Enveloped virus membrane fusion

However, in the case of the paramyxovirus F the HRB sequences wrap around the HRA coiled-coil forming an extremely stable six-helix bundle 6HB in the post-fusion hairpin. Formation of this 6HB provides most of the energy required to overcome membrane repulsion. The 6HB structure is shared by other class I fusion glycoproteins, such as the gp41 chain of the HIV envelope glycoprotein.

While activation of influenza HA requires exposure to the endosomal low pH probably by protonation of key amino acid residues , the event that triggers paramyxovirus F proteins is still ill-defined. Cell-cell fusion of transfected cells that express paramyxovirus F requires in most cases co-expression of the homotypic attachment protein, suggesting that an interaction of the two proteins is needed for membrane fusion.

The clamp model postulates that HN or the equivalent attachment protein depending on the virus is complexed with F in the virus particle, retaining the latter in the metastable configuration. Conformational changes in HN upon receptor binding release F from the complex to initiate membrane fusion. Alternatively, the provocateur model postulates that HN and F do not interact in the virus before contacting the cell. Concomitantly to the structural changes induced in HN upon receptor binding, HN binds to F and this interaction triggers F for fusion [ 25 ].

Intriguingly, the F protein of viruses belonging to the Pneumovirinae subfamily of paramyxoviruses e. Furthermore, deletion mutant viruses have been obtained in which the entire G gene is obliterated. These mutants still infect cells in vitro , although less efficiently than the wild type virus and are attenuated in animal models of infection [ 27 ]. Activation of the F protein of those deletion mutants cannot rely on interactions with the G protein and therefore alternative regulatory mechanisms should control membrane fusion.

Of note, a unique characteristic of the RSV F protein is the presence of two proteolytic cleavage sites instead of one, as in all other paramyxovirus in the F0 protein precursor [ 26 ]. The presence of a double cleavage site in F has been found to influence membrane fusion activation by a still poorly understood G independent mechanism [ 28 ].

In contrast to class I fusion proteins, the so-called class II fusion proteins Table Both proteins fold co-translationally with a companion or regulatory protein, termed p62 for alphaviruses and prM for flaviviruses [ 9 ]. In alphavirus, the pE1 complex is transported to the plasma membrane where they are incorporated into new budding icosahedral virus particles as dimers of pE1. E2 mediates binding to the cell surface receptor.

In contrast, flavivirus particles bud into the endoplasmic reticulum as immature virions containing prM-E protein complexes. The immature viruses are then transported to the exterior through the exocytic pathway where prM is processed and separated from E [ 29 ].

The latter protein is then arranged in E-E homodimers at the virion surface with icosahedral symmetry. The flavivirus fusion E protein is additionally responsible for receptor binding. The first structure of any class II glycoprotein, solved by X-ray crystallography, was that of the tick-borne encephalitis TBE flavivirus E protein ectodomain [ 30 ], solubilized from virions by limited trypsin digestion.

Similar structures have now been solved for the E ectodomain of dengue virus types 2 and 3 [ 9 ]. At the tip of domain II is the hydrophobic fusion loop which remains buried in the virion from the hydrophilic environment by interaction with domain III blue of the adjacent monomer in the E-E dimer. Domain I is also connected to domain III which bridges the E ectodomain with the so-called stem region that extends to the TM region of the protein.

Membrane fusion mediated by a class II fusion protein Flavivirus. Ribbon representation of the atomic structures of the dengue virus E protein dimer in the pre-fusion conformation [ 54 ] upper left and the E protein trimer in the post-fusion conformation [ 55 ] lower right.

In the last two steps, two E protein molecules are represented to indicate the cooperation needed to drive the fusion process. Unlike the class I fusion proteins, which are trimeric in their pre- and post-fusion conformations, class II fusion glycoproteins undergo major oligomeric transformations during fusion.

As in the case of influenza virus, the flavivirus E protein first binds to a cell surface receptor which induces endocytosis of the virion. Once in the acidic endosome, the E-E homodimer dissociates, resulting in disassembly of the icosahedral scaffold. The individual subunits swing outward by the hinge region that connects domains I and II, and the fusion loops insert into the target membrane. Lateral interactions between monomers facilitates reclustering into trimers [ 31 ].

These rearrangements lead to the formation of an extended trimeric structure, analogous to the pre-hairpin intermediate of class I fusion glycoproteins, in which two different regions of each E polypeptide are inserted into the two membranes to be fused.

Collapse of the extended intermediate can proceed by rotation of domain III in each subunit about the segment that links it to domain I and zipping up of the stem alongside the clustered domains II. This refolding brings the two membranes together to initiate formation of the lipid stalk, the hemifusion diaphragm and the fusion pore. The structure of the fusion E1 glycoprotein of alphaviruses SFV was found unexpectedly very similar to that of the flavivirus E protein, despite the lack of detectable sequence conservation.

E1 has also three discernible domains, equivalent to those of flavivirus E. The only significant difference is the association of E1 with E2 in the virus particle. E2 interacts with the cell surface receptor to initiate the endocytic internalization of the SFV virion [ 32 ]. In the acidic endosome, E2 separates from E1 and it is probably degraded. Upon low pH exposure, E1 undergoes similar conformational changes to those of the flavivirus E protein, leading to fusion of the viral and endosomal membranes.

Electron microscopy and X-ray crystallography results provide support for interactions between adjacent E1 trimers when the fusion loops are inserted in the target membrane to produce rings of five or six trimers. It has been postulated that these fivefold interactions would act at the fusion site to induce the formation of a nipple-like curvature in the viral and target membranes, favoring membrane fusion [ 33 ].

Although there is no direct evidence, it is likely that the flavivirus E protein forms similar rings of trimers during fusion. The best characterized members of the so-called class III fusion viral glycoproteins are the rhabdovirus e.

Class III fusion glycoproteins are expressed from individual mRNAs and do not require proteolytic processing of either a protein precursor as in class I proteins or an accompanying protein as in class II proteins for activity. Class III proteins are trimeric before and after fusion and share structural characteristics with both class I and class II fusion glycoproteins, as described below.

The rhabdovirus G protein possesses both receptor binding and fusion promoting activities. As in the case of influenza virus, binding of rhabdovirus G to a poorly characterized receptor at the cell surface induces endocytosis of the virus particle. Acidification of the endosome triggers G for membrane fusion. However, and in contrast with all other fusion proteins, the low pH inactivation of rhabdovirus G is reversible. This reversibility may be required to allow G to be transported through the acidic Golgi apparatus and to recover its native fusion-competent state when incorporated to new virions [ 39 ].

Given this reversibility, it is believed that the energy released during the structural transition of a single trimer from the pre-fusion to the post-fusion conformation is probably small, compared with the energetic barrier of the fusion reaction. In agreement with this hypothesis, the estimated number of rhabdovirus spikes required for fusion is higher at least 15 trimers than for other enveloped viruses.

Several domains could be observed in both structures that are rearranged in their relative orientations during transit from the pre- to the post-fusion structure Fig.

In the pre-fusion conformation, the fusion domain contains two fusion loops reminiscent of class II proteins that are oriented downward towards the viral membrane. After low pH exposure, the fusion domain moves upward by flipping relative to the central core of the trimer. Thus, an intermediate equivalent to the pre-hairpin structure of class I proteins is formed. This is followed by the reversal of the molecule around a central rigid block formed by lengthening of the central helix and refolding of the three C-terminal segments into helices that position themselves in the grooves of the central core in an anti-parallel manner.

This six-helix bundle has obvious resemblance with that of the class I proteins. Ribbon representations of the VSV glycoprotein G , in the pre- upper left and post-fusion lower right conformations.

Domains are colored similarly in all images. The fusion domain is colored in yellow and the fusion loops in green. In the last two steps, two G protein molecules are represented to indicate the cooperation needed to drive the fusion process. It is also likely that cooperativity between G glycoproteins is needed to overcome the energy barrier, as mentioned above. As for class II glycoproteins, lattices of G proteins have been observed in virions, particularly in the planar base of the rhabdovirus bullet-shape particle, which may act to induce nipple-like deformations in the viral and target membranes.

Although it has not been reported, it is likely that low pH exposure also leads to reversible inactivation of the baculovirus gp64 glycoprotein.

In contrast, membrane fusion mediated by herpesvirus gB can occur either at the plasma membrane or in endosomes, depending on the virus and the target cell type. In either case, attachment of herpesvirus to host cells follows a complex mechanism in which several viral glycoproteins interact with cell surface molecules.

Some of these interactions trigger fusion, whereas others simply serve to tether the virus to the cell and are dispensable for fusion.

In any case, the gB protein, shared by all viruses of the Herpesviridae family, is responsible for fusion [ 42 ]. Poxviruses vaccinia virus is the best known member represent an extreme case among enveloped viruses, regarding the number of viral glycoproteins required for entry.

As for herpes virus, entry can occur by fusion at the plasma membrane or in a low pH-dependent manner from within an intracellular particle, depending on the virus strain and the cell type. Vaccinia virus internalization is believed to occur by macropinocytosis a type of non-specific endocytosis. Four vaccinia virus proteins are involved in attachment to cell surface proteoglycans or laminin [ 43 ].

Eleven or 12 other relatively small glycoproteins, ranging in size between 35 and amino acids, form the so-called entry fusion complex EFC that mediates membrane fusion [ 44 ]. These proteins have N- or C-terminal transmembrane domains but no sequence similarity with the fusion peptide of other viral fusion glycoproteins has been found in any of them.

Therefore, the actual mechanism of vaccinia virus membrane fusion remains to be elucidated but it seems to be different from that of other enveloped viruses. By using conditional lethal mutants of each of the 11 proteins that make the EFC, it was found that eight of them were required to reach the hemifusion step and the other three were needed for completion of virus entry [ 44 ]. It is likely that hydrophobic regions of several proteins may assemble in the EFC to form a hydrophobic surface that could bind to the target membrane and drive membrane fusion by some novel mechanism.

Finally, the fusion-associated small transmembrane FAST proteins of reoviruses are brought here -despite not being involved in virus entry and reovirus being a non-enveloped virus- because they induce cell-cell fusion and therefore facilitates dissemination of virus to neighboring cells.

The FAST proteins are small non structural proteins 98— amino acids, depending on the viral strain that are expressed on the surfaces of virus-infected cells, where they induce cell-cell fusion and syncytia multinucleate cells formation. Purified FAST proteins, when reconstituted into liposome membranes, induce fusion indicating that they are bona fide fusogens [ 45 ]. The orientation of the FAST polypeptides in the cell membrane is also unique among viral fusogens, with a relatively short N-terminal ectodomain followed by a transmembrane region and a long C-terminal cytoplasmic tail.

Although they lack a fusion peptide, a relatively hydrophobic region near the N-terminus which is additionally myristoylated seems to insert into the target membrane to drive membrane fusion, at least for certain FAST proteins [ 46 ]. Structures for both pre- and postfusion conformations of illustrate the beginning and end points of a process that can be probed by single-virion measurements of fusion kinetics.

Keywords: Fusion mechanism; Fusion protein; Virus entry. Published by Elsevier Inc. The viral fusogens experience drastic structural rearrangements during fusion, liberating the energy required to overcome the repulsive forces that prevent spontaneous fusion of the two membranes.

This chapter describes the different types of viral fusogens and their mode of action, as are currently known. The field of enveloped virus fusion in endosomes has come a long way since the inaugural paper by Helenius and coworkers 1. As elaborated above, detailed fusion mechanisms — encompassing key structural elements and key structural changes in the fusion protein, and key environmental triggering cues low pH, receptors and proteases — are now known for representatives of class I, II and III fusion proteins.

Many questions remain. For examples: Are there additional classes of enveloped viral fusion proteins? What are the common principles by which proteases trigger fusion proteins post receptor binding? How is fusion triggering orchestrated in multicomponent fusion machines?

For their part, some viruses, notably HIV, have evolved means to counter attempts by cells to thwart virus—cell fusion , How exactly are these battles between enveloped viruses and cells enacted? We thank numerous colleagues for helpful discussions, and we thank Kathryn Schornberg for preparing the figures.

We apologize for citation omissions. The authors have no conflicts of interest to report. National Center for Biotechnology Information , U.

Published online Apr 7. Judith M. White 1 and Gary R. Whittaker 2. Gary R. Author information Article notes Copyright and License information Disclaimer. Corresponding author. White, ude. It can be used for unrestricted research re-use and analysis in any form or by any means with acknowledgement of the original source, for the duration of the public health emergency.

This article has been cited by other articles in PMC. Abstract To initiate infection, enveloped viruses must fuse with a cell membrane, a process mediated by a dedicated viral fusion protein. Keywords: enveloped virus, fuse, low pH , membrane, prime, proteases, trigger, viral fusion protein, virus entry, virus receptors.

Abstract Ari Helenius launched the field of enveloped virus fusion in endosomes with a seminal paper in the Journal of Cell Biology in Open in a separate window. Figure 1. Table 1 Definition of terms pertinent to viral membrane fusion proteins. Fusion protein The transmembrane protein on the surface of an enveloped virus that engages the target bilayer to mediate virus—cell membrane fusion.

All characterized viral fusion proteins contain both a fusion peptide or fusion loop that engages the target membrane and a transmembrane domain that anchors the protein in the viral membrane. Fusion subunit Certain viral fusion proteins e. In all of these cases the fusion subunit contains both the fusion peptide or fusion loop and a transmembrane domain.

It is the portion of the fusion protein that engages the target membrane. Like a fusion peptide, it is the region of the fusion protein that engages the target membrane. Prefusion conformation The conformation of the viral fusion protein as it appears on the viral membrane after priming, but before fusion triggering.

Priming All characterized class I and class II viral fusion proteins are primed to a state capable of responding to a fusion trigger. This involves a proteolytic cleavage event in the fusion protein precursor or in a companion protein. Triggering All viral fusion proteins must be triggered for fusion. In most cases, a single trigger is sufficient, but in some cases e. Viral Membrane Fusion Proteins: General Considerations Enveloped viruses vary in the number of different types of glycoproteins that protrude from their membranes.

Table 3 Examples of viral membrane fusion proteins. Its single glycoprotein GP is cleaved by furin to GP1 and GP2 , this cleavage is important for fusion 34 and GP2 is postulated to possess fusion activity Figure 2. Figure 3. Table 4 Examples of endosomal viral fusion triggers. Andes Gc Andes virus fusion requires high levels of cholesterol in the target membrane. Fusion may be triggered at neutral pH or under low pH conditions.

See text for references and details. Triggering by Binding to a Receptor Followed by Exposure to Low pH or the Action of a Protease Below, we will discuss how engagement of a host cell receptor can play an active, albeit not exclusive, role in triggering certain viral fusion proteins that function in endosomes, As a prelude, we review how viruses that fuse at neutral pH are activated by their host cell receptors.

Cases for Which the Triggering Mechanism Is Not Yet Clear There are several viruses that enter cells through endosomes for which the mechanism of fusion triggering is unclear. Concluding Remarks The field of enveloped virus fusion in endosomes has come a long way since the inaugural paper by Helenius and coworkers 1.

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