Structural biology

An alphavirus puzzle solved

Alphaviruses infect their host by binding to cellular receptors and fusing with cell membranes. New studies define the receptor-binding protein of these viruses and its regulation of the membrane-fusion reaction. See Letters p.705 & p.709

Many viruses are enclosed in an envelope — a membrane that is derived from the infected host cell during virus exit. To infect a new host cell, specialized membrane-fusion proteins on the virus envelope fuse it with a membrane of the host cell, delivering the viral genome into the cell. This fusion activity must be deployed at precisely the right time during virus entry, and must also be silenced during viral assembly and exit. In this issue, Li et al.1 and Voss et al.2 provide structural insights into the regulation of the membrane-fusion proteins of enveloped alphaviruses during the viruses' entry into and exit from the host cell.

Many alphaviruses are medically relevant; chikungunya virus, for example, is an emerging human pathogen responsible for major recent epidemics3. There are currently no treatments for alphavirus infections, and detailed information on the structure and life cycle of these viruses is crucial for developing antiviral strategies and vaccines.

But first, a quick glance at what is already known. The membrane-fusion protein of alphaviruses is E1, and its fusion activity is triggered by the mildly acidic pH of intracellular vesicles4. Structural studies have defined the architecture of the E1 molecule5,6, its arrangement on the virus particle5,6,7,8, and conformational changes in it that drive membrane fusion9. E1 is tightly associated with another membrane protein, E2, which has been an elusive missing piece of the virus's structural puzzle. The alphavirus envelope is covered by an organized lattice composed of E2/E1 pairs arranged into 80 trimers, or 'spikes'.

The alphavirus infection cycle begins when E2 binds to receptors on the surface of a host cell. This allows the virus to be internalized and transported into the acidic intracellular vesicles. The low pH induces a rearrangement of the E2/E1 pair, unleashing E1's fusion activity10 (Fig. 1). E1 inserts its hydrophobic fusion loop into the membrane of the host-cell vesicle, forms E1 trimers, and refolds to pull the host-cell and viral membranes together, thereby causing membrane fusion and virus infection9.

Figure 1: The alphavirus infection cycle.
figure1

a, The fusion proteins of an alphavirus are connected to the viral membrane and can consist of three components: E1, which contains domains I, II and III, and a fusion loop (star); E2, which contains domains A, B and C, and a ribbon-like connector region; and E3. The alphavirus envelope is peppered with trimers of E2/E1 pairs, although for simplicity only one E2/E1 pair is shown. E3 is a by-product of the cleavage of the E2 precursor protein p62 by the enzyme furin (not shown), and is released in some alphavirus species. b, On exposure to low pH, domain B and the connector region of E2 move, exposing the E1 fusion loop. c, E1 can then detach from E2 and insert into the host cell membrane to form an extended trimer. d, Finally, to drive membrane fusion, E1 refolds into a hairpin-like structure through the movement of its domain III and the stem region, which lies adjacent to the membrane. For simplicity, only the final fused membrane is shown.

In addition to binding to host-cell receptors, E2 is an essential component throughout the alphavirus life cycle. During viral replication, this protein is synthesized as a precursor called p62 (or PE2) and acts to chaperone the folding of its E1 partner. Like the vesicular entry pathway, the exit pathway involves transport through cellular compartments that have an acidic pH. The p62/E1 pair is more acid-resistant than the E2/E1 dimer, and this property seems to protect E1 from premature fusion during transport through the exit pathway11. Late in transport, the cellular enzyme furin cleaves p62 to produce the mature E2 protein plus a small peripheral protein, E3 (ref. 12). The virus then exits by budding from the cell surface, with some alphavirus species retaining E3 and others releasing it.

Li et al.1 (page 705) and Voss et al.2 (page 709) present the molecular structure of the E2/E1 pair and define the mechanisms by which E2 both silences E1 during virus exit and regulates E1's triggering at low pH during virus entry. Focusing respectively on Sindbis virus1 and chikungunya virus2, the authors generated modified versions of p62/E1 proteins that were joined together by flexible linkers and lacked their membrane-anchoring domains; such modifications were critical for stabilizing E2 for structural studies.

Each team determined the crystal structures of these protein pairs for their virus and fitted them into the molecular outline of the alphavirus particle, previously established8,13 by electron microscopy. Voss and colleagues' chikungunya virus structures define the immature p62/E1 pair and the mature E2/E1 complex with the retained E3, whereas the Sindbis virus structures of Li et al. reveal the mature E2/E1 pair (without E3), associated in trimeric spikes as on the surface of the virus. The new structures show that the mature E2 protein is an elongated molecule containing three domains with immunoglobulin-like folds: the amino-terminal domain A, located at the centre; domain B at the tip; and the carboxy-terminal domain C, located close to the viral membrane.

The chikungunya E2 covers much of its E1 partner on the virus surface, with the hydrophobic fusion loop of E1 clamped in the groove between domains A and B of E2 (Fig. 1). The Sindbis virus E2/E1 pair was crystallized at acidic pH. Although it closely resembles the chikungunya E2/E1 structure, the E2 domain B that 'caps' the E1 fusion loop is disordered and not visualized. This structure suggests that an early intermediate in the low-pH-triggered fusion process is formed by the release of E2 domain B, exposing the E1 fusion loop. This E2/E1 rearrangement seems to occur through changes in a flexible, ribbon-like connector that links domain B to domains A and C, and packs tightly against the underlying E1 protein.

The immature p62/E1 and mature E3/E2/E1 complexes are very similar apart from the tether region that links E3 to E2 and is the site of cleavage by furin. This suggests that the main difference leading to the increased acid resistance of the immature spike is that the connector ribbon that maintains the domain-B cap in place is stabilized by interactions of the tethered E3.

The new structures1,2 illuminate key aspects of the alphavirus life cycle. In addition, the exposed regions of E2 domains A and B contain several sites to which neutralizing antibodies bind, as well as sites implicated in virus–receptor interaction. The structures of these domains can therefore now be used to clarify the mechanisms of virus receptor binding and neutralization, and to exploit these processes for antiviral and vaccine strategies.

The structures of the p62/E1 and E2/E1 pairs identify specific residues that may control their dissociation at low pH and explain how p62 and E2 regulate virus fusion. Knowing the details of the p62/E1 interaction will also help to determine how much of the requirement for p62 during E1 synthesis is to protect E1 from low pH and how much is to directly assist with E1 folding. The intriguing 'uncapped' structure of the alphavirus spike highlights how little is known about the downstream fusion intermediates, which must involve considerable movements of E2 and E1 on a highly organized virus particle, and which will be an exciting area for future work.

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Kielian, M. An alphavirus puzzle solved. Nature 468, 645–646 (2010). https://doi.org/10.1038/468645a

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