The reversible activation of norovirus by metal ions.

Murine norovirus (MNV) undergoes extremely large conformational changes in response to the environment. The T = 3 icosahedral capsid is composed of 180 copies of ~58-kDa VP1 comprised of N-terminus (N), shell (S), and C-terminal protruding (P) domains. At neutral pH, the P domains are loosely tethered to the shell and float ~15 Å above the surface. At low pH or in the presence of bile salts, the P domain drops onto the shell and this movement is accompanied by conformational changes within the P domain that enhance receptor interactions while blocking antibody binding. While previous crystallographic studies identified metal binding sites in the isolated P domain, the ~2.7-Å cryo-electron microscopy structures of MNV in the presence of Mg2+ or Ca2+ presented here show that metal ions can recapitulate the contraction observed at low pH or in the presence of bile. Further, we show that these conformational changes are reversed by dialysis against EDTA. As observed in the P domain crystal structures, metal ions bind to and contract the G'H' loop. This movement is correlated with the lifting of the C'D' loop and rotation of the P domain dimers about each other, exposing the bile salt binding pocket. Isothermal titration calorimetry experiments presented here demonstrate that the activation signals (bile salts, low pH, and metal ions) act in a synergistic manner that, individually, all result in the same activated structure. We present a model whereby these reversible conformational changes represent a uniquely dynamic and tissue-specific structural adaptation to the in vivo environment.IMPORTANCEThe highly mobile protruding domains on the calicivirus capsids are recognized by cell receptor(s) and antibodies. At neutral pH, they float ~15 Å above the shell but at low pH or in the presence of bile salts, they contract onto the surface. Concomitantly, changes within the P domain block antibody binding while enhancing receptor binding. While we previously demonstrated that metals also block antibody binding, it was unknown whether they might also cause similar conformational changes in the virion. Here, we present the near atomic cryo-electron microscopy structures of infectious murine norovirus (MNV) in the presence of calcium or magnesium ions. The metal ions reversibly induce the same P domain contraction as low pH and bile salts and act in a synergistic manner with the other stimuli. We propose that, unlike most other viruses, MNV facilely changes conformations as a unique means to escape immune surveillance as it moves through various tissues.

Environmentally-triggered contraction of the norovirus virion determines diarrheagenic potential.

Noroviruses are the leading cause of severe childhood diarrhea and foodborne disease worldwide. While they are a major cause of disease in all age groups, infections in the very young can be quite severe with annual estimates of 50,000-200,000 fatalities in children under 5 years old. In spite of the remarkable disease burden associated with norovirus infections in people, very little is known about the pathogenic mechanisms underlying norovirus diarrhea, principally because of the lack of tractable small animal models. We recently demonstrated that wild-type neonatal mice are susceptible to murine norovirus (MNV)-induced acute self-resolving diarrhea in a time course mirroring human norovirus disease. Using this robust pathogenesis model system, we demonstrate that virulence is regulated by the responsiveness of the viral capsid to environmental cues that trigger contraction of the VP1 protruding (P) domain onto the particle shell, thus enhancing receptor binding and infectivity. The capacity of a given MNV strain to undergo this contraction positively correlates with infection of cells expressing low abundance of the virus receptor CD300lf, supporting a model whereby virion contraction triggers infection of CD300lflo cell types that are responsible for diarrhea induction. These findings directly link environmentally-influenced biophysical features with norovirus disease severity.

Structural Studies on the Shapeshifting Murine Norovirus.

Noroviruses are responsible for almost a fifth of all cases of gastroenteritis worldwide. The calicivirus capsid is composed of 180 copies of VP1 with a molecular weight of ~58 kDa. This coat protein is divided into the N-terminus (N), the shell (S) and C-terminal protruding (P) domains. The S domain forms a shell around the viral RNA genome, while the P domains dimerize to form protrusions on the capsid surface. The P domain is subdivided into P1 and P2 subdomains, with the latter containing the binding sites for cellular receptors and neutralizing antibodies. Reviewed here are studies on murine norovirus (MNV) showing that the capsid responds to several physiologically relevant cues; bile, pH, Mg2+, and Ca2+. In the initial site of infection, the intestinal tract, high bile and metal concentrations and low pH cause two significant conformational changes: (1) the P domain contracts onto the shell domain and (2) several conformational changes within the P domain lead to enhanced receptor binding while blocking antibody neutralization. In contrast, the pH is neutral, and the concentrations of bile and metals are low in the serum. Under these conditions, the loops at the tip of the P domain are in the open conformation with the P domain floating on a linker or tether above the shell. This conformational state favors antibody binding but reduces interactions with the receptor. In this way, MNV uses metabolites and environmental cues in the intestine to optimize cellular attachment and escape antibody binding but presents a wholly different structure to the immune system in the serum. To our knowledge, this is the first example of a virus shapeshifting in this manner to escape the immune response.

Multiple Signals in the Gut Contract the Mouse Norovirus Capsid To Block Antibody Binding While Enhancing Receptor Affinity.

Human norovirus is the leading cause of gastroenteritis worldwide, with no approved vaccine or antiviral treatment to mitigate infection. These plus-strand RNA viruses have T = 3 icosahedral protein capsids with 90 pronounced protruding (P) domain dimers, to which antibodies and cellular receptors bind. We previously demonstrated that bile binding to the capsid of mouse norovirus (MNV) causes several major conformational changes; the entire P domain rotates by ∼90° and contracts onto the shell, the P domain dimers rotate about each other, and the structural equilibrium of the epitopes at the top of the P domain shifts toward the closed conformation, which favors receptor binding while blocking antibody binding. Here, we demonstrate that MNV undergoes reversible conformational changes at pH 5.0 that are nearly identical to those observed when bile binds. Notably, at low pH or when metals bind, a cluster of acidic resides in the G'-H' loop interact and distort the G'-H' loop, and this may drive C'-D' loop movement toward the closed conformation. Enzyme-linked immunosorbent assays with infectious virus particles at low pH or in the presence of metals demonstrated that all tested antibodies do not bind to this contracted form, akin to what was observed with the MNV-bile complex. Therefore, low pH, cationic metals, and bile salts are physiological triggers in the gut for P domain contraction and structural rearrangement, which synergistically prime the virus for receptor binding while blocking antibody binding. IMPORTANCE The protruding domains on the calicivirus capsids are recognized by cell receptors and antibodies. We demonstrated that MNV P domains are highly mobile, and bile causes contraction onto the shell surface while allosterically blocking antibody binding. We present the near-atomic cryo-electron microscopy structures of infectious MNV at pH 5.0 and pH 7.5. Surprisingly, low pH is sufficient to cause the same conformational changes as when bile binds. A cluster of acidic residues on the G'-H' loop were most likely involved in the pH effects. These residues also bound divalent cations and had the same conformation as observed here at pH 5. Binding assays demonstrated that low pH and metals block antibody binding, and thus the G'-H' loop might be driving the conformational changes. Therefore, low pH, cationic metals, and bile salts in the gut synergistically prime the virus for receptor binding while blocking antibody binding.

A Norovirus Uses Bile Salts To Escape Antibody Recognition While Enhancing Receptor Binding.

Noroviruses, members of the Caliciviridae family, are the major cause of epidemic gastroenteritis in humans, causing ∼20 million cases annually. These plus-strand RNA viruses have T=3 icosahedral protein capsids with 90 pronounced protruding (P) domain dimers to which antibodies and cellular receptors bind. In the case of mouse norovirus (MNV), bile salts have been shown to enhance receptor (CD300lf) binding to the P domain. We demonstrated previously that the P domains of several genotypes are markedly flexible and "float" over the shell, but the role of this flexibility was unclear. Recently, we demonstrated that bile causes a 90° rotation and collapse of the P domain onto the shell surface. Since bile binds distally to the P-shell interface, it was not at all clear how it could cause such dramatic changes. Here, we present the near-atomic resolution cryo-electron microscopy (cryo-EM) structure of the MNV protruding domain complexed with a neutralizing Fab. On the basis of previous results, we show here that bile salts cause allosteric conformational changes in the P domain that block antibody recognition of the top of the P domain. In addition, bile causes a major rearrangement of the P domain dimers that is likely responsible for the bile-induced collapse of the P domain onto the shell. In the contracted shell conformation, antibodies to the P1 and shell domains are not expected to bind. Therefore, at the site of infection in the gut, the host's own bile allows the virus to escape antibody-mediated neutralization while enhancing cell attachment. IMPORTANCE The major feature of calicivirus capsids is the 90 protruding domains (P domains) that are the site of cell receptor attachment and antibody epitopes. We demonstrated previously that these P domains are highly mobile and that bile causes these "floating" P domains in mouse norovirus (MNV) to contract onto the shell surface. Here, we present the near-atomic cryo-EM structure of the isolated MNV P domain complexed with a neutralizing Fab fragment. Our data show that bile causes two sets of changes. First, bile causes allosteric conformational changes in the epitopes at the top of the P domain that block antibody binding. Second, bile causes the P domain dimer subunits to rotate relative to each other, causing a contraction of the P domain that buries epitopes at the base of the P and shell domains. Taken together, the results show that MNV uses the host's own metabolites to enhance cell receptor binding while simultaneously blocking antibody recognition.

The Dynamic Life of Virus Capsids.

Protein-shelled viruses have been thought as "tin cans" that merely carry the genomic cargo from cell to cell. However, through the years, it has become clear that viruses such as rhinoviruses and caliciviruses are active and dynamic structures waiting for the right environmental cues to deliver their genomic payload to the host cell. In the case of human rhinoviruses, the capsid has empty cavities that decrease the energy required to cause conformational changes, resulting in the capsids "breathing", waiting for the moment when the receptor binds for it to release its genome. Most strikingly, the buried N-termini of VP1 and VP4 are transiently exposed during this process. A more recent example of a "living" protein capsid is mouse norovirus (MNV). This family of viruses have a large protruding (P) domain that is loosely attached to the shell via a single-polypeptide tether. Small molecules found in the gut, such as bile salts, cause the P domains to rotate and collapse onto the shell surface. Concomitantly, bile alters the conformation of the P domain itself from one that binds antibodies to one that recognizes receptors. In this way, MNV appears to use capsid flexibility to present one face to the immune system and a completely different one to attack the host tissue. Therefore, it appears that even protein-shelled viruses have developed an impressive array of tricks to dodge our immune system and efficiently attack the host.

Bile Salts Alter the Mouse Norovirus Capsid Conformation: Possible Implications for Cell Attachment and Immune Evasion.

Caliciviruses are single-stranded RNA viruses with 180 copies of capsid protein comprising the T=3 icosahedral capsids. The main capsid feature is a pronounced protruding (P) domain dimer formed by adjacent subunits on the icosahedral surface while the shell domain forms a tight icosahedral sphere around the genome. While the P domain in the crystal structure of human Norwalk virus (genotype I.1) was tightly associated with the shell surface, the cryo-electron microscopy (cryo-EM) structures of several members of the Caliciviridae family (mouse norovirus [MNV], rabbit hemorrhagic disease virus, and human norovirus genotype II.10) revealed a "floating" P domain that hovers above the shell by nearly 10 to 15 Å in physiological buffers. Since this unusual feature is shared among, and unique to, the Caliciviridae, it suggests an important biological role. Recently, we demonstrated that bile salts enhance cell attachment to the target cell and increase the intrinsic affinity between the P domain and receptor. Presented here are the cryo-EM structures of MNV-1 in the presence of bile salts (∼3 Å) and the receptor CD300lf (∼8 Å). Surprisingly, bile salts cause the rotation and contraction of the P domain onto the shell surface. This both stabilizes the P domain and appears to allow for a higher degree of saturation of receptor onto the virus. Together, these results suggest that, as the virus moves into the gut and the associated high concentrations of bile, the entire capsid face undergoes a conformational change to optimize receptor avidity while the P domain itself undergoes smaller conformational changes to improve receptor affinity.IMPORTANCE Mouse norovirus and several other members of the Caliciviridae have been shown to have a highly unusual structure with the receptor binding protruding (P) domain only loosely tethered to the main capsid shell. Recent studies demonstrated that bile salts enhance the intrinsic P domain/receptor affinity and is necessary for cell attachment. Presented here are the high-resolution cryo-EM structures of apo MNV, MNV/bile salt, and MNV/bile salt/receptor. Bile salts cause a 90° rotation and collapse of the P domain onto the shell surface that may increase the number of available receptor binding sites. Therefore, bile salts appear to be having several effects on MNV. Bile salts shift the structural equilibrium of the P domain toward a form that binds the receptor and away from one that binds antibody. They may also cause the entire P domain to optimize receptor binding while burying a number of potential epitopes.

The Dynamic Capsid Structures of the Noroviruses.

Noroviruses are responsible for almost a fifth of all cases of gastroenteritis worldwide. New strains evolve every 2⁻4 years by escaping herd immunity and cause worldwide epidemics. In the US alone, noroviruses are responsible for ~20 million cases and more than 70,000 hospitalizations of infected children, annually. Efforts towards a vaccine have been hindered by a lack of detailed structural information about antibody binding and the mechanisms of antibody escape. Caliciviruses have 180 copies of the major capsid protein (VP1; ~58 kDa), that is divided into the N-terminus (N), the shell (S) and C-terminal protruding (P) domains. The S domain forms a shell around the viral RNA genome, while the P domains dimerize to form protrusions on the capsid surface. The P domain is subdivided into P1 and P2 subdomains, with the latter containing the binding sites for cellular receptors and neutralizing antibodies. There is increasing evidence that these viruses are extremely dynamic and this flexibility is critical for viral replication. There are at least two modes of flexibility; the entire P domain relative to the shell and within the P domain itself. Here, the details and possible roles for this remarkable flexibility will be reviewed.

Glutamate Dehydrogenase, a Complex Enzyme at a Crucial Metabolic Branch Point.

In-vitro, glutamate dehydrogenase (GDH) catalyzes the reversible oxidative deamination of glutamate to α-ketoglutarate (α-KG). GDH is found in all organisms, but in animals is allosterically regulated by a wide array of metabolites. For many years, it was not at all clear why animals required such complex control. Further, in both standard textbooks and some research publications, there has been some controversy as to the directionality of the reaction. Here we review recent work demonstrating that GDH operates mainly in the catabolic direction in-vivo and that the finely tuned network of allosteric regulators allows GDH to meet the varied needs in a wide range of tissues in animals. Finally, we review the progress in using pharmacological agents to activate or inhibit GDH that could impact a wide range of pathologies from insulin disorders to tumor growth.

Norovirus Escape from Broadly Neutralizing Antibodies Is Limited to Allostery-Like Mechanisms.

Ideal antiviral vaccines elicit antibodies (Abs) with broad strain recognition that bind to regions that are difficult to mutate for escape. Using 10 murine norovirus (MNV) strains and 5 human norovirus (HuNoV) virus-like particles (VLPs), we identified monoclonal antibody (MAb) 2D3, which broadly neutralized all MNV strains tested. Importantly, escape mutants corresponding to this antibody were very slow to develop and were distal to those raised against our previously studied antibody, A6.2. To understand the atomic details of 2D3 neutralization, we determined the cryo-electron microscopy (cryo-EM) structure of the 2D3/MNV1 complex. Interestingly, 2D3 binds to the top of the P domain, very close to where A6.2 binds, but the only escape mutations identified to date fall well outside the contact regions of both 2D3 and A6.2. To determine how mutations in distal residues could block antibody binding, we used molecular dynamics flexible fitting simulations of the atomic structures placed into the density map to examine the 2D3/MNV1 complex and these mutations. Our findings suggest that the escape mutant, V339I, may stabilize a salt bridge network at the P-domain dimer interface that, in an allostery-like manner, affects the conformational relaxation of the P domain and the efficiency of binding. They further highlight the unusual antigenic surface bound by MAb 2D3, one which elicits cross-reactive antibodies but which the virus is unable to alter to escape neutralization. These results may be leveraged to generate norovirus (NoV) vaccines containing broadly neutralizing antibodies. IMPORTANCE The simplest and most common way for viruses to escape antibody neutralization is by mutating residues that are essential for antibody binding. Escape mutations are strongly selected for by their effect on viral fitness, which is most often related to issues of protein folding, particle assembly, and capsid function. The studies presented here demonstrated that a broadly neutralizing antibody to mouse norovirus binds to an exposed surface but that the only escape mutants that arose were distal to the antibody binding surface. To understand this finding, we performed an in silico analysis that suggested that those escape mutations blocked antibody binding by affecting structural plasticity. This kind of antigenic region-one that gives rise to broadly neutralizing antibodies but that the virus finds difficult to escape from-is therefore ideal for vaccine development.

Identification of a Novel Activator of Mammalian Glutamate Dehydrogenase.

Glutamate dehydrogenase (GDH) catalyzes the oxidative deamination of l-glutamate and in animals is highly regulated. GDH in hyperinsulinism/hyperammonemia syndrome patients lacks GTP inhibition, resulting in hypersecretion of insulin upon protein consumption. This suggests insulin secretion could be stimulated with GDH activators. A high-throughput screen yielded one potent activator, N1-[4-(2-aminopyrimidin-4-yl)phenyl]-3-(trifluoromethyl)benzene-1-sulfonamide (75-E10). 75-E10 is ∼1000-fold more efficacious than the synthetic activator, BCH, and is at least as effective as ADP. 75-E10 compound is highly effective at alleviating GTP inhibition and may be binding to the ADP site. Unlike ADP, 75-E10 is activated over a broad range of conditions.