Astrovirus Structure and Assembly
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Recent structural studies on the astrovirus virion and viral proteins have yielded exciting new insights into the molecular mechanisms of the astrovirus life cycle. The 25 Å-resolution cryo-electron microscopy (Cryo-EM) reconstructions of the astrovirus virion reveal a solid capsid shell studded with spikes. Proteolytic maturation of the virus particle results in capsid conformational changes, most prominently at the spikes. High-resolution crystal structures of the human and avian astrovirus capsid spike domains have shed light on potential host receptors and species specificity. Together, both the structural studies on the astrovirus virion and capsid spike domains have revealed similarities to hepatitis E virus, suggesting an evolutionary relationship. The only other structural information on astrovirus is from the high-resolution crystal structure of the protease that is involved in nonstructural polyprotein processing. Overall, these structural studies have led a better understanding of the astrovirus life cycle, including astrovirus assembly, virus release, maturation, receptor binding, antibody neutralization, and nonstructural polyprotein processing.
KeywordsDime Interface Trypsin Cleavage Projection Domain Capsid Shell Surface Spike
Structural studies on the astrovirus virion and viral proteins are important to understand the molecular details of the astrovirus life cycle. Recent structural studies of the astrovirus virion using cryo-electron microscopy have yielded important insights into the proteolytic maturation and corresponding conformational changes in the astrovirus virion. Additionally, recent high-resolution X-ray crystallographic studies on human and avian astrovirus capsid spikes have revealed insights into receptor binding, host cell entry, and species specificity. Together, the studies on the astrovirus virion and capsid spike have uncovered striking structural similarities to hepatitis E virus, suggesting that these viruses have a closer phylogenetic relationship than previously recognized. Finally, the high-resolution X-ray crystallographic structure of the astrovirus protease revealed a number of distinctive features that provide insight into its role in nonstructural polyprotein processing events. Overall, astrovirus structural studies provide a foundation for future mutagenesis studies to probe protein functions and roles in the astrovirus life cycle and pathogenesis. Furthermore, these structural studies will have a large impact on the development of antivirals and vaccines targeting astrovirus.
Cryo-EM Structure of Immature and Mature Human Astrovirus
Astroviruses are assembled from the structural protein VP90, an 87–90 kDa capsid precursor protein, which is encoded by the 3′-terminal one-third of the genome (ORF-2). This capsid protein has a highly conserved N-terminal domain and hypervariable C-terminal domain and must undergo a cascade of cleavages to become infectious. The final size and number of structural proteins that form the capsid vary depending on the serotype (reviewed by Krishna ). VP90 undergoes an initial caspase cleavage at the C-terminus to generate VP70 (a 70–79 kDa protein), which is required for particle release from cells . Viral infectivity is further dependent upon trypsin cleavage and the genesis, usually, of three structural proteins, VP34, VP27 (27–29 kDa), and VP25 (25–26 kDa) [4, 27, 33]. The current consensus is that the cleavage product VP34 contains the conserved, N-terminal domain, while VP27 and VP25 contain the variable domain, which mediate the attachment of virions to cell surface receptors and contain epitopes for neutralizing antibodies . VP25 is generated upon additional trimming at the N-terminus of VP27.
Astroviruses were initially named for the distinctive star-like appearance of the viral surface that was seen in ∼10% of the fecally shed viral particles evaluated by negative-stain electron microscopy (EM) [2, 11]. EM ultrastructural analysis of infectious HAstV generated in cell culture showed spherical particles with a surface studded with spikes and an external diameter of 41 nm . The characteristic star-like surface appearance was inducible after brief alkaline (pH 10) treatment of the particles, which made them less stable, and suggested that particles with star-like morphology may represent an intermediate step toward viral disassembly. More recently, the structures of immature (VP70) and mature virions have been calculated to moderate 25 Å-resolution by electron cryomicroscopy (cryo-EM) and image analysis . The majority of the variable domain (a.a. 425–646) has been crystallized  and is discussed in detail later in this chapter.
Structural Characteristics and Similarity to Hepatitis E Virus
The distal layer of density is composed of discrete domains and represents the spikes that have been observed directly by electron microscopy (Fig. 3.1, upper). These globular domains have very poorly defined connections to the underlying capsid, implying that they may be flexible or have thin linkers. The densities are ∼40 Å in diameter, although more elliptical than spherical, and extend to an outer radius of ∼215 Å making the overall diameter of the particles ∼430 Å. These densities are attributed the C-terminal, variable domain. Details of the astrovirus spike structure have been further elucidated from the crystal structure of the dimerized VP25 fragment .
Like HEV, the overall structural organization of HAstV polyprotein can be similarly described, with the astrovirus conserved domain corresponding to the arginine-rich and S-domain of HEV, and the variable domain corresponding to the P1-, P2-, and cleaved C-terminal domains of HEV. Sequence alignment of the two viruses has 21, 13, and 14% identity for the S-, P1-, and P2-domains, respectively . The major noticeable difference between the HAstV and HEV structures is that the astrovirus spikes extend to a greater radius than those of HEV, which has more compact spikes and an outer radius of ∼190 Å (Fig. 3.2c, arrow). The HEV atomic model docks exceedingly well into the HAstV cryo-EM density maps without any scaling (Fig. 3.2c). The subunit organization of astrovirus can be inferred by analogy, and Dong et al. used the HEV atomic model to build a corresponding model for HAstV . As with HEV, the spike stoichiometry and orientation suggests that they are dimers with contributions from subunits situated on different trimeric facets (Fig. 3.1, lower left). Using the nomenclature of HEV, AB dimers encircle the fivefold axes, while neighboring C subunits form dimers across the icosahedral twofold axes. In the case of HEV, and perhaps more so for HAstV, this arrangement leads to different interactions for the peripentonal dimers versus the dimers on the twofold axes. The spike bases for the twofold dimers are in much closer proximity than those around the fivefold, which are situated at nearly twice the distance across the local twofold groove. Extrapolating further from the HEV structure, it is likely that there is an underlying difference in the hinge angles between these dimers, which may reflect the presence or absence of RNA in close contact on the interior surface .
Partial Loss of Visible Spikes upon Trypsin Cleavage
While the capsid shells of both the immature and mature HAstV particles are essentially the same, there is a dramatic loss of spikes during trypsin cleavage (Fig. 3.1, upper). The reconstruction of the immature, uncleaved particles has 90 globular, or spike, densities. In the reconstruction of the mature particles, however, only the 30 spike densities located at the icosahedral twofold axes remain visible. Trypsin cleavage of the immature particles produces three products: we presume that VP34 forms the capsid shell and the two different C-terminal products, VP27 and VP25, form the surface spikes. Although a first assumption would be that the trypsin cleavage has removed two-thirds of the spikes from the particles, previously published SDS-PAGE gels show that the amount of VP34 present in infectious particles was comparable to that of VP27 and VP25 [4, 27, 33], indicating that most of the VP27 and VP25, although covalently independent, remain associated with the particle. Therefore, an alternative explanation is that the stoichiometry of the spikes may be conserved, and trypsin cleavage results in conformational flexibility so that the density is lost upon image averaging.
Proteolytic cleavage is a common mechanism for activation in both enveloped and nonenveloped viruses, including influenza virus [21, 24], coronavirus , rotavirus , reovirus , and alphaviruses . A recurrent theme is that cleavage triggers a conformational change in a surface spike or other cell-attachment domain. In the case of rotavirus, the spike is initially completely unstructured, and therefore not observed by image analysis, until trypsin cleavage, at which point the polypeptides form a stable structure [12, 34]. It is possible that for astroviruses, the reverse takes place, and that fairly stable densities visible before cleavage become completely disordered at some positions. When the virus moves into the stomach of its host and is exposed to protease, the two arrangements of the spikes formed by AB and CC capsid protein dimers are likely to present different cleavage sites, giving rise to the two different C-terminal cleavage products, VP27 and VP25. The peripentonal spikes have dimer contributions that must extend nearly twice the distance of those at the icosahedral twofold axes, which may uncover sites for the additional trimming that generate VP25, and VP27 would be comprise the remaining visible spikes. The spikes located on the icosahedral twofolds are likely to be VP27 homodimers, which have restricted movement just like the spikes in unprocessed capsid and thus can be visualized in a population-averaged reconstruction. There are several potential ways the conformational change could confer infectivity—by exposing the capsid surface, allowing some variable domains to be flexible, or exposure of binding sites.
Crystal Structure of the Human Astrovirus Capsid Spike
Among the four small, nonenveloped positive-sense RNA viruses (i.e., picornavirus, calicivirus, astrovirus, and hepevirus) that infect animals, astrovirus is the only one whose atomic structure is not yet fully resolved. Based on primary sequence analysis, it was hypothesized that VP34 builds up the continuous capsid shell, while VP27 and VP25 form the dimeric projections seen in the cryo-EM reconstruction image [14, 23]. In an attempt to express the projection/spike domain, Dong et al. made five constructs containing residues 394–646, 501–658, 415–628, 501–628, and 415–646, respectively . Of these five constructs, only P2415–646 produced soluble proteins in E. coli, presumably because it encompasses an intact spike domain, and its two ends both terminate in flexible domain linkers. Considering that the N-terminal ends of VP27 and VP25 start from Gln394 and Ser424, respectively , P2415–646 is a little longer than VP25 but a bit shorter than VP27 at the N-terminus. P2415–646 exists as a dimer in solution as judged by gel filtration chromatography .
Purified P2415–646 dimer was crystallized in two different crystal forms of space group P3221 and I213, respectively. The I213 crystals, grown at pH 4.6, diffracted to only 2.8 Å possibly due to its high solvent content (82.8%). The P3221 crystals, which were grown at pH 9.5, diffracted far better to 1.8 Å resolution (solvent content = 41.0%). Both structures have been determined, but it was found that the two structures were essentially the same with a RMSD of only 0.548 Å for all Ca atoms, despite the fact that these two crystals were grown at very different pHs and had different crystal packing interactions. Amino acid residues 430–645 of P2415–646 were modeled in the crystal structure. As in solution, P2415–646 formed dimers in both crystal forms around the twofold crystal symmetry axes. As discussed below, the crystal structure of the astrovirus P2415–646 spike provides a first glimpse into the astrovirus capsid structure at the atomic level (PDB ID: 3QSQ).
The P2415–646 dimer appears to be very stable. The buried surface area at the dimer interface is ∼3,490 Å2, which counts for 20.2% of the solvent-accessible area of the complex. Therefore, the dimer interaction mediated by the projection domain is likely to play an important role in stabilizing the viral capsid. Because the astrovirus projection domain is connected to the capsid shell at its N-terminus, the thinner end of the projection dimer (where both the N- and C-termini are located) should directly abut the capsid outer surface, whereas the opposite, flat end would point away from the capsid (Fig. 3.3a). When visually compared to a cryo-EM reconstruction image of an astrovirus (Fig. 3.1), the overall morphology of the P2415–646 dimer matches that of the dimeric surface spikes.
The projection dimer interface is primarily mediated by loops L3 and L4 and strands β1 and β11 (Fig. 3.3). In particular, two L3 loops from the two subunits of a dimer reach over to the opposing subunit like two crossing arms, making extensive interactions along their entire length. Sitting on top of the projection domain dimer, L3 also makes close contacts with the L1 and L4 loops from both subunits. A total of 51 residues from each subunit are observed at the dimer interface, among which 29 of the residues are highly conserved. Among the nonconserved interface residues, 12 residues interact with the other subunit via backbone atoms. Except the protruding L3 loop, residues at the dimer interface create a nearly flat surface in between the two molecules that is mostly hydrophobic in nature. The protruding L3 loop, however, contains mostly polar/charged residues and thus predominantly makes hydrogen bonds at the dimer interface. For example, 20 of the 26 intermolecular hydrogen bonds involve residues from the protruding L3 loop. Intermolecular hydrogen bonds are mediated by either main-chain atoms or side-chain atoms of highly conserved residues (i.e., Gln450, Tyr555, Arg558, and Arg572). The highly conserved nature of these inter-subunit hydrogen bonds indicates an important functional role and suggests that they may determine the specificity of dimer interaction.
Putative Receptor Binding Site(s)
A close inspection of the P-site shows that it is largely hydrophilic in nature, and is abundant in hydrogen bond donors and acceptors (Fig. 3.4d). In addition to the strictly conserved residues Lys455, Ser 554, Thr575, and Glu610, other residues found in the vicinity are Thr456 (Pro in serotype 1), Tyr552 (His in serotypes 5, 6, and 7), and Asn613 (Thr in serotype 1, Ser in serotype 2), together with main-chain carbonyls from Asp573 and Leu611. These residues mainly come from the three loops L1, L3, and L4 (Fig. 3.4d). The polar residue composition, especially the positively/negatively charged pair Lys455 and Glu610, is also observed in the receptor binding sites of several viruses that use oligosaccharide moieties as receptors (e.g., influenza A virus and norovirus) [10, 17], suggesting that human astroviruses are likely to use a polysaccharide molecule as a cell receptor. Further molecular modeling and docking indicate that the P-site may be able to accommodate a di- or trisaccharide moiety. Consistent with structural analysis, the addition of heparin, heparan sulfate, or dextran sulfate was able to partially block HAstV-8 infectivity in a consistent manner . Among the three polysaccharides used, heparin had the most pronounced effect.
Structure Comparison with HEV
In addition to the similar structural folds, several major structural differences were also observed between the two viral spikes (Fig. 3.5b). First, the loop L2 that runs across the bottom of the core β-barrel of astrovirus P2415–646 is replaced by two β-strands (β4 and β5) and a one-turn α-helix in HEV. Second, the long β-hairpin β6 and β7 in astrovirus P2415–646 is replaced with two short strands (β7 and β9) with a loop in between that contains an extra β-strand (β8) in HEV. Third, there are two extra β-hairpin insertions in astrovirus, one between β1 and β4 and the other between β7 and β10. Fourth, β6, β7, and β10 in astrovirus CP are much longer than their counterparts in HEV P2. In astrovirus, these three β-strands form a small protruding knob on each side of the surface spikes. Because of these insertions and also because of its longer β-strands, the astrovirus P2 domain is substantially larger in size compared to the HEV P2 (232 vs. 143 residues), which could account for the extended radius of the HAstV spikes (Fig. 3.2).
The P2 dimer interaction is mediated by similar structural elements in both astrovirus and HEV, as structure superposition based on P2 monomers also placed the P2 dimers on top of each other, with the two dimer axes differing by only ∼14°. Therefore, the inclination angle of the β-barrel with respect to the icosahedral twofold symmetry axis must be similar in astrovirus and HEV, but the azimuthal rotation of the dimeric spike about the icosahedral twofold axis is rather different in these two viruses. Like in the HEV CP, both N- and C-termini of the astrovirus P2 are located at the bottom of the spike close to the particle surface.
Structure Modeling of the Human Astrovirus Capsid and Insights into Maturation
Based on the noted structural similarity between the astrovirus and HEV CPs, an attempt was made to create an atomic structure of the astrovirus CP by homology modeling . Indeed, homology structure recognition based on the astrovirus CP sequence alone indicated that the HEV CP is the best structural homolog . In particular, residues 82–266 and 267–407 of the human astrovirus (serotype 8) CP were matched to the S (aa118–313) and P1 (aa314–453) domains of the HEV CP, respectively. Based on this alignment, a homology model of the human astrovirus CP was created with the HEV CP structure as a template  (Fig. 3.6). By aligning the S and P1 domains of the astrovirus CP model onto the three quasi-equivalent HEV CP molecules from the T = 3 EM model , a T = 3 astrovirus capsid structure was generated . The astrovirus capsid model closely resembles the cryo-EM reconstruction of astrovirus in terms of particle diameter and the capsid landscape variation. The close resemblance of the two structures further confirms the notion that astroviruses are phylogenetically related to HEV.
During maturation, the immature viral particles are processed by extracellular proteases to produce smaller peptides species that are observed in infectious particles. VP34 is derived from the conserved region, while VP27 and VP25 are from the hypervariable region of the astrovirus CP  (Fig. 3.6). The N-terminal end of VP27 and VP25 starts from Gln394 and Ser424, respectively . Based on the astrovirus capsid model, the N-terminal cleavage site of VP27 (Gln394) was mapped to the unstructured loop right before the last β-strand in the P1 domain. Therefore, VP27 should be able to remain associated with the viral particle through main-chain H-bonds mediated by this last β-strand in the P1 domain. The N-terminal cleavage site of VP25, however, is located in an extended linker (the P1–P2 linker) between the P1 and the P2 domain. Therefore, VP25 may need to dimerize with a VP27 to remain stably bound to the viral particle.
While the astrovirus CP is larger than HEV CP by ∼120aa, there may be additional similarities in their capsid assembly processes. HastV-8 has been shown to have caspase processing at the TYVD657 sequence , which removes the last ∼125 amino acids for the VP90 conversion to VP70 and is required for viral release. Although this particular sequence is not conserved in all astroviruses, they all contain potential caspase cleavage sequences within amino acids 650–700 of their CP polypeptides. Because HEV assembly has only been achieved in vitro with the deletion of ∼50 C-terminal amino acids, HEV may have a similar cleavage requirement for particle release. As very little is known about HEV infectivity, it is tempting to speculate that those viruses may also require extracellular protease cleavage for activation, which may be similarly asymmetric to that observed for astrovirus.
Avian Astrovirus Capsid Spike Domain Structure
Human Astrovirus Protease Structure
The astrovirus genome open reading frames ORF1a and ORF1b encode the nonstructural proteins nsp1a and nsp1b, respectively, which are involved in viral replication. The nonstructural proteins are synthesized as polyprotein precursors that become proteolytically processed. Although the exact processing sites remain undefined, a 3C-like serine protease motif was found to be encoded at the 3′-end of ORF1a and is thought to play an important role in the processing events . Further studies show that mutation of the astrovirus protease motif resulted in deficient or altered processing of nsp1a translated in vitro  or in BHK cells .
However, the astrovirus protease has several structural features that distinguish it from other viral proteases . First, two loops near the active site, loop eI-fI and loop cII-dII, are shorter than most viral proteases. The result of this is a more open and solvent-exposed active site. Second, the active site residue Asp489 is oriented away from His461 and does not form the hydrogen bond normally seen in trypsin-like proteases. Instead, a water molecule forms the hydrogen bond. The authors note that Asp489 likely undergoes a conformational change during substrate binding, as seen in hepatitis A virus protease [6, 38], although the authors do not rule out the possibility of an atypical catalytic mechanism. Last, the astrovirus protease substrate binding pocket S1 is more shallow than other viral proteases and has a positive electrostatic potential. Consistent with this structural observation, hydrolytic activity assays show that the astrovirus protease S1 pocket has specificity for negatively charged Asp and Glu residues. Furthermore, the hydrolytic activity is dependent on a functional astrovirus protease active site because the Ser551Ala mutant was inactive.
Taken together, the astrovirus protease structure reveals a trypsin-like serine protease with a specificity for cleavage at negatively charged residues Asp and Glu. Several distinctive structural features of the astrovirus protease may allow for further specificity during cleavage of the nonstructural polyprotein processing events.
Astrovirus structure and assembly are particularly intriguing in several aspects. First, a ∼125aa C-terminal domain of the viral CP is removed by host caspase following assembly. The exact function of this C-terminal domain is not known but it may facilitate the sorting/trafficking of the viral CP to promote assembly. Additionally, extensive extracellular protease processing of the viral capsid is required for virus infectivity and maturation, which is rather unusual for small, positive-sense RNA viruses. In this chapter, we have primarily focused our discussion on the cryo-EM reconstructions of the immature and mature astrovirus, the crystal structures of the human astrovirus and turkey astrovirus capsid spikes, and the crystal structure of the human astrovirus protease. Altogether, these new findings have led a better understanding of many facets of the astrovirus replication, including astrovirus assembly, virus release, maturation, receptor binding, antibody neutralization, and nonstructural polyprotein processing. Of course, these findings would not be possible without many years of pioneering virological work that is not fully discussed here. We also realize that the ultimate understanding of the astrovirus assembly and maturation will require further characterization of the viral capsid/capsid proteins by structural as well as biochemical, genetic, and cell biology approaches.
Although there is a clear structural homology between the astrovirus and HEV capsid proteins, the replication mechanism of the astrovirus appears to differ from that of HEV. Unlike HEV, which is known to encode both methyltransferase and guanylyltransferase activities within the 5′ half of its ORF1, no capping enzymes have been identified in the astrovirus nonstructural proteins. Only three enzymes, the viral protease, a viral polymerase, and a putative helicase, have been found in the astrovirus genome. Interestingly, phylogenetic analysis suggests that the astrovirus genome might encode a VPg with sequence similar to the VPgs from Caliciviridae and Potyviridae . We expect that further studies on astrovirus and other small, nonenveloped RNA viruses, both on their capsid and their replication, would help to establish their phylogenetic relationships, which, in turn, would greatly facilitate further structural and functional characterization of these viruses.
The authors are supported by the Welch Foundation (C-1565 to YJT) and the National Institutes of Health (R01 GM066087 to MY).
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