Structural Studies on the Shapeshifting Murine Norovirus

Abstract

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.

Keywords: antibodies; bile; neutralization; norovirus.

Figure 1

Overall architecture of the caliciviruses. (A) This figure shows the entire capsid of murine norovirus (MNV) observed in the cryo-EM structures in the presence of bile [11] and at low pH [20]. The subunits A, B, and C are shown in blue, green, and red, respectively. The P domain dimers are composed of A and B subunits around the 5-fold axes and of C dimers at the 2-fold axes. Also highlighted are the A’-B’ (cyan) and E’-F’ (tan) loops discussed in the text. (B) Shown here is one copy of the capsid protein colored from blue to red as the chain extends from the amino to carboxyl termini. Note that this represents the contracted form of the capsid seen at low pH [20] or in the presence of bile [11].

Figure 2

Cross sections of MNV-1 at pH 7.5 in the absence and presence of GCDCA [11]. Shown here are the central sections of the cryo-EM image reconstructions of MNV-1 in the absence (A) and presence (B) of GCDCA. The density surfaces are colored according to the distance from the center of the particle. Note that the P domain in the apo structure is more diffuse, suggesting mobility, and is >15 Å above the shell surface compared to the structure in the presence of GCDCA. The contracted structure observed in the presence of GCDCA was essentially identical to MNV-1 at pH 5.0 [20].

Figure 3

High-resolution structures of MNV-1 P domain complexed with a neutralizing antibody (A6.2) and the receptor CD300lf. (A) The ~3 Å cryo-EM structure of the isolated P domain complexed with neutralizing Fab A6.2 [37]. The two P domains are colored tan and light green and the antibody heavy and light chains are colored orange and light blue, respectively. The loops at the tip of the P domain, A’B’, C’D’, E’F’, and G’H’, are colored mauve, red, black, and blue, respectively. Note that the P domain is in the open conformation and that the CDR3 loop of the antibody reaches down between the A’B’ and E’F’ loops. (B) The crystal structure of the P domain complexed with GCDCA and the receptor CD300lf [23]. The color scheme of the P domain is the same as in (A) and CD300lf is shown in yellow. (C) Stereo figure of the Fab A6.2 bound to the open conformation of the P domain. The transparent image is the structure of the P domain in the closed conformation. The yellow arrow highlights the structural clashes between Fab A6.2 and the closed P domain structure. (D) Stereo figure of the P domain/CD300lf complex. With the transparent ribbon diagram of the P domain from the Fab complex.

Figure 4

Environmentally driven changes in the G’H’ loop may control the P domain conformation. Shown here are ribbon diagrams of the tip of the P domain with the same color scheme as the previous figures. (A) There are three acidic groups on the G’H’ loop (D440, D443, and E447). At a pH of 7.5, these are expected to be deprotonated and therefore charged and repulsive. Indeed, under these conditions, the more vertical conformation of the loop allows the acidic sidechains to be well separated [31,37]. In this position, there is now room for the C’D’ loop to be in the downward conformation and thus allowing the A’B’/E’F’ loops to be in the open conformation. (B) At acidic conditions [20] or at neutral pH but in the presence of metals [40], these acidic groups are allowed to move into proximity and cause the G’H’ loop to distort, filling the space normally occupied by the C’D’ loop. This starts a chain reaction that leads to the closed conformation of the P domain.

Figure 5

Rotation of the A/B P domains and resulting contraction onto the shell. (Panel A) shows a stereo ribbon diagram of the unrotated A/B dimer (open conformation) modeled onto the A/B dimer in the contracted, closed conformation observed in the low pH and bile complex structures. The open conformation (unrotated) is shown as a transparent image and the closed conformation in solid colors. Note that the P1 domains of the A subunit (light green) were used for the alignment process and therefore match well. However, this places the P1 domain of the B subunit in the open conformation too close to the shell and, hence, why the P domain in the open conformation (unrotated) cannot rest on the shell. (Panel B) shows a schematic representation of the structures shown in (A). Before the A/B subunit rotation, the surface of the P domain base is not complementary to the shell surface and the P domain is not able to rest upon the shell (red X). This changes upon rotation of the A/B subunits, where the base of the P domain dimer changes and forms a complementary surface to the top of the shell, thus allowing the P domain to rest upon the shell.

Figure 6

Structural details of the transition from the extended to the contracted MNV-1 states. (Panel A) shows the structural changes of the whole P domain transitioning from the extended to contracted structure. (Panel B) shows a stereo view of the concomitant conformational changes within the P domain. For clarity, the two panels are presented at different viewing angles. There are likely multiple structural equilibriums at work, and therefore, this figure represents one possible series of events. Step 1 is where three different environmental stimuli (bile, low pH, metals), alone or in concert, start the cascade of conformational changes. In step 2, the C’D’ loop moves up away from the virion surface. This process likely occurs in conjunction with step 3 where the G’H’ loop becomes distorted since they switch locations during the conformational change. When the C’D’ loop is in the up position, this leads to step 4 where the A’B’ and E’F’ loops are pushed together to the closed conformation. From our various structures, it seems likely that the movement of the C’D’ and G’H’ loops then lead to step 5 where the two subunits rotate about each other. This rotation then causes the entire P domain to rotate ~90° (step 6) and contraction of the P domain onto the shell surface (step 7). (B) Shown here is a stereo figure magnifying the conformational changes in the loops. The opaque ribbon diagram represents the open conformation, and the transparent figure is the closed conformation.

Figure 7

Schematic of the various states of MNV as it travels through the alimentary canal. Where available, the numbers cited are for the murine where the parenthetical values are for humans for comparison. There are three signals that cause the contraction of the P domain onto the shell and the transition of the P domain loops to the closed conformation: low pH, binding of bile salts, and the binding of metals. These conditions are found throughout the alimentary canal. In the stomach, the low pH not only causes contraction but also solubilizes ingested metal salts. In the duodenum, the pH increases to 4.8 [44], but large amounts of bile salts are deposited via the gall bladder. Here the concentration comes from measurements on humans but the flux of bile in mice is significantly greater [47]. Throughout the small intestine, the pH remains acidic, and the metal and bile concentrations remain high. In the ileum, most of the bile salts and metals are absorbed. Finally, in the colon, there are still significant metal concentrations in the ascending colon and the pH remains significantly acidic. Interestingly, the pH of the feces is also rather acidic. Therefore, the conditions throughout the alimentary canal favor the contracted, closed conformation of MNV that blocks antibody binding while enhancing receptor binding. This reverses once the infection spreads outside the intestine where the neutral pH, low metal, and low bile concentrations favor the extended conformation with the open conformation in the P domain that favors antibody over receptor binding. Not drawn to scale.