Class II fusion proteins are not proteolytically cleaved and have internal, rather than N-terminal fusion peptides^30,31. They are synthesized as a complex with a second membrane glycoprotein, and the activation of the fusogenic potential of the class II fusion proteins involves the cleavage of this accessory protein. X-ray crystallography of three class II fusion proteins — the E proteins of TBE and dengue virus, and the E1 protein of SFV — has revealed a common fold for these proteins, which is structurally unrelated to the class I viral fusion proteins. Class II fusion proteins fold as heterodimers with the companion (chaperone) glycoprotein — for example, pE2 with pE1 in alphaviruses and prM with pE in flaviviruses — and form a protein network at the viral surface. The fusogenic conformational changes lead to a reversible dissociation of the dimers to release monomers, followed by irreversible reassociation into stable homotrimers in the presence of membranes (Fig. 5). Although the alphaviruses SFV and SIN require the membrane components cholesterol and sphingolipids for the binding and initiation of conformational changes, the flavivirus TBE does not require these membrane components — but binding to target membranes and trimerization of the TBE E protein might involve interactions with cholesterol⁶¹.

Unlike class I and class II fusion proteins, the conformational changes of the G proteins of rabies virus and the vesicular stomatitis virus (VSV) (family rhabdoviridae) that are induced by a low pH are reversible, which indicates that the low pH does not trigger a transition through a high-energy intermediate state^62,63. However, interactions with membranes could induce irreversible conformational changes. VSV and rabies virus can both fuse with pure lipid vesicles, although neither virus requires any specific lipids for fusion. The transition between equilibrium states of the entry proteins could provide free energy to overcome the membrane fusion barrier, but formation of the fusion site might require many more trimers acting cooperatively than the 5–6 that have typically been estimated to be involved⁶⁴. The G proteins of rhabdoviruses have several characteristics in common with class I fusion proteins (for example, they have an internal fusion peptide and are not complexed with other proteins at the virion surface) and class II fusion proteins (for example, they are not cleaved and do not have heptad sequences that are predictive of coiled-coils). The X-ray crystal structure of the rhabdovirus G protein has not been solved, and it remains to be seen whether it will become the founding member of a new class III fusion protein family.

Enveloped viruses: membrane fusion. An important role of the conformational changes that all classes of fusion proteins undergo is to overcome energy barriers to enable membrane destabilization and the formation of fusion pores. The energy barrier for the fusion of VSV with a cell is estimated to be about 42 kcal mol⁻¹ (Ref. 65). For protein-free model lipid bilayers, the activation energies are comparable — estimated values of 37, 27 and 22 kcal mol⁻¹ were reported for the formation of a reversible first intermediate, its conversion to a second, semi-stable intermediate and irreversible fusion-pore formation, respectively⁶⁶, which indicates that the basic molecular mechanisms of viral fusion might involve similar lipid molecular rearrangements to those observed in the fusion of model lipid membranes.

How the energy that is released by conformational changes of the viral protein is used to overcome the membrane fusion energy barriers is unclear. For class I fusion proteins it was recently found that the formation of six-helix bundles stabilizes fusion-pore formation⁶⁷. A spring-loaded conformational change⁶⁸ (Fig. 5) is required, but might not be sufficient, for HA-mediated fusion, and the transition to the membrane fusion pore could depend, in part, on the subsequent action of the HA fusion peptide and transmembrane domain⁶⁹. For class II fusion proteins it was proposed that the pH-triggered conformational changes result in insertion of class II protein β-barrels into the host membrane, which, in turn, leads to fusion-pore formation²⁴. It seems that once the viral protein conformational changes have provided sufficient energy to enable close membrane apposition (less than 1 nm) and destabilization, formation of the fusion pore can proceed through intermediates, stalks and hemifusion diaphragms, which might be common to all known viral membrane fusion processes^70,71. Data obtained for baculovirus gp64 are consistent with this hypothesis⁷².

A number of models have been suggested for fusion-pore formation by lipid–protein complexes^52,70. To ensure the transfer of the viral nucleoprotein complex, which is typically stable in solution, it seems that the internal lining of the fusion pore must be hydrophilic. Fusion-pore formation requires a minimal number (5–6) of oligomeric viral entry proteins to form a supramolecular lipid–protein complex. It is a dynamic process and small pores can open and close reversibly. Models of fusion-pore formation are mainly based on fusion mediated by influenza HA, but might prove generally valid. The enlargement of the fusion pore that allows transfer of the genome into the cell occurs by an unknown mechanism.

Non-enveloped viruses: membrane penetration. The mechanism of penetration of non-enveloped viruses remains poorly understood, although they are relatively small and simple in structure¹⁸. Fusion of the more complex enveloped viruses is better understood owing to the similarities between enveloped viruses and cells — both are surrounded by membranes and their fusion does not necessarily require a significant reorganization of the viral nucleoprotein complex. By contrast, either the whole non-enveloped virus must cross the membrane, or it must undergo important conformational changes and transfer the genome through the membrane. In the latter case, the energy of the metastable state must be used not only to destabilize the cell membrane, but also to reorganize the nucleoprotein complex so that the genome can be released through the destabilized membrane either through pores or other structures. Although the structures of several key intermediates in the conformational changes of viral entry proteins have now been solved to atomic resolution, it is clear that there is a great deal left to learn owing to the difficulties that are associated with studying the rapid conformational changes of only a few molecules of each virion against a background of large numbers of surface molecules on the virion that undergo no structural changes.

Entry proteins and pathogenesis

Viral proteins can destabilize not only plasma and endosomal membranes during entry, but can also destabilize other membranes inside cells to which the protein is in close proximity if there is a suitable trigger, such as a receptor, low pH or low calcium concentration. For example, such conditions arise during transportation of viral proteins after synthesis in the host cell in acidic Golgi vesicles. It seems that viruses have developed strategies to cope with this problem. For example, the G protein of rhabdoviruses, such as rabies and VSV, can exist in native, activated and inactive conformations. It was proposed that the inactive state helps to avoid membrane fusion during the transport of the G protein⁷³. However, the HIV-1 Env can induce cell lysis after interaction with receptors — probably through disruption of important intracellular membranes — so methods to cope with destabilization of cell functions are not universal⁷⁴. The significance of membrane fusion effects in human pathogenesis is unclear. Perhaps viruses that are well adapted to their hosts do not induce significant pathogenic effects that could lead to a reduction of virus production. However, viruses that infect a new host, such as HIV and SARS, might not be well adapted to this host and their entry proteins could cause CYTOPATHIC EFFECTS. Cell fusion — perhaps mediated by Envs — by endogenous retroviruses could contribute to cancer⁷⁵.

Entry inhibitors, antibodies and vaccines

Entry is an attractive target for inhibition because the entry machinery is extracellular and it is therefore easier for drug molecules to reach than intracellular targets. Any step(s) of the entry process can be targeted by an entry inhibitor. Various types of molecules, such as proteins, peptides, carbohydrates, small organic molecules, nucleic acids and supramolecular structures, including liposomes and phage, have been found to inhibit entry. Yet, out of more than 30 antiviral drugs⁷⁶, there are only two entry inhibitors — Synagis⁷⁷ and T-20 (Ref. 78) — that have been approved by the US Food and Drug Administration (FDA) for clinical use (excluding human immune globulin for use against hepatitis A and measles, and virus-specific polyclonal human immune globulins for use against cytomegalovirus, hepatitis B, rabies, RSV, vaccinia and varicella-zoster⁷⁹). Only T-20 (which is marketed as enfuvirtide) is used for the treatment of ongoing viral (HIV-1) infection. The humanized monoclonal antibody Synagis (which is also known as palivizumab) is used for the prevention of RSV infections in neonates and immunocompromised individuals. T-20 is not a small molecule — regarded as the 'gold standard' for a drug — but is a peptide that cannot be taken orally. A small organic molecule entry inhibitor (pleconaril) showed promising results for the treatment of infections caused by the picornaviruses that cause the common cold⁸⁰, but was not approved by the FDA owing to concerns about potential interactions with other drugs — although different formulations are presently being evaluated for use in life-threatening disease. At present, a number of compounds are in clinical trials, including small organic molecules that bind to the HIV-1 co-receptor CCR5, and entry inhibitors are also being tested for efficacy as microbicides.

Challenges in the development of virus entry inhibitors. Major problems in the development of effective entry inhibitors include the generation of virus mutants that are resistant to such drugs, a low potency in vivo and toxic side effects. Although these problems are common to the development of other antiviral (and cancer) drugs and limit their efficacy, the development of entry inhibitors might also face specific challenges. Viruses have evolved to recognize cellular receptors and enter cells despite the presence of the host immune system. Antibodies that exhibit inhibitory effects on virus entry must bind viruses that have already developed a number of strategies for immune evasion, including the use of immunodominant variable loops and incomplete complementary binding surfaces, oligomeric occlusion, glycosylation⁸¹, conformational masking⁸² and multivalent interactions. Some problems that are inherent to the design of any inhibitor are especially challenging for those targeted to virus entry, such as the inhibition of high-affinity protein–protein interactions by small molecules. Another strategy used by viruses to counteract the action of antibodies and other entry inhibitors is the direct cell-to-cell transmission of viruses, which avoids or reduces the exposure of the virus to inhibitors that are not designed to penetrate through membranes or other barriers. This could be one important reason for the low efficacy that is observed in vivo of otherwise potent in vitro inhibitors. Low inhibitor potency combined with a high virus mutation rate is perhaps the most challenging problem in the development of entry inhibitors.

Although the rapid progress in our understanding of the structural mechanisms of virus entry promises new approaches that could 'outsmart' the virus, no entry inhibitors or treatment protocols in clinical use have so far been developed on the basis of predictions made by structural models, and the main source of new inhibitors is still from screening large libraries of small organic molecules, natural products, peptides and antibodies. However, structures of entry proteins have been invaluable for the development of our understanding of the mechanisms of inhibition and should allow further improvement of the inhibitors. It seems likely that sooner or later structure-based design will yield entry inhibitors that will be in clinical use.

Conserved entry intermediates as targets for inhibitors; multivalent inhibitors and other approaches. One direction of research that could hasten the arrival of entry inhibitors to the clinics is the development of compounds that interact with conserved intermediates of the entry process or with the protein structures that, on binding to receptors, trigger the conformational changes that lead to the formation of these entry intermediates. Typically, such intermediates are only transiently exposed, so viruses might not have evolved strategies to avoid inhibitors targeted to these structures. In addition, conserved intermediates are usually important for virus entry and presumably cannot easily be substituted by other structures after mutation.

An example of the successful design of an entry inhibitor that shows proof of the concept is the 5-Helix protein, which interferes with a conserved intermediate in the entry of HIV-1 (Ref. 83). A related example is a class of peptides that could have broad applications to several viruses containing class I fusion proteins. The peptides are derived from regions of fusion proteins (heptad repeats) that have a propensity to form coiled-coils and which serve as fusion intermediates and enable oligomerization of proteins. T-20 (also known as DP178) is derived from the carboxy-terminal heptad region of the HIV-1 gp41 and showed potent inhibitory activity in vivo⁷⁸. Similar peptides from other viruses, including HTLV-1, RSV, measles virus, Nipah virus, Hendra virus, Ebola virus and SARS-CoV, are also promising entry inhibitor candidates. However, Ebola virus is inhibited only at very high concentrations, influenza HA-mediated fusion is not inhibited and SARS-CoV infection might not be inhibited either (K. Bossart and C. Broder, personal communication), perhaps owing to the endocytic entry route of these viruses. Although peptides have certain promising features, including a relatively small size that might ensure good penetration combined with high binding affinity, the lack of oral formulations, short half-life, possible toxicity and immunogenicity might limit their application.

Recently, a small molecule, BMS-378806, was identified that inhibits the entry of a broad range of HIV-1 isolates by a mechanism which was attributed to competition with the CD4 receptor for binding to gp120 (Ref. 84). However, this compound inhibited the entry of one isolate of HIV-1 (HIV-1_JRFL) with an IC₅₀ of 1.5 nM, and the binding of CD4 to gp120 from the same isolate with an IC₅₀ of 100 nM. So, at inhibitory concentrations, BMS-378806 does not interfere with CD4 binding, indicating a different inhibitory mechanism. By analogy with inhibitors of picornavirus entry, such as pleconaril, BMS-378806 might inhibit the entry of HIV-1 by binding to conserved structures that are important for the conformational changes which gp120 must undergo for viral entry⁸⁰. Potent virus-specific inhibitors of the viral-membrane-merging step have not been identified yet.

Another promising direction is the development of multivalent inhibitors that can overcome problems caused by mutation of viral proteins to escape inhibition because multivalent inhibitors bind to several regions of the same (or different) protein(s) on the viral surface. One example of a multivalent inhibitor is the multimeric soluble receptors of influenza and HIV-1 (Ref. 82), which are potent inhibitors of entry in vitro and, to some extent, in vivo. The use of entry inhibitors in combination or as fusion proteins could also result in increased efficiency. Finally, improvement of current methods for structure-based design by accounting for protein flexibility and dynamics in binding to ligands⁸⁵, and screening methods for inhibitors^86,87 would certainly expand the range of possible inhibitors that can be tested.

Neutralizing antibodies and vaccine immunogen design. Neutralizing antibodies usually inhibit virus entry by preventing attachment of the virus to the cell or by binding to entry intermediates^88,89,90. Human immunoglobulin composed of concentrated antibodies collected from pooled human plasma has been successfully used as a preventative treatment for virus infections, including rabies, hepatitis A and B, measles, mumps, varicella, cytomegalovirus and arenaviruses. Antibodies can completely prevent infection, but once infection is established they are a much less efficient treatment. The only monoclonal antibody in clinical use today to treat a viral disease — Synagis (MEDI-493) — is more potent than the polyclonal immunoglobulin that is presently in use, and is broadly active against numerous RSV type A and B clinical isolates⁹¹. It binds to the F protein of RSV with high affinity (3 nM) and inhibits virus entry and cell fusion in vitro with an IC₅₀ of approximately 0.1 μg ml⁻¹. It seems that the efficacy of Synagis in vivo is correlated with the high affinity of binding and potency of this antibody in vitro⁹⁴. However, Synagis had no measurable clinical efficacy after administration as a single 15 mg kg⁻¹ intravenous dose to infants that were hospitalized with established RSV infection, although it did significantly reduce RSV concentrations in tracheal aspirates⁹².

The X-ray crystal structures of rhinovirus²¹ and poliovirus²² indicate a possible mechanism by which picornaviruses can avoid neutralization by antibodies through the mutation of non-conserved amino acid residues surrounding the receptor-binding site — a 2 nm deep and 2 nm wide canyon (Fig. 4c). It was initially hypothesized that the conserved amino acid residues of the canyon are not accessible by antibodies; however, it was later shown that a strongly neutralizing antibody, Fab17, can penetrate deep within the receptor-binding canyon by undergoing a large conformational change without inducing conformational changes in the virus^90,93. Unusually, not only the hypervariable residues but also residues from the framework region of Fab17 contact the canyon. Yet another remarkable mechanism of immune recognition of viruses is the recently discovered receptor mimicry by post-translational modification (tyrosine sulphation) of antibodies (Ref. 94; Huang, C. et al., manuscript in preparation). It seems that any accessible viral surface can be recognized by antibodies. Rapidly mutating viruses can escape neutralizing antibodies even if they bind to structures that are essential for virus replication, such as receptor-binding sites, unless they bind with energetically identical profiles⁹⁵. Whether a virus will escape neutralization by antibodies depends on the interplay between the antibody affinity (avidity) and kinetics of binding, generation rate, concentration and the viral mutation rate and fitness. Mutations of immunodominant structural loops that form antibody-binding sites and mutations leading to changes in oligosaccharide attachment to viral entry proteins are common mechanisms by which viruses avoid neutralization^90,96.

Mutations of conserved residues that have a role in the entry mechanism typically result in reduction or loss of infectivity. Antibodies or their derivatives that bind to epitopes where residues contribute most of the binding energy could have potential as entry inhibitors. Epitopes that are exposed after virus binding to receptors are typically well conserved — for example, the 17b³⁹ and X5 (Ref. 97) epitopes on HIV-1 gp120. One potential problem with using antibodies as entry inhibitors in this case could be limited access to the post-receptor-binding state of the viral entry protein due to the relatively large size of the whole antibody. Solving this, and other problems, could lead to the development of potent broadly neutralizing antibodies which could limit the generation of resistant viruses, especially if these inhibitors are used in combination with other antibodies or inhibitory molecules.

Many viruses, especially RNA viruses such as HIV-1, exist as swarms of virions inside an infected individual, and might significantly differ in sequence between isolates. So, elicitation of potent, broadly neutralizing antibodies is an important goal for vaccine development. Potent, broadly neutralizing antibodies for HIV-1 Env do exist — for example, b12, 2G12, 447-52D, X5, 2F5 and 4E10/Z13. However, elicitation of these antibodies in vivo has not been successful. Identification of broadly neutralizing antibodies and the characterization of their epitopes could help to design vaccine immunogens that would be able to elicit these neutralizing antibodies in vivo — so-called retrovaccinology⁸⁹. At present, all vaccines that elicit antibodies against entry proteins have been developed empirically using an antigen, rather than by designing an immunogen on the basis of the antibodies produced.

The important advantages of human antibodies as therapeutics are low or negligible toxicity combined with high potency and a long half-life. However, drawbacks include the generation of neutralization-resistant virus mutants, limited access of the large antibody molecules to the site of virus replication, lack of oral formulations and the high cost of production and storage.

Virus entry physiology and retargeting

Viruses are usually associated with disease. However, some viruses can be beneficial. Human endogenous retrovirus W (HERV-W) might be essential in human physiology — for the formation of the placenta. The HERV-W Env, known as syncytin, is fusogenic and has a role in human trophoblast cell fusion and differentiation⁹⁸. Retroviral particles have been observed in the placenta, along with fused placental cells, which are morphologically reminiscent of virally induced syncytia. These studies led to the proposal that an ancient retroviral infection might have been a pivotal event in mammalian evolution⁹⁹.

Viruses have long been used to transfer genes into cells. During the last decade, another important application has been the viral delivery of genes and drugs to treat cancer. A major challenge has been to develop virus entry proteins to deliver molecules to specific cells with high efficiency. To achieve this goal it is often desirable to engineer viruses that do not infect cells expressing the native receptor, but instead target a cell of choice. Engineering of entry proteins in this way is known as transductional retargeting¹⁰⁰.

A conceptually simple approach to transductional retargeting is to incorporate the protein that determines cell tropism into the infecting virion of choice — known as virus 'pseudotyping'. This has been used in both retroviruses and adenoviruses, and does not require prior knowledge of specific virus–receptor interactions. In a related approach, viral entry proteins are used to produce drug and gene delivery vehicles, for example, the F protein of Sendai virus has been incorporated into liposomes to form virosomes¹⁰¹ and the L protein of hepatitis B has been incorporated into yeast-derived lipid vesicles¹⁰². Retargeting of retroviruses, adenoviruses and AAVs has been achieved by conjugation of entry proteins with molecular adaptors, such as bi-specific antibodies that have particular receptor-binding properties. Modification of the entry proteins so that the normal receptor-binding property is abolished, or a ligand for alternative receptor binding is incorporated has also been successful at redirecting adenovirus tropism in cell culture, but is unlikely to work for the entry of viruses that require receptor-induced conformational changes, such as retroviruses, unless detailed molecular mechanisms of those conformational changes are better understood. A related approach is based on screening libraries of chimaeric Envs from different strains of MLV¹⁰³, or randomized peptides inserted at tolerant sites in viral proteins, such as VP3 of AAV¹⁰⁴. This approach seems promising for the selection of specific retargeting vectors. Understanding the structure of AAV¹⁰⁵ and other viruses could help to further improve the specificity and efficiency of retargeting. Retargeting viruses with complex entry mechanisms that involve several proteins, such as those of herpes viruses and poxviruses, remains challenging.

Challenges and perspectives

Elucidation of the molecular mechanisms and the dynamics of the conformational changes driving virus entry remains a significant challenge. It requires the development of new approaches to study the rapid conformational changes of a small number of membrane-interacting protein molecules that are surrounded by many more non-interacting molecules. A more realistic goal is the determination of the structures of proteins that mediate the entry of all human viruses and the identification of the cognate cellular receptors. If research continues at the present pace, this goal could be accomplished within the next decade. Identification of all the cellular receptors for human viruses would be an important contribution to our understanding of virus tropism and pathogenesis. The various, and in many cases unexpected, ways that entry proteins can affect pathogenesis could offer new opportunities for intervention. Rational structure/mechanism-based design of entry inhibitors and vaccine immunogens that are capable of eliciting potent, broadly neutralizing antibodies of known epitopes is expected to contribute towards the development of clinically useful therapeutics and vaccines. The development of panels of human monoclonal antibodies against every entry-related protein of all pathogenic human viruses could accelerate our understanding of entry mechanisms and help to fight viral diseases. Recent progress in virus retargeting also raises hopes for the possibility of designing entry machines that can deliver genes and other molecules to any cell of choice.