Introduction 1,2 Human astroviruses (HAstVs) are an important cause of gastroenteritis in children, the elderly, and immunocompromised adults. The virus was first identified in 1975 in infants who developed diarrhea in hospital nurseries in the United Kingdom. HAstV is a small, single-stranded, positive-sense RNA virus with an ~6.8-kb genome. The 5'-terminal two-thirds of the genome [open reading frame (ORF)-1a and ORF-1b] encodes two major nonstruc- tural proteins, a serine protease whose structure has been solved by X-ray crystallography³ and an RNA- dependent RNA polymerase. The 3'-terminal one- third of the genome (ORF-2) encodes the structural protein VP90, which is translated as an 87- to 90-kDa capsid precursor protein. VP90 has a highly con- served N-terminal domain and a variable C-terminal domain among astrovirus serotypes. To date, eight serotypes have been identified of which type 1 is the most prevalent, and type 8 has been studied extensively. While their genomes are fairly well conserved, 4-6 the mature virions of different serotypes vary somewhat in the exact size and number of structural proteins that form the capsid (reviewed by Krishna 7). The current consen- sus is that morphogenesis of infectious particles relies on a series of proteolytic cleavages of the capsid precursor protein (Fig. 1). VP90 assembles in infected cells and undergoes C-terminal cleavages by caspases to generate VP70 (a 70- to 79-kDa protein) for release from the cells as immature virus particles. Generation of infectious particles is dependent upon further trypsin cleavages usually resulting in three structural proteins, VP34, VP27, and VP25, each of which range in actual size and name depending on the serotype. VP34 contains the conserved N-terminal domain and is ascribed to the capsid, while the overlapping VP27 and VP25 subunits contain the variable C-domain and are 8 9-11 11 ascribed to the viral spikes. 7,12 VP25 is generated by additional trimming at the N-terminus of VP27." Astroviruses were initially named for the distinc- tive star-like appearance of their viral surface observed in ~10% of the fecally shed viral particles evaluated by negative-stain electron microscopy (EM). EM ultrastructural analysis of infectious HAstV generated in cell culture showed spherical particles with a surface that was studded with spikes and an external diameter of ~410 Å. 13 A recent milestone was determination of a high- resolution X-ray structure of the P-domain of the astrovirus spike VP25 (ordered residues 430-645). 12 Similarities in sequence and domain organization of hepatitis E virus (HEV) and HAstV enabled the building of a homology model of HAstV. In this study, we sought to gain direct insight into the structure and assembly of astrovirus by electron cryomicroscopy (cryoEM) of immature and proteo- lytically activated particles. We examined (1) imma- ture HAstV-8 particles composed of the 70-kDa intermediate protein, which has undergone caspase but not trypsin cleavage and is therefore not infectious, and (2) fully cleaved, mature infectious particles of HAstV-1. The sequences of the two strains are extremely similar, with 83% identity in the conserved domain and 60% identity in the variable domain. Therefore, we expect that our three-dimensional (3D) reconstructions are repre- sentative of the Astroviridae family. Although the capsid shells of both immature and mature HAstVs are nearly identical, there is a striking difference in the apparent stoichiometry of the surface spikes. Immature, uncleaved particles, which are strikingly similar in appearance to HEV-like particles (HEV-lp)," VP90 VP70 VP34 conserved region variable region intracellular cleavage 787 A XXXXX 657 extracellular cleavage VP27 394 648 313 VP25 424 Fig. 1. Protein organization and proteolysis. Schematic diagram showing proteolytic processing of the HAstV coat protein. Sites for caspase and trypsin cleavage are indicated with open and filled arrowheads, respectively. Trypsin cleavage of the coat protein between the conserved (white boxes) and variable (shaded boxes) domains is required for viral maturation. The specific pattern of peptide products varies between serotypes. In these experiments, we examined uncleaved particles of HAstV-8 formed by VP70 and fully cleaved particles of HAstV-1.