Structure of the measles virus H glycoprotein sheds light on an efficient vaccine

Prevention by vaccination is the most efficient and economical way of fighting viral disease, as exemplified by the successful eradication of smallpox in the late 1980s. In the 1960s, many attenuated vaccines were developed, and such vaccines are among the most efficient for preventing human (such as smallpox and measles) and animal disease. Attenuated vaccines have been empirically obtained by growing viruses in various cell types, including cells from species that are not naturally infected. However, development of successful vaccines against many other enveloped viruses has found limited success (influenza) or still awaits convincing proof of concept (HIV). Both influenza and HIV escape neutralizing immunity by mutating the coding regions of their envelope glycoproteins to escape from recognition by neutralizing antibodies. The underlying molecular mechanism, i.e., why measles virus behaves monotypically from the point of view of the immune response, has now been elucidated with the crystal structure of the viral H glycoprotein (1).

The Immunosuppressive Measles Virus and Animal Morbilliviruses Use CD150 as a Receptor

Measles remains a leading cause of death for young children (345,000 deaths in 2005 among an estimated 30 million people infected; data available at www.who.int/mediacentre/factsheets/fs286/en). Measles is one of the most contagious respiratory diseases known. It is caused by an RNA virus from the Morbillivirus genus. This genus also houses several animal pathogens severely affecting cattle and/or wild animals: canine distemper virus (CDV), rinderpest virus (RPV), peste des petits ruminants virus (PPRV), cetacean morbillivirus, and phocine distemper virus. Morbilliviruses share many structural and biological properties. The diseases that they induce are characterized by a severe, but fortunately transient, cellular immunodepression that, in the case of measles, can be biologically as severe as what is observed in patients suffering from AIDS. This immunodepression is linked to the use of the CD150 (or SLAM) receptor, which is expressed only on activated cells of the immune system. The ability to mount a sterilizing lifelong immunity against measles despite this severe but transient immunodeficiency constitutes what is called the “immunologic paradox of measles” (2).

RNA Viruses Have a Very High Mutation Rate

The replicating enzymes of RNA viruses, such as measles, influenza, and HIV, make approximately one mistake per every 10,000 nucleotides (3). Measles virus stores its biological information in an ≈16,000 ribonucleotide-long

Despite the error-prone viral polymerase, the amino acid sequence of H is strongly conserved.

RNA genome, which means that, on average, every offspring RNA genome has one or two mutations and that, during the infection, a significant sequence variation in the pool of viruses is generated. In particular, mutations in the glycoproteins of the viral envelope could allow escape from a neutralizing antibody response, as is the case for influenza virus and HIV. So how can the measles virus vaccine still function after >40 years, whereas the influenza virus vaccine needs to be replaced almost every year, and why has it not been possible to produce an HIV vaccine? The answers lie in the peculiar structure of the measles H glycoprotein, against which neutralizing antibodies are produced.

Influenza and HIV Glycoproteins Accommodate Mutations

The influenza virus hemagglutinin (HA) glycoprotein, which binds to the viral receptor sialic acid, is richly covered with sugar chains. The receptor binding site at the top of the molecule is a shallow hollow (4). Most of the amino acids around this hollow and others on the surface of HA can mutate without significantly affecting receptor binding. However, HA has a rather rigid structure, and many mutations are not allowed because they would destabilize the molecule or lead to a nonfunctional molecule. The accumulation of mutations, selected and directed by the immune pressure in the host population, is such that the vaccine strains have to be frequently replaced to optimize protection. The HIV surface glycoprotein, against which neutralizing antibodies should be made, is GP120. This molecule is much more flexible than influenza virus HA. GP120 has a number of highly variable surface loops that do not seem to have a specific three-dimensional structure. Moreover, GP120 does not have a single structure but appears to have two or three structures. One is the native structure of the molecule and is not bound to its receptor (5). The second structure exists when GP120 is bound to its receptor, CD4 (6). A third structural form likely exists when GP120 is bound to its secondary receptor, leading to membrane fusion between the viral and host cell membranes. These structural changes are very large and are possible because the molecule is plastic. The combination of the sugar chains on the surface of GP120 together with its plastic structure and the variable loops that have no sequence constraint results in a molecule that is too flexible and variable for the production of neutralizing antibodies.

Crystal Structure of Measles Virus H Glycoprotein Reveals Constraint Receptor Binding Site

The work by Hashiguchi et al. (1) in a recent issue of PNAS provides the crystal structure of the measles virus hemagglutinin (H), which is the envelope glycoprotein responsible for the attachment to CD150. The surface of this glycoprotein also is covered by sugar chains, but, interestingly, a large surface area is free from sugar chains and hosts a rather extensive binding site for CD150. Mutations in this region are not allowed because they interfere with receptor binding. Therefore, the extreme sequence restriction of this site allows for very efficient production of neutralizing antibodies that by definition block binding of the virus to its receptor. For this reason, the original vaccine strain developed in the 1960s is still effective. Moreover, a variant genotype that escapes neutralization by a monoclonal antibody still uses CD150 as a receptor and is neutralized by sera from vaccinated or convalescent humans (7). Despite the error-prone viral polymerase, the amino acid sequence of H is strongly conserved, with 60% of the residues being identical or similar among 494 available sequences, meaning that any mutation changing the nature of these conserved residues forbids viability of the virus. From a detailed analysis of site-specific mutants (8), Hashigushi et al. predict several CD150-contacting residues E503, D505 (G505), D507, Y529 (D529), D530, T531, R533, E535, F552 (S552), Y553 and P554, which are highly conserved. Interestingly, the sequence of H is also highly conserved within the entire Morbillivirus genus, with 42% of the residues identical or similar, including the putative CD150-contacting amino acids. Because CDV and RPV use canine and bovine CD150, respectively, the cellular receptors for cetacean morbillivirus and phocine distemper virus are likely to be the CD150 counterparts of their natural hosts.

Due to only a few point mutations, H of the measles vaccine strain has acquired a binding site for a second cellular receptor, CD46 (or MCP), which is expressed on all human-nucleated cells. This binding site is located in another area not covered by sugar moieties. CD46 binds to H with lower affinity than CD150. Intriguingly, this difference is mirrored by the difference in the avidity of neutralizing antibodies directed to these two binding sites. Sera from infected humans and monkeys are prevalently directed against the CD150 binding site rather than against the CD46 binding site (9, 10), as if the two surfaces are inherently designed for high- and low-affinity contacts with another protein surface (9). Furthermore, the CD150 binding site, but not the CD46 binding site, is sensitive to chemical substitution by formalin, as shown by its ability to induce neutralizing antibodies in monkeys (10), possibly because of formalin modification of the primary amino group of the crucial R533 residue. This observation is informative from a historical point of view because tests with chemically fixed measles virus as a vaccine were conducted in infants in the 1960s but were immediately stopped because of the induction of exacerbated measles disease after secondary natural infection (11).

The Structure of H Paves the Way for Better Understanding Morbillivirus Entry and Receptor Retargeting

Compared with wild-type strains, the vaccine strains for CDV and RPV have also acquired the ability to use another cellular receptor (12, 13). Modeling the structure of their H onto that of measles may lead to the prediction of key residues responsible for binding to these alternate receptors. The same group (14) has provided evidence for the use of a third receptor by wild-type measles virus to infect human epithelial cells, and the present structure of H will hopefully boost the identification of this third receptor. Measles virus H is particular in another way because it is the only viral glycoprotein to be successfully engineered for retargeting to another cellular receptor with the aim of developing oncolytic vectors (15). Future structural studies of H in complex with natural and artificial receptors or docking modeling with the crystal structures that are available for CD46-related (16) and CD150-related (17) receptors should soon provide more information.

H is intimately associated with a companion molecule, the fusion protein F, which ensures measles virus entry into the cytoplasm of the host cell (18, 19). The attachment of H to the cellular receptor is thought to initiate conformational changes in F, thereby activating membrane fusion at the right time and place. The knowledge of the crystal structure of the head domain of H will be helpful in our understanding of this molecular cross-talk with the fusion protein. In particular, it is important to answer the question of how the configuration of H bound to various natural or artificial (retargeting) cellular receptors can give rise to a similar molecular cross-talk with the fusion protein. More answers will come from solving other structures, such as the stalk connecting the head of H to the transmembrane anchor and H in complex with the fusion protein.