus-1 also makes use of certain dynamin- and lipid raft- dependent endocytic pathway (Gerondopoulos et al., 2010; Perry et al., 2010). Additionally, although den-gue virus-2 enters A549 cells by CME, its infectious entry into Vero cells occurs by a non-classical endocytic path-way independent of clathrin, caveolae and lipid rafts, but dependent on dynamin (Acosta et al., 2009).
II. Dynamin-independent endocytosisVirus may also enter cells by dynamin-independent endocytic pathways. Macropinocytosis is an impor-tant dynamin-independent endocytic pathway used by vaccinia virus (Schmidt et al., 2012), influenza virus (de Vries et al., 2011), adenovirus type 3/35 (Meertens et al., 2006; Kalin et al., 2010) and Ebola virus (Yang et al., 2004). Macropinccytosis is associated with the formation of membrane ruffles that take the form of lamellipodia,
circular ruffles or blebs and generate macropinosomes by backfolding of membrane extensions and fusing with the plasma membrane. In the case of virus cargo uptake, membrane scission that separates macropinosomes from plasma membrane does not require dynamin, but it does need CtBP-1/BARS as a fission factor (Mercer et al., 2009).
Recently, the dynamin-independent CLIC/GEEC endocytic pathway was associated with the entry of adeno-associated virus 2 (Nonnenmacher et al., 2011). In this pathway, cargos such as cholera toxin binding subunit (CTB) and glycosylphosphatidylinositol (GPI)-anchored proteins (APs) are internalized by tubular clathrin-independent carriers (CLICs) into GPI-AP-enriched early endosomal compartments (GEECs). Dynamin is not involved in this pathway and the scis-sion factors responsible for the GEEC generation are still unknown (Lundmark et al., 2008; Mercer et al., 2010b).
III. Penetration of viruses in endosomal systemOnce inside the cell, primary endocytic vesicles (PEVs) containing the incoming virus cargos are trafficked to dif-ferent endosomal compartments. The endosomal system is composed of different classes of endosomes, including early endosomes (EE), maturing endosome (ME), late endosomes (LE), and recycling endosomes (RE), which are heterogeneous with regard to composition, function and trafficking pathways. The endosome system is tightly connected with the secretory pathway by vesicles that shuttle between endosomes and the trans-Golgi net-work (TGN), and the plasma membrane. Most endocytic routes converge, directly or indirectly, onto conventional endosomes (Gruenberg et al., 2006; Gruenberg, 2009). Virus cargos in PEVs are sorted into EE and then into more matured ME and LE. In most endocytic pathways, the penetration and release of the viral genomes may occur in different endosomal compartments depending on the viruse type. EE, ME and LE have been revealed to be the penetration sites of certain viruses. In the caveo-lin pathway, however, virus cargos often pass through EE and LE and traffic to the endoplasmic reticulum (ER), where they penetrate into the cytosol (Schelhaas et al., 2007; Mercer et al., 2010b). Both enveloped and non-enveloped can use energy of metastable states in vrial entry proteins to expose hydrophobic sequences that destabilize host cell endosomal membrane. The subsequent formation of different intermediates leads to the development of fusion pores (in the case of envel-oped viruses) or membrane pores (in the case of non-enveloped viruses). Dynamin has been involved in the membrane fusion step in the case of human immunode-ficiency virus (HIV-1) (Miyauchi et al., 2009) and vaccinia virus (Laliberte et al., 2011). The presumed mechanisms are discussed below.
Biochemical properties of dynamins
Dynamin belongs to a large superfamily of proteins whose members are found from bacteria to mammals.
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Within the superfamily, three isoforms of mammalian dynamin, dynamin (1, 2, and 3) are considered classi-cal dynamins. Other members of the superfamily are known as the dynamin related proteins (DRPs) or the dynamin-like proteins (DLPs). In this review, we use the term “dynamin” to describe any one of these three clas-sical dynamins. Among them, dynamin 1 is enriched in the brain, especially in the presynapse; dynamin 2 is ubiquitous; and dynamin 3 is found in the testis as well as the brain where it is abundant in the postsynapse (Praefcke et al., 2004).
Members of the dynamin superfamily are usually classified as large GTPase because besides having a large GTPase domain, they also have certain charac-teristics distinct from those of the small Ras family of GTPases. Additionally, they have a low GTP-binding affinity, can self-assemble into oligomers, and are able to bind to lipid membranes (Song et al., 2003). In vitro, purified dynamin has been found to form tetramers in high salt solutions (Muhlberg et al., 1997; Binns et al., 1999), and spontaneously self-assemble in vitro under low salt conditions into rings and short spirals (Hinshaw et al., 1995). The quantity and length of these assemblies are enhanced by inclusion of transition state mimics of the GTP hydrolysis reaction (e.g. GDP-AlF4−) (Carr and Hinshaw, 1997). In the absence of nucleotides, dynamin has been found to bind via its pleckstrin homology (PH) domain to negatively charged phosphatidylserine- or phosphatidyl-4,5-bisphosphate (PI4,5P2)-containing liposomes and assemble into long, tightly packed spirals that squeeze the liposomes into narrow, dyna-min-coated tubules 50 nm in diameter that are rep-resentative of the structures observed at the necks of clathrin-coated pits (CCPs) (Iversen et al., 2003). Self-assembly led to 100-fold increase in dynamin GTPase activity (Leonard et al., 2005). Surprisingly, GTP, but not of its non-hydrolysable analogue GTP-γS, stimulates these dynamin-encircled phosphatidylserine tubules to fragment into small vesicles, which potentially mim-ics the function of dynamin at the CCP necks (Sweitzer et al., 1998; Stowell et al., 1999).
Dynamin crystal structures
I. Crystal structure of the PH domain of dynaminClassical dynamins have five domains: the N-terminal is the GTPase (G) domain, followed in order by the middle domain (MD), the pleckstrin homology (PH) domain, the GTPase effector domain (GED), and the proline rich domain (PRD) (Figure 2A). The PH domain of human dynamin 1, through which dynamin binds membranes, was the first to have its crystal structure determined (Achiriloaie et al., 1999; Takamori et al., 2006). It was shown to be a anti-parallel β sandwich, with two nearly orthogonal sheets that form a positively charged pocket that can engulf negatively charged lipids (Ferguson et al., 1994; Timm et al., 1994).
II. Crystal structure of the GTPase domain of dynaminThe crystal structure of the GTPase domain of rat dyna-min 1 in the nucleotide-free state (Reubold et al., 2005) was determined more than 10 years after the PH domain was solved. The structure suggested that the conforma-tion of the active center, which consists of four guanine nucleotide binding motifs (G1, the P-loop; G2, switch 1; G3, switch 2; and G4), resembles that of other GTPases, such as Ras in the GTP binding state as opposed to GDP the binding state or the transition state. Only slight shifts are needed for switches 1 and 2 to achieve full coordi-nation with Mg2+-GTP. The structure also lends support to a mechanism in which the dynamin GED functions as an intramolecular GTPase-activating protein (GAP): the N-terminal and the C-terminal helix of the GTPase domain form a hydrophobic groove that could be a GED docking site.
III. Crystal structure of dynamin GTPase domain fused to GEDA study of the crystal structure of the GTPase domain, which was fused to the GED (GG) in the transition-state mimic GDP·AlF4− binding state (Chappie et al., 2010), revealed that GTPase domain dimerization plays a critical role in dynamin catalytic mechanism. The crystal structure revealed that active sites of the GG monomers are positioned close to the dimer interface with the bound nucleotides parallelly oriented relative to one another. Both the G4 loop and switch 2 of the GTPase active site contribute to dimer interface stabilization. For GTP hydrolyis, catalytic water for a nucleophilic attack on the γ-phosphate is correctly positioned by both cis interactions within the individual monomer and trans interactions across the dimer interface. Besides, the negative charge that develops between the β- and γ-phosphates is neutralized by a sodium ion in stead of the charge-compensating arginine finger observed at other GTPase active sites. Comparison of the GG struc-ture with the rat dynamin 1 GTPase domain revealed many obvious conformational changes that coordinate the catalytic mechanisms during binding transition mimic GDP·AlF4−. The structure also exposed a three-dimensional domain termed the bundle signalling element (BSE) consisting of the carboxyl termini of the GTPase domain and the GED, and the amino terminus of the GTPase domain (Figure 2A and 2B), which could pivot a rigid body around a conserved proline residue that kinks the C
GTPase helix.
The same group of investigators recently resolved the crystal structure of GG complexed with GMPPCP, a non-hydrolyzable CTP analog (Figure 2C; Chappie et al., 2011). Superposition of the crystal structure of GG
GDP·AlF4−
and GGGMPPCP
identifies a dramatic conformation change in the BSE. There is a 68.81° rigid-body downward rota-tion of the BSEs of GG
GDP·AlF4− about an axis perpendicular
to the CGTPase
helix that was coupled with a slight counter-clockwise twist (Figure 2D).
IV. Crystal structures full-length dynaminRecently, crystal structures of the assembly-deficient full-length human and rat dynamin 1 lacking the proline-rich domain (PRD) in their nucleotide-free state were resolved nearly simultaneously by two independent groups (Faelber et al., 2011; Ford et al., 2011). In both of these structures, dynamin monomers form extended architectures with the GTPase and PH domains that are separated by a stalk consisting of an anti-parallel helical bundle of the MD and a helix from the GED. As in the GG structure, the C
GTPase helix, the N
GTPase helix and the helix
at the C-terminus of the GED (CGED
) form three-helix BSE in full-length dynamin 1 (Figure 2E). In addition, the dynamin monomers stalks are positioned in a criss-cross pattern to form an liner filament oligomer. Two dynamin monomers interact with each other through an interface at the center of stalk and form a dimer that constitutes the building block of the liner higher-order oligomer.
Cryo-EM structures of dynamin-coated tubule assembly
cryo-EM of the dynamin-coated tubules provide a direct view of how dynamin mediates constriction of tubular mem-brane and scission of invaginated vesicles. The cryo-EM
showed that human dynamin 1 lacking PRD domain (ΔPRD dynamin)-coated tubules, but not wild type dynamin coated tubules, constricted from a diameter of ~50 nm to ~40 nm when non-hydrolysable GTP analogue GMPPCP or GMPPNP was added. The structure of ΔPRD dynamin in constricted state (Zhang et al., 2001) is a continuous helical line wrapping around, with 13–15 asymmetric units per turn. It repeats along the start helix consisted of a T-shaped dimer of ΔPRD dynamin extending from lipid membrane. The T-shaped ΔPRD dynamin dimer contains three prominent densities: leg, stalk and head. Within the asymmetric unit or dimer, two legs, corresponding to the PH domain, protrude from the lipid bilayer and intersect within the stalk, which is formed by both the middle domain and the GED, which then extends into the head region, GTPase domain.
The above three-dimensional reconstruction of ΔPRD dynamin in the constricted state with the nonhydrolyz-able GTP analog, GMPPCP, was generated by helical reconstruction. Later, three-dimensional reconstruction of ΔPRD dynamin in nonconstricted state was deter-mined by iterative helical real space reconstruction (Chen et al., 2004). The most obvious difference between these two states is in the stalk region. It forms a zigzag pattern in the constricted state, but is fairly straight in the non-constricted state.
Figure 2. Dynamin domain organization and crystal structures. (A) The primary sequence of dynamin consists of five domains including a GTPase domain, a middle domain, a pleckstrin homology (PH) domain, a GTPase effector domain (GED) and a proline rich domain (PRD). The three-dimensional structure of dynamin 1 can be divided into the GTPase domain, the bundle signal element (BSE) and the stalk. (B) The crystal structure of human dynamin 1-derived GTPase-GED fusion protein binding GDP·AlF4− (GG
GDP·AlF4−, PDB ID: 2X2E). The GTPase domain is shown in green, and the bundle signalling element (BSE) is shown in red. (C) The crystal structure of dynamin 1-derived GTPase–GED fusion protein binding GMPCCP (GG
GMPCCP, PDB ID: 3ZYC). The color labels are the same as in (C). (D) Superpositioning of the crystal
structures of GGGDP·AlF4− and GG
GMPCCP shows a large conformation change of the BSE. The structure of GG
GDP·AlF4− is shown in orange, and the structure of GG
GMPCCP is shown in cyan. (E) The crystal structure of rat dynamin 1 lacking PRD (Dyn1 G397D △PRD) in nucleotide-free state
(PDB ID: 3ZVR).
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By fitting the X-ray structures of rat dynamin 1 GTPase and PH domains of dynamin to cryo-EM structures of human ΔPRD dynamin tubeles in both non-constricted and constricted states, conformational changes during dynamin constriction were defined and a corkscrew model for dynamin constriction was proposed (Mears et al., 2007). Fitting the GTPase domain to cryo-EM maps revealed that in both the non-constricted and con-stricted states, two GTPase monomers were successfully fit to a dimer. Thirteen such dimers were fit to a complete turn of the constricted ΔPRD dynamin helix while 14 dimers were fit to a complete turn of the non-constricted ΔPRD dynamin helix. GTPase dimers were further away from each other in non-constricted state. Upon nucleo-tide binding, the dimers packed more closely when constricted. Fitting the PH domain to constricted and non-constricted maps revealed that the arrangement also shifted during constriction. This motion coincides with the conformational changes observed in the stalk and GTPase region. The PH structures were also closer together in the constricted state as expected based on the tighter packing. These observations led to the proposal of a corkscrew model for dynamin constriction. According to this model, changes in the structure and orientation of the GTPase domain upon GTP binding (and later hydrolysis) are likely to be propagated to the GED/middle domain, leading to the kinked structure observed for the stalk region in the constricted state, which allows for tighter GTPase domain packing in the constricted state. Overall, the GTPase/middle/GED subunit motions mimic a corkscrew normal to the helical axis.
Dynamin as a regulatory GTPase in controlling early CCP assembly in CME
In vivo dynamin function is best studied in the context of CME (McMahon et al., 2011). The pathway begins when cargoes such as virus-carrying cellular receptors are captured at CCPs that are assembled from cytosolic coat proteins. The CCPs capture transmembrane cargo molecules, invaginate, and then pinch off to form CCVs. The successful genesis of CCVs is coordinated through an orchestra of lipids, coat proteins, adaptor proteins, and accessory proteins in temporal and spatial scales. Typically, specific motifs in the cargoes are recognized by the AP2 adaptor, and the latter recruits clathrin and accessory proteins (Collins et al., 2002; Kelly et al., 2008; Jackson et al., 2010). The clathrin then assembles (Fotin et al., 2004; Wilbur et al., 2010) and N-BAR or BAR domain-containing proteins such as amphiphysin (Peter et al., 2004) and sorting nexin 9 (SNX9) (Pylypenko et al., 2007; Wang et al., 2008) coordinate to facilitate mem-brane bending and invagination until CCP maturation and CCV scission are completed.
The concept that dynamin functions as a regulatory GTPase in endocytosis was first suggested by Sever et al. (1999). Their experimental results revealed that the GED of dynamin was an intramolecular GAP that stimulated
GTPase activity but inhibited dynamin self-assembly and receptor-mediated endocytosis. Overexpression of dyna-min with GED mutations (K694A and R725A) impair-ing self-assembly stimulated endocytosis, suggesting that dynamin is a regulatory molecule that affects early stage of the endocytic process. Additional evidence of dynamin regulating endocytosis came from Drosophila shibire mutants. The G146S mutation in shibire in switch 2 of the GTPase domain caused a temperature-sensitive endocytic defect. Two second-site mutations in the GED, dynamin’s putative GAP, fully rescue the defect. These second-site mutations impaired dynamin’s basal and assembly-stimulated GTPase activities without altering self-assemble ability, suggesting that accelerated GTP hydrolysis, which is dependent on GED function, nega-tively regulates dynamin function in vivo (Narayanan et al., 2005).
Dynamin was shown to be recruited along with clath-rin and AP2 early in CCP maturation, and the early role played by dynamin was suggested to be that of a regula-tory GTPase. In contrast to its membrane fission function during the late stage of CCV genesis, the regulatory func-tion of dynamin is performed by its unassembled GTP binding forms and is dependent on the basal GTPase hydrolysis activity (Sever et al., 2000). Using total inter-nal reflection fluorescence microscopy (TIR-FM), cell surface CCPs were classified into three types: early abor-tive, late abortive and productive CCPs; and dynamin was revealed to regulate their relative distributions and lifespan. The evidence suggests the existence of a check-point at the early stage of CCV formation and progress through this checkpoint is controlled in part by dyna-min. This regulatory role of dynamin during the early phase of CME might be achieved through monitoring local concentrations of cargoes and AP2 adaptors. If the local concentrations are low, resulting in an insufficient amount of SH3-containing partner dynamin recruitment during cargo loading and CCP assembly, early and late abortive CCPs are produced as they fail to pass through the restriction point. Aulixin/Hsc70 was then recruited to disassemble clathrin coat (Loerke et al., 2009).
The PRD of dynamin can interact with numerous SH3 domain-containing molecules, such as amphi-physins, endophilin, intersectins 1 and 2, and SNX9 in the CME pathway (Schmid et al., 2007). Besides hav-ing a C-terminal SH3 domain, both amphiphysins and endophilin also possess an N-terminal lipid interacting BAR domain that forms a crescent-shaped dimer. They can sense membrane curvature or bend the membrane via hydrophobic insertion and scaffolding mecha-nisms (Powell, 2009). Intersectins are multi-domain proteins that serve as scaffolds during the initiation of clathrin-coated pit formation (O’Bryan, 2010). The SH3-containing and lipid binding protein SNX9 has a C-terminal BAR domain that has also been proposed to be involved in membrane remodeling (Lundmark et al., 2009). The signal from the interaction between these SH3 domain-containing proteins and PRD of dynamin may
be transmitted through the BSE in dynamin (Chappie et al., 2010), which then regulates basal GTP hydrolysis activities and enables unassembled dynamin to directly or indirectly control the termination or progression of early endocytic intermediates.
Dynamin as a mechanochemical enzyme in mediating CCV scission in endocytosis
I. Biochemical evidence of dynamin-mediated membrane fissionSeveral recent lines of biochemical evidence suggest that dynamins could independently mediate membrane fis-sion. For example, dynamin in the continuous presence of GTP causes small, wave-like fluctuations in the con-ductivity of the sub-micrometer tubules that precedes fission events. Short dynamin scaffolds assemble around the tubules and squeezed them to a critical diameter to allow the stochastic formation of a hemi-fission inter-mediate, which leads to membrane fission (Bashkirov et al., 2008). In addition, dynamin induces vesicle forma-tion from planar lipid templates. Recently, an assay was developed using supported lipid bilayers with an excess membrane reservoir on 5 μm silica beads, or SUPER templates, to visualize dynamin’s effects on the adsorbed lipid bilayer and to quantitatively measure membrane fission by the release of fluorescently-labeled lipids into the supernatant during a low speed spin. When dynamin was added to these SUPER templates in the constant presence of GTP, it stimulated multiple rounds of mem-brane fission that released small vesicles into the super-natant (Pucadyil et al., 2008).
II. Mechanochemical models of dynamin-mediated membrane fissionIn biochemical studies, GTP binding and hydrolysis is invariably required for dynamin-catalysed membrane fission both in vivo and in vitro. Subsequently, several mechanochemical models of GTP hydrolysis-driven conformational changes were proposed to explain dyna-min-mediated membrane fission. Hinshaw and Schmid (Hinshaw et al., 1995) proposed that during the process of endocytosis, GTP binding triggered dynamin redistri-bution to the neck of invaginated pits and self-assembly into the constricted collar. Subsequent GTP hydrolysis resulted in a concerted conformational change that tightened the ring around the constricted neck and stim-ulated membrane fission and vesicle budding. Stowell et al. (Stowell et al., 1999) used electron microscopy to reveal that a GTP binding and hydrolysis led to a ‘spring-like’ conformational change in the dynamin helix around the neck of invaginated pits. Roux et al. (Roux et al., 2006) concluded that GTP hydrolysis led to helix turn twisting that produced a longitudinal tension in dynamin tubules.
As mentioned above, Chappie et al. resolved the crystal structures of GG
GDP·AlF4– transition-state com-
plexes (Chappie et al., 2010) and ground-state GGGMPPCP
(Chappie et al., 2011). A comparison of these two
structures revealed a large conformational change in BSE. Docking of these structures into the cryo-EM map of GMPPCP-bound, constricted ΔPRD dynamin tubules suggested that G domain dimerization only occurred in the presence of transition-state mimics and between adjacent helix rings. Chemical cross-linking suggested that the dynamin tetramers, which are basic units of the dynamin helix around the lipid tube, were made of two dimers in which the G domain of one molecule inter-acted in trans with the GED of another. Based on these data, a six-step model of GTP hydrolysis-dependent powerstroke for dynamin-mediated membrane fission was proposed (Chappie et al., 2011): (i) At the late stage of endocytosis, dynamin tetramers in solution bind to the membrane, and conformational changes occur so that their assembly interfaces are exposed. (ii) Dynamin tetramer assembly leads to the cooperative assembly of a helical dynamin collar at the neck of an invaginated CCP. (iii) GTP binding and architectural changes in the stalk constrict of the neck and promote G domain dimer-ization between tetramers in adjacent helical rungs to optimally position dynamin’s catalytic machinery. (iv) Subsequent GTP hydrolysis drives a major rotation of the BSE in the transition state that constitutes the dynamin powerstroke, which leads to further neck constriction. (v) The dynamin helix coat detacheds from the membrane surface, resulting in membrane remodeling and mem-brane fission. (vi) The dissociation dynamin scaffold dis-assembles upon release of the hydrolyzed γ-phosphate.
On the other hand, conformational changes of BSE in different nucleotide binding states were also analyzed by Ford et al. (Ford et al., 2011). When docking the crystal structure of dynamin 1 G397D ΔPRD in nucleotide-free state onto a cryo-EM reconstruction of GMPPCP-bound dynamin, they found that the latter had a BSE with a more open conformation relative to the GTPase domain. Thus, GTP hydrolysis could induce BSE closure via a transition state represented by the structure of the GTPase domain–BSE fusion in the presence of GDP·AlF4−.
Implication of dynamin in membrane fusion of enveloped viruses and possible mechanisms
I. Evidence of dynamin facilitating membrane fusion between the virus and its host cellEndocytosis is the common step and route shared by both non-enveloped and enveloped viruses to enter host cells. However, membrane fusion is the distinctive entry step of enveloped viruses following endocytosis. Although dynamin plays an obvious role in membrane fission, it has also occasionally been shown to be associ-ated with membrane fusion. Using lipid-mixing assay of membrane fusion, a recent study (Miyauchi et al., 2009) revealed that dynamin was essential in the process of membrane fusion in HIV-1 infection. The results showed that HIV entered into the cellular endosome through the clathrin-dependent endocytic pathway and then fused
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with the endosome. HIV-endosome fusion was inhibited by dynasore, the small molecule inhibitor of dynamin GTPase activity. These results suggested that membrane fusion between HIV and endosome requires dynamin GTPase.
Vaccinia virus membrane fusion has also been shown to be dynamin-dependent. The endocytic pathway of vaccinia virus is controversial. Although most studies showed that vaccine virus enters cells via dynamin-independent macropinocytosis (Mercer et al., 2008; 2010a), one publication provided the evidence that the virus uses dynamin-dependent macropinocytosis, or “fluid phase endocytosis” (Huang et al., 2008). A more recent study used a specific lipid mix analysis to show that dynamin is a critical factor in promoting vaccinia virus entry into HeLa cells at the membrane fusion step (Laliberte et al., 2011). Herpesviruses (HSVs) also make use of dynamin-dependnet entry pathways. Depending on the cell line, HSV-1 can enter cells either by direct fusion of the viral envelope with the plasma membrane or by endocytic pathways (Connolly et al., 2011). In αVβ3-Integrin–expressing Chinese hamster ovary (CHO) cells, HSV-1 utilizes an endocytic pathway that is lipid raft- and dynamin-dependent (Gianni et al., 2010). HSV-1 enters human keratinocytes via both endocytosis and cell sur-face fusion, and dynamin inhibition reduces HSV-1 the infectivity, suggesting that dynamin may function either in endocytosis as a fission factor or in membrane fusion process (Rahn et al., 2011).
In fact, DLPs have been associated with membrane fusion for several years. For example, mammalian Mfn1/Mfn2 and OPA1 or yeast Fzo1 and Mgm1, the highly con-served mitochondrial DLPs that are essential for outer and inner mitochondrial membrane fusion, respectively. Topology mapping and crystal structure studies have revealed that Mfn1/Mfn2 has three heptad repeat (NHR, HR1, and HR2) domains. And HR2 domains of two Mfn1/Mfn2 molecules are capable of forming a dimeric, anti-parallel coiled-coil structure via inter-molecular interactions (Koshiba et al., 2004). It also has two trans-membrane regions that anchor it in the mitochondrial outer membrane and place the GTPase domain and the HR domains in the cytosol. The inter-molecular interac-tions mediated by the HR2 domains of the two Mfn1/Mfn2 or Fzo1 molecules in the opposing outer mitochon-drial membranes tether the two membranes together and promote fusion. GTPase activity is indispensable during membrane fusion. Fzo1 or Mfn1/Mfn2 molecules may function as oligomers, and the GTPase and three HR domains in different Fzo1 or Mfn1/Mfn2 molecules within an oligomer may function together to mediate fusion (Griffin et al., 2006a,b; Hoppins et al., 2009).
Two different Mgm1/OPA1 isoforms are generated by divergent proteolytic mechanisms: long (l) isoforms are anchored via the N-terminus to the inner membrane, and short (s) isoforms are predicted to be soluble in the intermembrane space. Both l- Mgm1 and s-Mgm1 con-tain a GTPase domain and a GED domain, but l- Mgm1
is unique in that it has a transmembrane region. s-Mgm1 assembles into a parallel dimer that further assembles into higher-order structures. l-Mgm1 and s-Mgm1 can also form heterodimers. These interactions allow the two isoforms to act together to mediates mitochondrial fusion. Unlike s-Mgm1, l- Mgm1 GTPase activity of is undetectable in inner mitochondrial membrane com-position (IMC), but l- Mgm1 stimulates s-Mgm1 GTPase activity. It is likely that l-Mgm1, as the integral membrane protein, tethers inner membranes together and har-nesses GTP-dependent s-Mgm1 conformational changes of that are needed to destabilize lipid bilayers for fusion (DeVay et al., 2009).
Atlastin (ATL), another DRP, is essentially required in homotypic endoplasmic reticulum fusion. A recent study of the crystal structures of human ATL1 cytosolic frag-ment in the prefusion and postfusion states revealed a possible mechanism for ATL-mediated homotypic mem-brane fusion. The results showed that ATL1 structures were composed of a GTPase domain and a three-helix bundle connected by a linker region. In the prefusion state, the ATL molecules in opposing membranes inter-act through their GTPase domains to form a dimer with the nucleotides bound at the interface, while the three-helix bundles of the two ATL molecules undergo a major conformational change relative to the GTPase domains, which could pull the membranes together (Cheng et al., 2007; Liu et al., 2011).
II. A hypothetical model for dynamin-facilitated membrane fusionHow dynamin mediates viral and cellular membrane fusion must be very different from mitochondrial or ER membrane fusion because viral and cellular membranes are heterotypic and asymmetrical, and dynamin is only available in the cellular membrane prior to fusion initia-tion. We know that viral glycoproteins play a critical role in virus membrane fusion. They insert into the cellular membrane by their fusion peptide and then undergo drastic conformational changes to form a hairpin inter-mediate via two HR domains within the molecule so that the membranes of the virus and the cell are pulled closer (Xu et al., 2004; Zhu et al., 2005; Pang et al., 2008).
Anantharam et al. (2010; Anantharam et al., 2011) proposed a mechanism in which dynamin regulates fusion pore expansion to explain how dynamin functions in membrane fusion during the process of exocytosis. They used a polarized TIR-FM to directly detect the effect of dynamin on topological membrane changes in the expanding fusion pore. Their results showed that when dynamin GTPase activity was reduced, the expansion of the fusion pore slowed with concomitant long-lasting membrane deformations. Conversely, when dynamin GTPase activity was elevated, fusion pore expansion was accelerated with more rapid curvature transitions.
Here we suggest that although dynamin is involved in viral and cellular membrane fusion, as suggested by Miyauchi et al. (2009), it does not function in isolation.
Instead, it is an assistant molecule that facilitates the fusion process initiated by conformational changes in the viral fusogenic protein (Permanyer et al., 2010). As stated above, experimental evidence suggests that dynamin expands the fusion pores during the process of mem-brane fusion. Consequently, the role of dynamin in mem-brane fusion can be described as follows: during transit
through endosomes, hydrophobic fusion sequences in viral fusogenic proteins insert into the endosomal mem-brane, and then dramatic conformational changes occur in the viral fusogenic protein and the released free energy is used to force the membranes closer together at a focal site, resulting in fusion pore generation and the subse-quent membrane fusion. When the fusion pore forms,
Figure 3. A hypothetical model for dynamin-facilitated membrane fusion. (A) The fusion peptide of viral surface fusogenic proteins (e.g. gp41 of HIV) inserts into the cellular endosome membrane. (B) Dramatic conformational change in the fusogenic protein pulls the viral and cellular membranes together, forming the hemifusion state of the two membranes. Dynamin tetramers bind to the curved membrane in the hemifusion state. (C) When the fusion pore generates, dynamins assemble at the fusion pore edge. (D) Dynamins facilitate the expansion of the fusion pore and ultimately membrane fusion through GTP hydrolysis dependent constriction and conformational change.
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dynamin binds to the curved membrane at the edge of the fusion pore and assembles into polymers to prevent the spontaneous closure of the pore. Then, through its mechanochemical enzyme activities, namely, GTPase domain dimer formation, GTP hydrolysis and conforma-tion changes, dynamin drives the fusion pore expansion and ultimately influences viral and endosome mem-brane fusion (Figure 3).
Perspective
Dynamin is a molecule with properties of a mechano-chemical enzyme and a regulatory GTPase. These dual characteristics make it a versatile actor that plays mul-tiple roles in virus entry.
The functions of dynamin are the best studied in CME. The process of virus entry via CME can be divided into six stages in terms of dynamin: (i) receptor binding and recruitment of clathrin coat proteins including clathrin, adaptors and accessory proteins; (ii) CCP maturation; (iii) CCV scission; (iv) CCV coat disassembly and virus entry into endosomes; (v) fusion pore formation and expansion; and (vi) fusion completion. By integrating the recent results discussed in this review, we propose that dynamin is involved in every stage of this virus entry pro-cess, playing alternate roles as a regulatory GTPase and a mechano-chemical enzyme (Figure 4).
Besides CME, a variety of virus entry pathways are dynamin-dependent. Although information on the roles of dynamin in these processes is very limited, the solved crystal structures of full-length dyanmin and cryo-EM
structure of dynamin-coated tubule assembly have led the proposal of precise mechanisms of dynamin-medi-ated membrane fission. However, the role of dynamin as a regulatory GTPase has only been identified in CME, and the involvement of dynamin in virus membrane fusion has only been demonstrated in two viruses: HIV-1 and vaccinia virus. The limited information available underscores the need to assess dynamin’s multiple func-tions in more viruses.
Acknowledgements
This work is supported by the grants from the National Basic Research Program of China (973 Program) (Nos. 2010CB530102, 2011CB504703), and by the National Natural Science Foundation of China (NSFC, Grant No. 81021003).
Declaration of interest
The authors report no declarations of interest.
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