Structural basis of viral invasion: lessons from paramyxovirus F

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The structures of glycoproteins that mediate enveloped virus entry into cells have revealed dramatic structural changes that accompany membrane fusion and provided mechanistic insights into this process. The group of class I viral fusion proteins includes the influenza hemagglutinin, paramyxovirus F, HIV env, and other mechanistically related fusogens, but these proteins are unrelated in sequence and exhibit clearly distinct structural features. Recently determined crystal structures of the paramyxovirus F protein in two conformations, representing pre-fusion and post-fusion states, reveal a novel protein architecture that undergoes large-scale, irreversible refolding during membrane fusion, extending our understanding of this diverse group of membrane fusion machines.

Introduction

Viruses have evolved a variety of architectures that are designed to protect and transmit their nucleic acid genomes, insuring their survival despite fundamental dependencies on their hosts for replication and spread [1]. For all viruses, recognition of and subsequent penetration into host cells are key steps in the viral life cycle, and many adaptations in structures, mechanisms, and entry pathways have evolved to overcome the fundamental barriers in this process, such as crossing the topological barrier presented by the cellular lipid bilayer [1, 2]. Many viruses themselves, the so-called enveloped viruses, are surrounded by a lipid bilayer that is acquired during prior budding from infected cells. In contrast to those viruses coated by a protein shell, enveloped viruses are presented with the problem of uniting two lipid bilayers during entry into cells, one membrane from the target cell and one from the virus. As more has been learned about the structural aspects of enveloped virus entry mechanisms, many common mechanistic features have been identified that appear to hold for proteins of widely different structures.

Pioneering studies of the influenza virus hemagglutinin (HA) carried out by the Wiley and Skehel laboratories, beginning in the 1970s and extending for over two decades, provided the first insights into the membrane fusion machinery of enveloped viruses [3, 4, 5, 6, 7]. The activation of membrane fusion by HA is triggered by low pH, after virus trafficking into endosomal compartments of the cell [2]. Structures of the HA in its pre-fusion and post-fusion conformations revealed a dramatic refolding of the C-terminal portion of the protein into a hairpin-like conformation that suggested how HA might lower the barriers to lipid bilayer fusion [4, 5, 6, 7]. At two ends of the ‘hairpin’ are two membrane-interacting segments within the fusion protein sequence—one of these being a fusion peptide initially sequestered in the core of the trimeric HA and the other being the C-terminal transmembrane anchor regions. Importantly, the observation of this hairpin and associated refolding changes within previously identified heptad repeat segments of HA led to the proposal of a ‘spring-loaded’ mechanism for HA-mediated membrane fusion, in which the hydrophobic fusion peptide would be projected toward the target cell bilayer by a coil–helix transition in HA, followed by a folding back of the C-terminal end of the protein, resulting in the close juxtaposition of the fusion peptides and transmembrane domains of the HA trimer [4, 5, 8]. Mutations in HA that affect the optimal pH of this refolding are located throughout the structure, suggesting that HA might function as a ‘global’ pH sensor [4], though it is possible that ionization of specific residues may play an important initiating role in the refolding process.

These structural insights into the HA fusion mechanism also suggested that aspects of this process might be extendable to other viral fusion proteins. In particular, it was recognized that the fusion proteins from unrelated viruses, such as Ebola, HIV, paramyxoviruses, and others, contained identifiable sequence motifs that appeared to be related to those in HA. These motifs include an internal, often furin-like, cleavage site near or adjacent to a stretch of 20–25 hydrophobic amino acids (a fusion peptide), followed by one or two heptad repeat regions (Figure 1). Subsequent structural, biochemical, and functional studies of the heptad repeat regions from these viral fusion proteins (Figure 2) demonstrated their assembly into trimeric hairpin-like structures similar to that of the low-pH induced HA, indicative of commonalities in the mechanism of membrane fusion [9, 10, 11, 12, 13, 14, 15, 16]. In all structures, three hydrophobic fusion peptides are located near the N-termini of helices forming a centralized coiled coil, while an antiparallel structure, often helical and deriving from a second heptad repeat in the sequence, positions the transmembrane anchors at the same end of a rod-like structure (Figure 2).

These similarities have been recognized by a nomenclature that places viral fusion proteins with these sequence and structural features into the so-called class I viral fusion protein group [17, 18, 19]. It is generally thought that the class I viral fusion proteins fold to a pre-fusion, metastable conformation, which is then activated to undergo a large conformational rearrangement to a lower energy state, thereby providing the energy needed to accomplish membrane fusion. The process is irreversible and independent of the use of ATP. A class II group, which includes flavivirus and alphavirus membrane fusion proteins, has a different, primarily beta-sheet architecture, and different mechanistic details involving changes in oligomerization state and domain repositioning that are distinct from the refolding transitions observed in HA and paramyxovirus F proteins, and that are presumed to occur in other class I viral fusion proteins. Nonetheless, class II fusion proteins are also thought to assemble into a hairpin-like conformation during membrane fusion [17, 20]. Recent structural studies of the VSV G protein in two conformations, and its surprising structural homology to the herpesvirus gB protein, suggest that these two proteins may define a third class of fusion protein [21, 22, 23].

Despite this classification scheme, it has been clear that class I fusion proteins from different viruses do not exhibit any significant sequence homologies, despite the presence of some similar architectural motifs, and that these class I proteins probably represent distinct structural subfamilies—much more different than the alphavirus and flavivirus fusion proteins. For example, the distribution of heptad repeats, cleavage sites, and fusion peptides in HA and paramyxovirus F are very different: In F, over 250 amino acids separate the two heptad repeats that assemble into a six-helix bundle (6HB), whereas in HA very few residues separate the heptad repeat regions that form the base of the hairpin structure (Figure 1, Figure 2). Despite overall analogous hairpin arrangements, the core region structures from different class I proteins are also quite different in many of their structural details (Figure 2). Finally, the mechanisms for activation of different viral fusion proteins are distinct. Some, like HA, are activated by low pH, others, like HIV env and paramyxovirus F, are activated by receptor-binding events, either by direct interactions (HIV env) or indirectly through a viral attachment protein (paramyxovirus F). Ebola GP-mediated fusion requires its trafficking through the endosomal pathway, but this probably reflects a need for further GP processing by lysosomal enzymes, such as cathepsins, to trigger virus entry, rather than simply low pH itself [24]. Thus, many questions regarding the structures, folding, and activation of class I viral fusion proteins remain to be addressed. Recent structural results on the paramyxovirus F protein have provided new insights into just how different these class I viral fusion proteins may be and suggest what structural parallels may apply across this class of membrane fusogens.

The paramyxoviruses are enveloped, negative-strand RNA viruses that cause both respiratory and systemic disease. The paramyxovirus family includes, among others, mumps virus, measles virus, Sendai virus, Newcastle disease virus (NDV), human respiratory syncytial virus (hRSV), parainfluenza virus 5 (PIV5; formerly known as SV5), human parainfluenza viruses 1–4 (hPIV) [25, 26, 27], and the deadly Nipah and Hendra viruses [28, 29]. Members of this viral family are among the most significant human and animal pathogens, being directly responsible for many human deaths and hospitalizations each year, and for infections of farm animals that have major economic consequences. For example, measles virus is still a major cause of death in children in developing countries, and hRSV is the primary cause of infant hospitalization for respiratory infection in the US, accounting for ∼70% of viral bronchiolitis cases [30].

Two viral glycoproteins are involved in the infection of cells—an attachment protein, called HN, H, or G, depending on the virus, and the fusion (F) protein. In all paramyxoviruses, the F protein catalyzes membrane fusion, after the attachment protein mediates binding of the virus to the cell surface [31, 32]. Although the F protein sequences can vary substantially between viruses, the majority of F cysteine residues involved in disulfide bonds are conserved. Given this and their similar biological activities, it is likely that representative F structures provide insight into the shared F function of membrane fusion, but there are likely to be important virus-specific sequence and structural differences. For many of the paramyxoviruses (NDV, hPIV1–4, PIV5, and others), the attachment protein is a hemagglutinin/neuraminidase (HN) protein, which binds to and can also cleave sialic acid structures. The morbilliviruses, such as measles virus, express a hemagglutinin (H) protein in place of HN, while the pneumoviruses (RSV) and henipaviruses (Nipah and Hendra) express a distinct attachment glycoprotein (G) [32, 33]. In contrast to HN, H from measles interacts with CD46 or CDw150/SLAM [34, 35], and RSV G has been shown to interact with heparin sulfate [32, 36]. In many of the paramyxoviruses, it is the attachment protein interaction with receptors that is thought to initiate conformational changes in F, thereby activating membrane fusion at the right time and right place. Thus, in contrast to influenza virus, paramyxoviruses carry out membrane fusion and entry at the cell surface and at neutral pH.

Section snippets

Crystal structures of paramyxovirus F proteins

Crystal structures of fragments of F proteins from different paramyxoviruses provided the first insights into the F structure and revealed a conserved six-helix bundle arrangement formed by two heptad repeat regions (HRA and HRB; Figure 1, Figure 2), whose assembly is tightly coupled to membrane fusion [11, 12]. Peptides spanning HRA and HRB regions are inhibitors of F-mediated membrane fusion and mechanistic studies identified two distinct F refolding intermediates during the process [31, 37].

Ethics and conflicts of interest

The authors declare that this manuscript represents their original work and that they have no conflicts of interest.

Acknowledgements

We thank past and present members of the Jardetzky and Lamb Laboratories. This research was supported partly by NIH research grants to TSJ and RAL. RAL is an investigator of the Howard Hughes Medical Institute.

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